UNIVERSIDAD DE CÓRDOBA Departamento de Genética

Mejora genética del contenido en glucosinolatos en semillas de mostaza etíope (Brassica carinata A. Braun) Angustias Márquez Lema

TITULO: Mejora genética del contenido en glucosinolatos en semillas de mostaza etíope (Brassica carinata A. Braum) AUTOR: Angustias Márquez Lema © Edita: Servicio de Publicaciones de la Universidad de Córdoba. 2008 Campus de Rabanales Ctra. Nacional IV, Km. 396 14071 Córdoba www.uco.es/publicaciones [email protected]

ISBN-13: 978-84-7801-921-2 D.L.: CO-186-2009

UNIVERSIDAD DE CÓRDOBA DEPARTAMENTO DE GENÉTICA

TESIS DOCTORAL

Mejora genética del contenido en glucosinolatos en semillas de mostaza etíope (Brassica carinata A. Braun)

Doctoranda:

Angustias Márquez Lema

Directores: Leonardo Velasco Varo Begoña Pérez Vich

Córdoba, Septiembre de 2008

Trabajo presentado por Angustias Márquez Lema para la obtención del grado de Doctor por la Universidad de Córdoba

Fdo.: Angustias Márquez Lema Córdoba, Septiembre de 2008

Directores:

Leonardo Velasco Varo

Begoña Pérez Vich

Científico Titular

Científico Titular

Instituto de Agricultura Sostenible (CSIC)

Instituto de Agricultura Sostenible (CSIC)

Córdoba

Córdoba

Fdo.: Leonardo Velasco Varo

Fdo.: Begoña Pérez Vich

Córdoba, Septiembre de 2008

D. Leonardo Velasco Varo, Científico Titular del Instituto de Agricultura Sostenible (CSIC) de Córdoba,

INFORMA:

Que el trabajo titulado “Mejora genética del contenido en glucosinolatos en semillas de mostaza etíope (Brassica carinata A. Braun)”, realizado por Angustias Márquez Lema, bajo la dirección del Dr. Leonardo Velasco Varo y de la Dra. Begoña Pérez Vich, puede ser presentado para su exposición y defensa como Tesis Doctoral en el Departamento de Genética de la Universidad de Córdoba.

Considerando que se encuentra concluida, doy el VºBº para su presentación y lectura.

Fdo.: Leonardo Velasco Varo Córdoba, Septiembre de 2008

Dña. Begoña Pérez Vich, Científico Titular del Instituto de Agricultura Sostenible (CSIC) de Córdoba,

INFORMA:

Que el trabajo titulado “Mejora genética del contenido en glucosinolatos en semillas de mostaza etíope (Brassica carinata A. Braun)”, realizado por Angustias Márquez Lema, bajo la dirección del Dr. Leonardo Velasco Varo y de la Dra. Begoña Pérez Vich, puede ser presentado para su exposición y defensa como Tesis Doctoral en el Departamento de Genética de la Universidad de Córdoba.

Considerando que se encuentra concluida, doy el VºBº para su presentación y lectura.

Fdo.: Begoña Pérez Vich Córdoba, Septiembre de 2008

Dña. Teresa Millán Valenzuela, Profesora Titular de la Universidad de Córdoba, tutora de esta Tesis,

INFORMA:

Que el trabajo titulado “Mejora genética del contenido en glucosinolatos en semillas de mostaza etíope (Brassica carinata A. Braun)”, realizado por Angustias Márquez Lema, bajo la dirección del Dr. Leonardo Velasco Varo y de la Dra. Begoña Pérez Vich, puede ser presentado para su exposición y defensa como Tesis Doctoral en el Departamento de Genética de la Universidad de Córdoba.

Considerando que se encuentra concluida, doy el VºBº para su presentación y lectura.

Fdo.: Teresa Millán Valenzuela Córdoba, Septiembre de 2008

“Hay que esperar cuando se está desesperado, y andar cuando se espera”

Gustave Flaubert (1821-1880)

A Manolo

Agradecimientos

A los doctores Leonardo Velasco Varo y Begoña Pérez Vich, directores de esta Tesis, por su inestimable ayuda en la dirección de este trabajo, disposición y dedicación a lo largo de la realización de este trabajo. Al Dr. José Mª Fernández Martínez, por ayudarme en cualquier cosa que he necesitado y por su dedicación que me ha servido como ejemplo. A la Dra. Teresa Millán Valenzuela, tutora de este trabajo, por su disposición en todo el período de realización de la Tesis. A los investigadores Mathilde Grelon, Fabien Nogué, Philippe Guergue, Ghislaine Gendrot, Christine Mézard, Liudmila Chelysheva y Daniel Venzon, por brindarme la oportunidad de trabajar en su equipo de la Station de Génétique et d´Amélioration des Plantes de l´Institut National de la Recherche Agronomique (INRA-Versailles, Francia). Al doctor Antonio Martín Muñoz por su colaboración e interés demostrado durante los análisis citológicos de Brassica. Sería muy largo mencionar a todas las personas a las que me gustaría agradecerle su amistad y apoyo, por ello os doy las gracias a todo el grupo de Oleaginosas del Instituto de Agricultura Sostenible (CSIC), y muy especialmente a mis compañeros becarios Yamen Hamdan, Mª José García, Lidia Del Moral, Álvaro Fernández y Rocio Pineda. A mis amigos los doctores Abdelghani Nabloussi y Behailu Guta, que han compartido conmigo tantos buenos momentos. A todo el equipo humano que ha colaborado en las largas y duras recolecciones de plantas durante los calurosos meses de verano, y muy especialmente a Alberto Merino y Plácida Nieto. A mis compañeros de despacho Juan Emilio Palomares, Azahara Carmen Martín y Sonia Álvarez, que durante estos años han compartido conmigo largos momentos de entusiasmo y preocupación común. Al Ministerio de Educación y Ciencia por la concesión de la beca predoctoral dentro del programa de Formación de Personal Investigador (FPI) y al proyecto AGL2001-2293 por su financiación. A mis abuelos, padres y hermanos, por no olvidarse de mí. Y a Manolo, mi marido, por darme la dosis de tranquilidad y optimismo que tantas veces he necesitado y que han sido claves para que esta Tesis haya sido terminada.

RESUMEN La mostaza etíope (Brassica carinata A. Braun) es una especie oleaginosa que tiene gran potencial agronómico como cultivo en zonas de clima semiárido. Sin embargo, B. carinata no se utiliza en la actualidad como cultivo oleaginoso debido al elevado contenido de compuestos tóxicos denominados glucosinolatos, presentes principalmente en sus semillas. Hasta la fecha no se ha descrito la obtención de líneas de B. carinata con niveles bajos y estables de glucosinolatos en semillas. La presente Tesis se enmarca en una línea de investigación dirigida al desarrollo de líneas de B. carinata con niveles reducidos de glucosinolatos en semillas y al estudio genético de la síntesis de glucosinolatos en esta especie. Los objetivos específicos de la Tesis fueron: (1) el estudio genético del contenido en glucosinolatos en semillas en una línea de B. carinata con niveles elevados de estos compuestos, (2) la evaluación de la transferibilidad de marcadores microsatélites (SSRs) desarrollados en B. nigra (BB) y B. napus (AACC) a B. carinata (BBCC) para su aplicación en programas de mejora genética de esta especie, (3) el estudio comparativo de varias líneas de B. carinata con niveles reducidos en glucosinolatos en semillas y selección de segregrantes transgresivos para éste carácter, y (4) reducción del contenido en glucosinolatos en semillas de B. carinata mediante hibridación interespecífica. En esta Tesis se ha determinado que el contenido elevado en glucosinolatos en semillas de la línea N2-6215 presenta una moderada heredabilidad (0.45 ≤ h2b ≤ 0.58; 0.35 ≤ h2n ≤ 0.50), y está controlado por un reducido número de genes, estimado entre dos y tres. Se ha puesto de manifiesto la buena transferibilidad y capacidad discriminatoria de los marcadores SSR de B. nigra (L.) Koch (genoma B) y B. napus L. (genoma AC) para su aplicación a la mejora de B. carinata (genoma BC). Como consecuencia de los cruzamientos realizados, se han generado nuevas líneas de B. carinata con niveles reducidos y estables en glucosinolatos totales en semillas que no habían sido obtenidos hasta ahora en esta especie. Así, mediante un programa de cruzamientos intraespecíficos se han identificado líneas segregantes con niveles de glucosinolatos en semillas inferiores al nivel mínimo encontrado en sus respectivos parentales (52 µmoles g-1 frente a 73 µmoles g-1), y se ha realizado mediante hibridación interespecífica la transferencia de genes para bajo contenido en glucosinolatos desde B. juncea doble cero (< 30 µmoles g-1 en semillas) a B.

carinata, dando lugar a una línea de B. carinata (BCH-1773) con niveles reducidos de glucosinolatos en semillas (42 µmoles g-1) y tipo de planta B. carinata, determinado tanto mediante descriptores fenotípicos como mediante los marcadores SSR transferidos a B. carinata en el transcurso de esta Tesis.

Introducción general

1

1. La mostaza etíope

3

2. Relación de la mostaza etíope con otras especies cultivadas del género

5

Brassica 3. Estado actual y perspectivas de futuro del cultivo de la mostaza etíope y otras

7

especies cultivadas del género Brassica 4. Mejora genética de la calidad de la semilla en la mostaza etíope y otras especies

8

cultivadas del género Brassica 4.1. Contenido en aceite y proteína

8

4.2. Contenido en glucosinolatos

10

4.3. Tamaño y color de la semilla

16

5. Biosíntesis y transporte de glucosinolatos

17

5.1. Biosíntesis de glucosinolatos

17

5.2. Mecanismos de transporte de los glucosinolatos

21

6. Estudios genéticos sobre el contenido en glucosinolatos en la mostaza etíope y otras 22 especies cultivadas de Brassica

CAPÍTULO 2

7. Uso de marcadores moleculares en Brassica

23

8. Objetivos

27

9. Referencias

28

Inheritance of very high glucosinolate content in Ethiopian mustard

45

seeds Abstract

47

Introduction

48

Materials and Methods

49

Plant material and genetic study

49

Analysis of total seed glucosinolate content

50

Statistical analyses

51

Results

51

Discussion

52

Acknowledgements

54

References

54

ÍNDICE E GENERAL ÍNDIC

CAPÍTULO 1

CAPÍTULO 3

Transferability, amplification quality and genome specificity

57

of microsatellites in Brassica carinata and related Brassica species

CAPÍTULO 4

Abstract

59

Introduction

60

Materials and Methods

61

Plant materials

61

DNA extraction and PCR analysis

62

Quality of SSRs transferability

62

Cluster analysis

62

DNA sequencing

63

Results

63

Discussion

68

Acknowledgements

70

References

70

Transgressive segregation for reduced glucosinolate content in

75

Brassica carinata A. Braun

CAPÍTULO 5

Abstract

77

Introduction

78

Materials and Methods

79

Plant materials

79

Crossing and selection scheme

79

Analysis of glucosinolate content

80

Results and Discussion

80

Acknowledgements

84

References

84

Development and characterisation of a Brassica carinata inbred line

87

incorporating genes for low glucosinolate content from B. juncea Abstract

89

Introduction

90

Materials and Methods

91

Plant materials

91

Crossing and selection scheme

92

Phenotypic characterisation of BC1F4 derived lines

93

Molecular characterisation of BC1F4 derived lines

93

Statistical analyses

94

Analyses of glucosinolate content

95

Mitotic analysis

97 97

Results Selection from BC1F2 to BC1F4 plant generations

97

Phenotypic and molecular characterisation of BC1F4 derived lines

98

Selection and evaluation of lines

101

Discussion

103

Acknowledgements

106

References

106

CONCLUSIONES FINALES

111

ANEXO

115 Justificante del editor jefe de la revista Plant Breeding para el artículo

117

aceptado: “Inheritance of very high glucosinolate content in Ethiopian mustard seeds”

Justificante del editor jefe de la revista Breeding Science para el artículo en revisión: “Transferability, amplification quality and genome specificity of microsatellites in Brassica carinata and related Brassica species”

118

CAPÍÍTULO 1 CAP

Introducción general

Márquez Lema A.

Capítulo 1

INTRODUCCIÓN GENERAL La mostaza etíope (Brassica carinata A. Braun) es una especie oleaginosa de gran interés para la agricultura de secano del sur de España, debido a que presenta una mejor adaptación y comportamiento agronómico a estas condiciones que otras especies oleaginosas del género Brassica como la colza (B. napus L.) y la mostaza india (B. juncea (L.) Czern.) (Fereres et al. 1983). Pese a estas cualidades, B. carinata no se utiliza en la actualidad como cultivo oleaginoso en Occidente debido principalmente al elevado contenido de glucosinolatos (compuestos tóxicos) en sus semillas (Downey y Röbbelen 1989). Por tanto, la reducción de los glucosinolatos mediante mejora genética es uno de los requisitos previos para hacer posible su utilización como cultivo innovador en zonas de secano del sur de Europa, y especialmente en Andalucía, donde actualmente existe un reducido número de cultivos incluidos en las rotaciones de secano (principalmente trigo y girasol).

1. La mostaza etíope La mostaza etíope (Brassica carinata A. Braun) (Figura 1.1) es una especie oleaginosa del género Brassica perteneciente a la familia Brassicaceae (Crucíferas). Se encuentra emparentada con otras especies del género Brassica como son la colza (B. napus L.), la mostaza india (B. juncea (L.) Czern.), la mostaza negra (B. nigra (L.) Koch), la nabina (B. rapa L.), y el grupo de las coles (B. oleracea L.), incluyéndose en el grupo de las mostazas. Brassica carinata es una especie anfidiploide de configuración genómica BBCC (n = 17), considerada resultado de la hibridación natural entre las especies diploides B. nigra (BB, n = 8) y B. oleracea (CC, n = 9) (U 1935). Aunque no se han encontrado formas silvestres de B. carinata, actualmente las tres especies conviven y se cultivan de forma tradicional en la meseta etíope, su probable lugar de origen (Vavilov 1951, Hemingway 1979, Tsunoda 1980). En esta zona, B. carinata se cultiva tanto para la obtención de aceite como para consumo de sus hojas como verdura (Tcacenco et al. 1985). Fuera de su lugar de origen, B. carinata presenta un enorme interés para la agricultura en zonas de clima semiárido, como se ha puesto de manifiesto en estudios realizados en California (Cohen y Knowles 1983), India (Anand et al. 1985, Katiyar et al. 1986, Singh et al. 1988, Malik

-3-

Márquez Lema A.

Capítulo 1

1990), Italia (Mazzoncini et al. 1993), y sur de España (Fernández-Martínez y Domínguez 1981, Fereres et al. 1983), debido a que muestra una mejor adaptación y comportamiento agronómico (Fereres et al. 1983, Getinet et al. 1996a), y una amplia resistencia a la mayoría de enfermedades (Anand et al. 1985, Sacristán y Gerdemann 1996, Choudhary et al. 2000) y plagas (Malik 1990, Rana 2005) que afectan a otros cultivos de Brassica.

Figura 1.1.- Plantas de Brassica carinata A. Braun cultivadas en Córdoba (España) en el año 2005

La alta productividad de B. carinata es consecuencia directa de las características morfológicas y fisiológicas que presenta esta especie respecto a otras especies cultivadas del género Brassica (Tabla 1.1). Una de las grandes ventajas de B. carinata es su gran capacidad de resistencia a condiciones de sequía y de régimen hídrico escaso, debido al mantenimiento de un prolongado período de floración y llenado del grano, una gran producción de silicuas por unidad de superficie, un desarrollo rápido y exuberante de materia verde en estado de roseta, y un poderoso sistema radicular (Fernández-Serrano 1991).

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Márquez Lema A.

Capítulo 1

Tabla 1.1.- Ventajas agronómicas de B. carinata respecto a B. napus y otras especies cultivadas del género Brassica.

Características morfológicas y fisiológicas de

Ventajas agronómicas

B. carinata

Sistema radicular poderoso con gran capacidad de

Superación de obstáculos

ramificación y tendencia natural a profundizar

Extracción de agua de las capas profundas del suelo (Fernández-Serrano 1991)

Desarrollo rápido y exuberante de materia verde

Mayor

intercepción

de

radiación

solar

(estado de roseta)

(Fereres et al. 1983), y por tanto, mayor tasa de fotosíntesis previa a la antesis (aumento de la producción de granos) (Tayo y Morgan 1975) Posible utilización para la producción de energía

Desarrollo

ramificaciones

secundarias,

terciarias,

Mayor

desarrollo

de

2

frutos/m

(mayor

cuaternarias, etc.

rendimiento) (Fereres et al. 1983)

Amplio período de floración

Área de superficie verde durante más tiempo

Resistencia a la dehiscencia de las silicuas

Evita pérdidas de semillas en la cosecha (Josefsson 1968) Resistencia o tolerancia a enfermedades que

Resistencia o tolerancia a hongos Leptosphaeria

maculans

(enfermedad

del

pie

negro),

atacan

comúnmente

a

las

especies

Alternaria brassicae (mancha negra o gris), Peronospora

cultivadas de Brassica (Anand et al. 1985,

parasitica (mildiu velloso), Albugo candida (roya blanca)

Sacristán y Gerdemann 1986, Choudhary et al. 2000)

Tolerancia a áfidos

Mayor tolerancia a áfidos que otras especies

Lipaphis erysimi (áfido de la mostaza)

cultivadas de Brassica (Malik 1990, Rana 2005).

2. Relación de la mostaza etíope con otras especies cultivadas del género Brassica Las relaciones citogenéticas entre las especies cultivadas del género Brassica se pusieron de manifiesto gracias a los estudios llevados a cabo por Morinaga (1928, 1929a, 1929b, 1929c, y 1931), basados en la observación del apareamiento meiótico de híbridos interespecíficos. Estos

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Márquez Lema A.

Capítulo 1

estudios mostraron que de las especies cultivadas de Brassica, tres son diploides elementales (B. rapa, B. nigra y B. oleracea), y las tres restantes son alotetraploides (B. carinata, B. juncea y B. napus). Estas relaciones se conocen como triángulo de U (1935) (Figura 1.2) en honor a este investigador que sintetizó por primera vez una de estas especies alotetraploides.

Figura 1.2.- Triángulo de U, relaciones de parentesco entre las seis especies cultivadas del género Brassica y su correspondiente número de cromosomas (U 1935).

B. carinata BBCC n = 17

B. oleracea

CC

BB

n=9

n=8

AACC

AA

AABB

n = 19

n = 10

n = 18

B. napus

Las

B. nigra

especies

B. juncea

B. rapa

alotetraploides

(aloploides

naturales)

surgieron

por

hibridaciones

interespecíficas espontáneas entre los progenitores diploides con duplicación del número cromosómico, y contienen cada una dos de los tres genomas elementales (A, B y C). Esta hipótesis se verificó al sintetizar artificialmente B. napus (U 1935, Frandsen 1947, Olsson 1960b, Song y Osborn 1992), y B. juncea (Frandsen 1943, Prakash 1973, Olsson 1960a). Otros estudios basados en la anatomía (Berggren 1962), hibridación de ADN (Verma y Rees 1974), espectroscopia de proteínas (Vaughan 1977, Uchimiya y Wildman 1978), y otras investigaciones (Olsson 1960a y 1960b, Prakash 1973, Song y Osborn 1992), confirmaron el origen de las especies anfidiploides del triángulo de U (B. carinata, B. juncea y B. napus). Mediante estudios

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Márquez Lema A.

Capítulo 1

moleculares también se han confirmado estas relaciones, y se ha propuesto que las especies diploides B. rapa (AA) y B. oleracea (CC) derivan de un antecesor común diferente al antecesor de la especie diploide B. nigra (BB), que estaría más próxima al género Sinapis (Pradhan et al. 1992).

3. Estado actual y perspectivas de futuro del cultivo de la mostaza etíope y otras especies cultivadas del género Brassica Las semillas de las especies del género Brassica se caracterizan por presentar un elevado contenido en aceite. El aceite procedente de estas especies constituye la tercera fuente mundial de aceites vegetales por detrás de la soja y la palma, y por delante del girasol (Figura 1.3). Las brassicas oleaginosas empleadas para la extracción de aceite son B. rapa, B. napus, B. juncea y B. carinata. Juntas totalizan a nivel mundial una superficie media anual que oscila entre 24 y 31 millones de hectáreas en los últimos cinco años, con una producción anual de 37 a 50 millones de toneladas métricas. Entre éstas, la colza destaca como la más importante, mientras que la mostaza etíope ocupa el último lugar (FAO 2008).

Figura 1.3.- Producción media mundial de aceites vegetales durante los últimos cinco años. Datos

28

47

2008)

métricas

soja 210

expresados en millones de toneladas

17 3

palma brassica girasol

(FAO 172

oliva sésamo

Entre los principales productores de aceite de Brassica destacan China, India, Canadá, Francia, Alemania y Reino Unido. En Etiopía, el aceite extraído de la mostaza etíope supone la segunda fuente de aceite comestible, por detrás de Guizotia abyssinica (Tcacenco et al. 1985). Además del uso de las brassicas para la producción de aceite comestible, diferentes estudios han puesto de manifiesto resultados prometedores en cuanto a su utilización para producción de biodiesel (Green y Ramans 1995, Körbitz 1995, Cardone et al. 2003), biofumigación (Kirkegaard y Saward 1998, Kirkegaard et al. 1998, Lazzeri et al. 2004, Bellostas et al. 2007), y

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Márquez Lema A.

Capítulo 1

recuperación de suelos contaminados por metales pesados (Nanda-Kumar et al. 1995, Del Río et al. 2000). Adicionalmente, numerosos estudios han demostrado que las especies de Brassica contienen compuestos con efecto positivo en la prevención contra el cáncer (Stoewsand 1995, Plumb et al. 1996, Jongen 1996, Murillo y Mehta 2001, Uhl et al. 2003). A pesar de las potencialidades agronómicas anteriormente citadas (Tabla 1.1), la mostaza etíope no se cultiva a gran escala. Los principales factores que limitan el desarrollo de su cultivo son la presencia de factores tóxicos y antinutritivos en su semilla como son el ácido erúcico, presente en el aceite (> 35%) (Gopalan et al. 1974), y los glucosinolatos (> 110 µmoles g-1 de semillas) (Downey y Röbbelen 1989). El estado actual de la mejora genética para ambos compuestos se describe en el siguiente apartado.

4. Mejora genética de la calidad de la semilla en la mostaza etíope y otras especies cultivadas del género Brassica 4.1. Contenido en aceite y proteína El contenido en aceite y el contenido en proteína son dos componentes de la semilla que están negativamente correlacionados (Grami y Stefansson 1977, Röbbelen 1981). En el caso de la mostaza etíope, el contenido en aceite que presentan sus semillas es menor que el de otras especies de oleaginosas, por tanto su incremento es uno de los objetivos de la mejora genética de esta especie (Röbbelen y Thies 1980, Fereres et al. 1983, Cohen y Knowles 1983). Sin embargo, la selección para alto contenido en aceite conlleva una reducción en el contenido en proteína, por lo que sería necesario realizar una selección combinada de ambos componentes (Grami y Stefansson 1977). Atendiendo a este criterio, Velasco et al. (1997) desarrollaron líneas de B. carinata con mayor contenido en ambos componentes (aceite y proteína), en relación a la línea empleada como control. Asimismo, estos autores encontraron una correlación negativa muy alta (r = - 0.96), entre el contenido en aceite y el contenido en proteína. La calidad del aceite está determinada principalmente por su composición en ácidos grasos y por el uso al que se destine: alimentario o no alimentario. En las especies de Brassica, el aceite se caracteriza por presentar una gran cantidad de ácidos grasos monoenoicos de cadena larga: ácido eicosenoico (20:1) y ácido erúcico (22:1). Desde el punto de vista industrial, los

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aceites vegetales ricos en ácido erúcico se utilizan para la producción de lubricantes, plásticos y biodiesel (Grewal et al. 1993, Green y Ramans 1995). En cambio, desde el punto de vista nutricional, se ha demostrado en varias especies animales que los aceites con elevado contenido en ácido erúcico tienen un efecto perjudicial sobre la salud. Los efectos más frecuentes son lipidosis cardíaca y lesiones necróticas en diversos órganos (Gopalan et al. 1974, Ackman y Loew 1977). Aunque no está bien definido el efecto del ácido erúcico en la salud humana (McDonalds 1982), todo el aceite de Brassica destinado al consumo humano en los países occidentales carece de este ácido graso. Mediante mejora genética, se han conseguido desarrollar materiales libres de ácido erúcico en B. napus (Stefansson et al. 1961), B. rapa (Downey 1964), B. juncea (Kirk y Oram 1981), y en B. carinata (Alonso et al. 1991, Getinet et al. 1994, Fernández-Martínez et al. 2001), aunque en esta última especie, a diferencia de las anteriores, la reducción del ácido erúcico hizo que el nivel de ácido linolénico (18:3) superase el 20%, lo que determinó problemas de enranciamiento del aceite que dificultaron su uso. También se ha desarrollado germoplasma con alto contenido en ácido erúcico (> 55%) en B. napus (Sasongko y Möllers 2005) y B. carinata (Velasco et al. 1998). Otros ácidos grasos de interés para usos alimentarios o industriales presentes en el aceite de Brassica son el ácido oleico (18:1), el ácido linoleico (18:2) y el ácido linolénico (18:3). Desde el punto de vista nutricional, los ácidos grasos linoleico y linolénico son esenciales y no pueden ser sintetizados por el hombre, por lo que es imprescindible que se tomen en la dieta diaria (Gunstone 1992). Su carencia provoca problemas en prácticamente todos los tejidos humanos, si bien un incremento de los niveles de estos dos ácidos grasos en la dieta juega un importante papel en la prevención de enfermedades cardiovasculares, ya que reducen los niveles de colesterol total y de lipoproteínas de baja densidad (LDL) en sangre, evitando su deposición en las paredes arteriales y, por tanto, disminuyendo el riesgo de arteriosclerosis (Vles y Gottenbos 1989). Asimismo, se ha demostrado que el ácido oleico es tan eficiente como los ácidos linoleico y linolénico en la reducción del colesterol y de las LDL en sangre (Mattsson y Grundy 1985, Mensik y Katan 1989). A pesar de sus propiedades nutricionales, el aceite con elevado contenido en ácido linolénico tiene tendencia a oxidarse. Dicha oxidación, favorecida por las altas temperaturas, como

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por ejemplo en las frituras, deteriora la calidad del aceite, y produce una serie de compuestos que pueden resultar tóxicos (Renard et al. 1992), por lo que para mejorar la estabilidad del aceite se recomienda disminuir el contenido en ácido linolénico, y aumentar el contenido en ácido oleico, puesto que este último presenta una elevada estabilidad oxidativa (Yodice 1990). Para usos industriales, los aceites ricos en ácido oleico son adecuados para la producción de lubricantes (Fick 1983, Röbbelen 1991, Harold et al. 1995), y biodiesel (Körbitz 1995). En cuanto al ácido linolénico, un alto nivel de este ácido graso en el aceite es útil para la fabricación de pinturas y productos relacionados (Young et al. 1994). Por tanto, dependiendo del uso final al que se destine el aceite (industrial o nutricional), los objetivos de mejora de la calidad del aceite irán encaminados hacia la reducción o aumento de uno o varios ácidos grasos. Atendiendo a diferentes objetivos de mejora del aceite, se ha conseguido desarrollar germoplasma con alto contenido en ácido oleico (> 75%) en B. napus (Wong y Swanson 1991, Auld et al. 1992, Rücker y Röbbelen 1997, Stoutjesdijk et al. 1999), B. rapa (Auld et al. 1992, Vilkki y Tanhuanpää 1995), y B. juncea (Stoutjesdijk et al. 1999). Asimismo, se ha obtenido material con niveles reducidos de ácido linolénico (< 3%) en B. napus (Rakow 1973, Röbbelen y Nitsch 1975, Wong y Swanson 1991, Auld et al. 1992, Rücker y Röbbelen 1997), B. rapa (Auld et al. 1992, Laakso et al. 1999) y B. juncea (Sivaraman et al. 2004). Por último, en B. carinata se ha conseguido desarrollar germoplasma con alto contenido en ácido oleico (> 80%, Velasco et al. 2003), bajo contenido en ácido linolénico (< 2%, Velasco et al. 2004), alto contenido en ácido linoleico (> 58%) y alto contenido en ácido linolénico (> 25%) (Nabloussi et al. 2008).

4.2. Contenido en glucosinolatos Tras la extracción del aceite a partir de semillas de Brassica, se obtiene una harina rica en proteínas (aproximadamente un 40%) (Getinet et al. 1997). Esta harina resultante presenta una equilibrada composición en aminoácidos, y por tanto una elevada eficiencia biológica, que supera notablemente a las proteínas presentes en otras oleaginosas y cereales (De Haro et al. 2006). Sin embargo, el uso de la harina de Brassica para alimentación animal está limitado por la presencia en la misma de unos compuestos tóxicos y antinutritivos denominados glucosinolatos (Griffiths et al. 1998).

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Los glucosinolatos son metabolitos secundarios de bajo peso molecular presentes en once familias de dicotiledóneas. Pero es principalmente en el género Brassica donde se encuentra una tercera parte de los 100 tipos de glucosinolatos conocidos, responsables del peculiar sabor amostazado de estas plantas. Estos compuestos se encuentran tanto en los tejidos vegetativos como en los tejidos reproductivos de la planta, y constituyen un mecanismo de defensa de la misma ante plagas y enfermedades (Mithen 1992). El contenido en glucosinolatos varía según la especie, el clima, el tipo de suelo y la fase de desarrollo en la que se encuentre la planta (Tapper y Reany 1973, Guillard y Allison 1989, Ciska et al. 2000, Bellostas et al. 2004 y 2007). La molécula de los glucosinolatos está constituida por un grupo β-D-tioglucósido unido a una oxima sulfonada y a una cadena lateral (R) derivada de un aminoácido (metionina, valina, alanina, leucina, isoleucina, fenilalanina, tirosina o triptófano) (Mikkelsen et al. 2002) (Figura 1.4).

Figura 1.4.- Estructura general de los glucosinolatos

S ⎯ β-D-glucosa R⎯C N-OSO3

Dependiendo de la cadena lateral (R), los glucosinolatos se dividen en tres grandes grupos: alifáticos (derivados de la metionina, alanina, valina, leucina o isoleucina), aromáticos (derivados de la fenilalanina o tirosina) e indólicos (derivados del triptófano) (Wittstock y Halkier 2002) (Tabla 1.2).

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Tabla 1.2.- Estructura de la cadena lateral R de los glucosinolatos más comunes

Estructura de R

1.

Nombre químico

Nombre común

Glucosinolatos alifáticos

CH3-

Metilglucosinolato

Glucocaparina

CH2=CH-CH2-

2-propenil o alil-glucosinolato

Sinigrina

CH3-CH2-CHOH-

1-Hidroxi-2-propil-glucosinolato

Glucosisimbrina

CH3-CHOH-CH2-

2-Hidroxi-2-metilpropil-glucosinolato

Glucoconringina

CH3 CH2=CH-CH2-CH2-

But-3-enilglucosinolato

Gluconapina

CH3-CH2-CHOH-CH2-

2-Hidroxi-2-metilbutil-glucosinolato

Glucocleomina

CH3 CH2=CH-CH2-CH2-CH2-

Pent-4-enil-glucosinolato

Glucobrasicanapina

CH2=CH-CHOH-CH2-

2-hidroxi-2-metilbutil-glucosinolato

Progoitrina

CH2=CHOH-CH2-

(2S)-2-Hidroxibut-3-enil-glucosinolato

Epiprogoitrina

CH2=CH-CH2-CHOH-CH2-

(2R)-2-Hidroxipent-4-enil-glucosinolato

Napoleiferina

CH2=CH-CH2-CHOH-CH2-

2-hidroxipent-4-enil-glucosinolato

Gluconapoleiferina

CH3-S-CH2-CH2-CH2-

3-metiltiopropil-glucosinolato

Glucoibervirina

CH3-S-CH2-CH2-CH2-CH2-

4-metiltiobutil-glucosinolato

Glucoerucina

CH3-SO-CH2-CH2-CH2-

3-metilsufinilpropil-glucosinolato

Glucoiberina

CH3-S-CH2-CH2-CH2-CH2-

4-metiltiobutil-glucosinolato

Glucoerucina

CH3-SO-CH2-CH2-CH2-CH2-

4-metilsulfinilbutil-glucosinolato

Glucorafanina

CH3-SO-CH=CH-CH2-CH2-

4-metilsulfinilbut-3-enil-glucosinolato

Glucorafenina

CH3-SO-CH2-CH2-CH2-CH2-CH2-

5-metilsulfinilpentil-glucosinolato

Glucoalisina

CH3-SO2-CH2-CH2-CH2-CH2-

4-metilsulfonilbutil-glucosinolato

Glucoerisolina

Bencil-glucosinolato

Glucotropaolina

Feniletil-glucosinolato

Gluconasturtina

p-Hidroxibencil-glucosinolato

Sinalbina

Indol-3-ilmetil-glucosinolato

Glucobrasicina

R1=OCH3, R4=H

N-metoxi-indol-3-ilmetil-glucosinolato

Neoglucobrasicina

R1=SO3-

N-Sulfoindol-3-ilmetil-glucosinolato

Sulfoglucobrasicina

R1=H, R4=OH

4-Hidroxi-indol-3-ilmetil-glucosinolato

4-Hidroxiglucobrasicina

R1=H, R4=OCH3

4-metoxi-indol-3-ilmetil-glucosinolato

4-Metoxiglucobrasicina

2.

Glucosinolatos aromáticos

-CH2-

-CH2-CH2

HO-

3.

-CH2-

Glucosinolatos indólicos

R4 -CH2-

N R1

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En todos los tejidos también se encuentra la enzima mirosinasa o tioglucósido glucohidrolasa, encargada de catalizar la hidrólisis de los glucosinolatos tras producirse la ruptura de la célula (Höglund et al. 1991). Los glucosinolatos y las enzimas mirosinasas se encuentran localizados en la misma célula, pero en compartimentos independientes (Kelly et al. 1998). Los glucosinolatos se encuentran almacenados en vacuolas junto al ácido L-ascórbico, mientras que las enzimas mirosinasas se encuentran en idioblastos especializados ricos en proteínas denominados células de mirosina, que se localizan en el tejido parenquimático (Bones y Rossiter 1996). El sistema glucosinolato-mirosinasa se identificó por primera vez en semillas de B. juncea (Bussy 1840), y posteriormente se encontró en todas las especies crucíferas (Brassicaceae), así como en las familias Capparaceae, Tropaeolaceae, y Caricaceae, entre otras (Rodman 1991). También se han encontrado enzimas mirosinasas en el hongo Aspergillus niger (Ohtsuru et al. 1973), en la bacteria Enterobacter cloacae (Tani et al. 1974), en tejidos de mamíferos (Goodman et al. 1959), y en áfidos que atacan a crucíferas, Brevicoryne brassicae y Lipaphis erysimi (McGibbon y Beuzenberg 1978). En cuanto a la modulación de la actividad de las enzimas mirosinasas, se sabe que el ácido L-ascórbico juega un importante papel en el proceso de degradación de los glucosinolatos (Nagashima y Uchiyama 1959, Ettlinger et al. 1961, James y Rossiter 1991). El daño mecánico, infecciones, o ataques de plagas producen, como respuesta de defensa de la planta tras la rotura celular, en primer lugar, la disminución de la concentración de ácido L-ascórbico que determinará la activación de las enzimas mirosinasas y, en segundo lugar, la exposición de los glucosinolatos almacenados a la acción de estas enzimas (Höglund et al. 1991). Dichas enzimas catalizan la hidrólisis de los glucosinolatos produciendo como resultado de la misma glucosa, sulfato y, dependiendo del pH y/o de otros factores, algunos de los siguientes productos: isotiocianatos, nitrilos, cianoepitioalcanos y tiocianatos (Bones y Rossiter 1996) (Figura 1.6). Estos productos de degradación son los responsables del sabor amargo atribuido a las brassicas (Fenwick 1983), así como de la acción biológica de los glucosinolatos.

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Figura 1.6.- Hidrólisis de los glucosinolatos catalizada por la enzima mirosinasa (Bones y Rossiter 1996). Productos de hidrólisis: isotiocianatos (1), nitrilos (2), cianoepitioalcanos (3) y tiocianatos (4). Glc: glucosa, ESP= proteína epitioespecificadora. Enzima mirosinasa

S -Glc

S

R⎯C

-

R⎯C 3

H2O

NOSO

-

NOSO3

Glucosinolato

Tiohidroxamato-O-sulfonato (producto intermedio)

pH 7.0

RCNS + SO42- + Glc (1)

pH 4.0

ESP + Fe

S

RCN + S + SO42- + Glc (2)

2+

CN + SO42- + Glc (3)

RSCN + SO42- + Glc (4)

En animales monogástricos que se alimentan de harina con elevado contenido en glucosinolatos se producen efectos tóxicos y antinutritivos, entre los que destacan la reducción de la palatabilidad y la hipertrofia del tiroides (Rosa 1999), aunque no hay evidencias de este efecto en la alimentación humana (Mithen 2001). Por el contrario, estudios epidemiológicos realizados en humanos han demostrado que la incorporación a la dieta de vegetales con glucosinolatos proporciona una protección natural frente a agentes cancerígenos, puesto que inhiben la activación de sustancias procarcinogenéticas y activan la acción de enzimas detoxificantes (NAD(P)H-quinona reductasa y glutatión S-transferasa) (Murillo y Mehta 2001). Por otra parte, numerosos estudios han demostrado que los glucosinolatos y los productos de su hidrólisis presentan una amplia actividad biocida ante organismos patógenos presentes en el suelo, como hongos y nematodos (Angus et al. 1994, Buskov et al. 2002, Lazzeri et al. 2004). La eliminación de los glucosinolatos o la inactivación de la enzima mirosinasa mediante el procesado de la harina para su uso como pienso animal resulta demasiado costosa, además de causar pérdidas en la calidad de las proteínas. Por este motivo, la reducción del contenido en glucosinolatos en B. carinata mediante mejora genética se considera la alternativa más viable,

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puesto que se ha empleado con éxito en otras especies oleaginosas como B. rapa (Kondra y Stefansson 1970), B. napus (Stefansson y Kondra 1975), y B. juncea (Love et al. 1990). En todas estas especies se han obtenido niveles de glucosinolatos por debajo de 30 μmoles g-1 de harina desengrasada, que es el límite superior tradicionalmente considerado, junto con la ausencia de ácido erúcico (< 2% en el aceite), para la denominación de calidad Canola (Downey 1990). Las primeras evaluaciones de germoplasma de B. carinata mostraron una escasa variabilidad para bajo contenido en glucosinolatos en semillas. Getinet (1987) describió un contenido mínimo en glucosinolatos de 76 μmoles g-1 de harina desengrasada a partir de una colección de 867 entradas de B. carinata. De forma similar, Getinet et al. (1996b) identificaron genotipos B. carinata con contenido mínimo en glucosinolatos de 70 μmoles g-1 en semillas pertenecientes a una colección de germoplasma procedente de Etiopía. Velasco et al. (1999) encontraron un rango de variación entre 82 y 178 µmoles g-1 en una colección de germoplasma de esta especie. Mediante selección a partir de las entradas de germoplasma con menor contenido en glucosinolatos, estos autores desarrollaron la línea N2-142, con un contenido medio en glucosinolatos de 82 µmoles g-1 y un rango de variación entre 63 y 114 μmoles g-1 (Velasco et al. 1999). Más tarde, Alemayehu y Becker (2002) describieron un rango de variación menor (entre 90 y 111 μmoles g-1 en semillas) tras la evaluación de germoplasma de B. carinata en tres ambientes diferentes. Debido a la escasa variabilidad genética para bajo contenido en glucosinolatos encontrada en las colecciones de germoplasma de B. carinata, se han seguido otras estrategias de mejora genética para la reducción de estos compuestos, tales como transferencia interespecífica y mutagénesis. Getinet et al. (1997) llevaron a cabo un programa de cruzamientos interespecíficos entre B. carinata y líneas de B. napus y B. juncea con niveles reducidos de glucosinolatos. Aunque los autores consiguieron reducir considerablemente los niveles de sinigrina (2-propenil glucosinolato) en cruzamientos (B. carinata x B. juncea) x B. carinata respecto a B. carinata, el contenido total en glucosinolatos se mantuvo por encima de 65 μmoles g-1 debido a que se produjo un incremento de los niveles de otros glucosinolatos alifáticos. Estos autores sugirieron la localización en el genoma B de los genes responsables de la síntesis de sinigrina (Getinet et al. 1997).

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Velasco et al. (1999) llevaron a cabo un programa de mutagénesis química a partir de la línea C-101 dirigido a la reducción del contenido en glucosinolatos. Estos autores desarrollaron varias líneas mutantes con contenidos medios en glucosinolatos en la generación M5 entre 90 y 101 µmoles g-1 en comparación con 125 μmoles g-1 en la línea C-101 cultivada en el mismo ambiente. El contenido mínimo en glucosinolatos en estas líneas mutantes fue de 58 μmoles g-1. Barro et al. (2002) realizaron un tratamiento mutagénico sobre microsporas de B. carinata, evaluando posteriormente el contenido en glucosinolatos de líneas dobles haploides. Los autores encontraron en la generación M2 plantas con niveles reducidos de hasta 36 µmoles g-1, aunque no se ha descrito la estabilidad genética de estos niveles reducidos de glucosinolatos en las siguientes generaciones. Desde el punto de vista no alimentario, otro de los objetivos de mejora de las especies de Brassica es el incremento de sus niveles de glucosinolatos con el fin de utilizar estas especies como biocidas naturales para reducir los niveles de patógenos en el suelo (Lazzeri et al. 2004, Bellostas et al. 2007). Concretamente, en B. carinata Velasco et al. (1996) desarrollaron una línea mutante (N2-6215) con un contenido medio en glucosinolatos en semillas de 160 µmoles g-1 en comparación con 116 μmoles g-1 en la línea C-101 cultivada en las mismas condiciones.

4.3. Tamaño y color de la semilla Estudios realizados en B. napus (Liu et al. 1995) y en B. carinata (Getinet 1987) han demostrado la relación existente entre el tamaño y la calidad nutritiva de la semilla. Las semillas de mayor tamaño tienen un contenido mayor en grasa, proteína y glucosinolatos, y un menor contenido en fibra que las semillas de menor tamaño. Teniendo en cuenta el efecto negativo que la fibra tiene sobre la digestibilidad, la reducción del porcentaje de la cubierta en la semilla mejora considerablemente la calidad nutritiva de la misma (Stringam et al. 1974). Por tanto, una mejora de la calidad nutritiva de las semillas de Brassica podría obtenerse mediante una selección dirigida hacia el incremento de su tamaño. En cuanto al color de las semillas, varios estudios han demostrado la relación existente entre el color y el grosor de la cubierta externa de la semilla (Stringam et al. 1974, Bell y Shires 1982, Slominski et al. 1995). Así, las semillas de color amarillo presentan una cubierta más fina y

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por tanto un menor contenido en fibra y lignina, y un mayor contenido en grasa y proteína que las semillas oscuras. Por tanto, la selección hacia genotipos caracterizados por semillas amarillas determina un incremento del valor nutritivo de las mismas (Slomiski et al. 1995). A diferencia de B. carinata y B. juncea, en B. napus no se encuentran de forma natural genotipos de semilla amarilla, por lo que en esta especie se han desarrollado mediante cruzamientos interespecíficos variedades de semilla amarilla con alto contenido en aceite y proteína, y bajo contenido en fibra (Rahman 2001).

5. Biosíntesis y transporte de glucosinolatos 5.1. Biosíntesis de glucosinolatos Los glucosinolatos provienen del metabolismo de los α-aminoácidos, en una serie de reacciones en las que el grupo carboxilo se pierde y el carbono α se transforma en el carbono central del glucosinolato (Mikkelsen et al. 2002). Estudios realizados en Arabidopsis thaliana (Chen et al. 2001, Petersen et al. 2002) y B. napus (Toroser et al. 1995a) sugieren que la síntesis de novo de los glucosinolatos presentes en semillas ocurre principalmente en las silicuas. Por otra parte, en B. nigra, B. juncea y B. oleracea, también se ha demostrado la translocación de ciertos glucosinolatos alifáticos (sinigrina) desde las silicuas y otros tejidos vegetativos de la planta hacia las semillas en desarrollo (Rangkadilok et al. 2002). La mayoría de los estudios sobre la biosíntesis de los glucosinolatos se han realizado en A. thaliana y en algunas especies del género Brassica. En la Figura 1.7 se muestra el modelo genético propuesto en estas especies para la biosíntesis de glucosinolatos (GSL) derivados de la metionina (glucosinolatos alifáticos).

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Figura 1.7.- Modelo genético de la biosíntesis de los glucosinolatos alifáticos en especies del género Brassica (Mahmood et al. 2003). GSL: glucosinolato, Cyt P450: citocromo P450, GSL-ELONG, GSL-OXID, GSL-OPH, GSL ALK, GSL-OH: genes mapeados en A. thaliana y en algunas especies del género Brassica. GSL-ELONG

homo-metionina

Metionina

GSL-ELONG

di-homometionina

Cyt P450

Cyt P450

tri-homometionina

Elongación de la cadena

Formación de

Cyt P450

la glucona

3-metiltiopropil GSL GSL-OXID

GSL-OXID

3-metilsulfinilpropil GSL GSL-OPH

4-metiltiobutil GSL

5-metiltiopentil GSL GSL-OXID

4-metilsulfinilbutil GSL

5-metilsulfinilpentil GSL

GSL-ALK

Modificación

GSL-ALK 3-hidroxipropil GSL

GSL-ALK

de la cadena

2-propenil GSL 3-butenil GSL GSL-OH

2-hidroxi-3-butenil GSL

4-pentenil GSL GSL-OH

3-hidroxi-4-pentenil GSL

En general, la biosíntesis de los glucosinolatos alifáticos se divide en las siguientes etapas (Figura 1.8): (a) elongación de la cadena lateral del aminoácido precursor, (b) formación de la estructura básica del glucosinolato (glucona), y (c) modificación de la cadena lateral del glucosinolato.

a) Elongación de la cadena lateral del aminoácido precursor A través de diferentes estudios bioquímicos en A. thaliana (Graser et al. 2001) y Eruca sativa (Graser et al. 2000), se ha demostrado que la elongación del aminoácido precursor (metionina) es similar a la síntesis de leucina a partir de valina y acetato, y requiere un total de cinco reacciones: transaminación inicial, condensación con acetil-CoA, isomerización, descarboxilación oxidativa y transaminación final (Figura 1.8).

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Figura 1.8.- Etapas de la biosíntesis de glucosinolatos alifáticos (Grubb y Abel 2006). (a) Elongación de la cadena lateral del aminoácido precursor, (b) formación de la estructura básica de los glucosinolatos (glucona) y (c) modificación de la cadena lateral del glucosinolato. GSL: glucosinolato; ?: enzima desconocida; MAM: enzimas metilalquilmalato sintetasas; CYP79 y CYP83: familias de enzimas monooxigenasas del citocromo P450; UGT74B1: enzima glucosiltransferasa; ST5: enzima sulfotransferasa; AOP: enzimas α-ketoglutarato dioxigenasas.

(a) Elongación de la cadena lateral del aminoácido precursor (metionina)

?

α-keto ácido

1. Transaminación inicial 2. Condensación

MAM

2-alquilmalato

?

3. Isomerización

3-alquilmalato ?

4. Descarboxilación oxidativa

homoketoácido ?

5. Transaminación final

homoamioácido

(b) Formación de la estructura básica del glucosinolato (glucona)

1. Oxidación

2. (i) Oxidación (ii) Conjugación

3. Rotura enlace C-S

4. Glucosilación 5. Sulfatación

CYP79

CYP83

?

C-S liasa

UGT74B1

ST5

aldoxima

compuesto aci-nitro S-alquiltiohidroxamato

tiohidroxamato

desulfo-glucosinolato (DSG) glucosinolato

(c) Modificación de la cadena lateral del glucosinolato AOP

1. Oxidación de los glucosinolatos derivados de la metionina

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La elongación de la cadena lateral del aminoácido comienza con la conversión de la metionina (aminoácido precursor) en un α-keto ácido mediante una reacción de transaminación. A partir del α-keto (ó α-amino) ácido se obtiene 2-alquilmalato, mediante una reacción de condensación catalizada por enzimas metilalquilmalato (MAM) sintetasas (Kroymann et al. 2001, Textor et al. 2004). Finalmente, mediante reacciones de isomerización, descarboxilación oxidativa y transaminación se produce la homometionina (precursora de la síntesis de 2-hidroxipropil glucosinolato y 2-propenil glucosinolato) (Figura 1.7 y 1.8). Por otra parte, la homometionina puede sufrir diferentes cambios estructurales que determinan la longitud de su cadena lateral, y por tanto la formación de di-homometionina (precursora de la síntesis de 2-hidroxi-3-butenil glucosinolato) o tri-homometionina (precursora de 3-hidroxi-4-pentenil glucosinolato) (Figura 1.7). Estudios realizados en A. thaliana y B. napus (Magrath et al. 1994) indican que la longitud de la cadena lateral de los glucosinolatos alifáticos está regulada por un único locus denominado GSL-ELONG, que podría también regular el contenido total de glucosinolatos alifáticos.

b) Formación de la estructura central del glucosinolato (glucona) Durante esta segunda etapa de la biosíntesis de los glucosinolatos alifáticos, las enzimas monooxigenasas de la familia CYP79 pertenecientes al complejo multienzimático citocromo P450 (Wittstock y Halkier 2002, Mikkelsen et al. 2002), catalizan la conversión de la homometionina, dihomometionina y tri-homometionina en sus correspondientes aldoximas, a partir de las cuales se sintetizarán

3-metiltiopropil

glucosinolato,

4-metiltiobutil

glucosinolato

y

5-metiltiopentil

glucosinolato, respectivamente, en la fase final de esta etapa (Figura 1.7). Una vez formadas las aldoximas, éstas serán convertidas en S-alquiltiohidroxamato mediante una reacción de oxidación catalizada por enzimas de la familia CYP83, y una posterior reacción de conjugación (Figura 1.8). A continuación, la enzima C-S liasa convertirá el Salquiltiohidroxamato en tiohidroxamato, que posteriormente será convertido en desulfoglucosinolato (Grubb et al. 2004) mediante la acción de la enzima glucosiltransferasa (UGT74B1). Finalmente,

la

sulfatación

del

desulfo-glucosinolato

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(DGS)

catalizada

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enzimas

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sulfotransferasas de la familia ST5, concluirá la fase final de la formación de la estructura básica de los glucosinolatos, la glucona.

c) Modificación de la cadena lateral del glucosinolato En esta tercera y última etapa de la biosíntesis de los glucosinolatos alifáticos, la cadena lateral de los glucosinolatos sufre modificaciones estructurales mediante reacciones de oxidación, eliminación, alquilación o esterificación (Kliebenstein et al. 2001, Li y Quiros 2003, Gao et al. 2004), que determinarán la síntesis final del glucosinolato en cuestión (Figura 1.8).

5.2. Mecanismos de transporte de los glucosinolatos En A. thaliana, los glucosinolatos sintetizados son transportados por proteínas transportadoras desconocidas desde el citosol hasta las vacuolas (donde se almacenan temporalmente), o bien son distribuidos a órganos y tejidos en desarrollo a través del floema (Grubb y Abel 2006) (Figura 1.9). También en esta especie se ha observado el transporte a través del floema de glucosinolatos presentes en las hojas maduras o envejecidas hacia las inflorescencias y frutos en desarrollo (Chen et al. 2001, Petersen et al. 2002). Durante este transporte, los glucosinolatos son convertidos en desulfo-glucosinolatos (DSG), una forma resistente a la acción de las enzimas mirosinasas. Estas formas resistentes presentes en el floema han sido observadas tanto en A. thaliana (Brudenell et al. 1999, Graser et al. 2001) como en B. napus (Thangstad et al. 2001). Aunque hasta la fecha se desconocen cómo ocurren los mecanismos de distribución y transporte celular de los glucosinolatos, la movilidad de estos metabolitos secundarios a través del floema se podría explicar mediante la hipótesis intermedia de la permeabilidad (Brudenell et al. 1999). Esta hipótesis se basa en el modelo matemático propuesto por Kleier (1988), y sugiere que los glucosinolatos son compuestos endógenos con propiedades fisico-químicas que determinarían su movilidad por el floema.

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Figura 1.9.- Modelo de síntesis y transporte de los glucosinolatos alifáticos en A. thaliana (Grubb y Abel 2006). AGSL: glucosinolatos alifáticos; DSG: desulfo-glucosinolato (resultado de la glucosilación catalizada por la enzima UGT74B1). En amarillo se representan las proteínas transportadoras desconocidas.

CLOROPLASTO Elongación cadena Met

Met

CYP79 UGT74B1 ST5b, ST5c

Retículo CYP83

endoplasmático

C-S liasa

DSG AGSL

Vacuola

AGSL

DSG

TRANSPORTE POR EL FLOEMA

AGSL

6. Estudios genéticos sobre el contenido en glucosinolatos en la mostaza etíope y otras especies cultivadas de Brassica El contenido total en glucosinolatos en las especies de Brassica es un carácter complejo en el que están implicados un número relativamente elevado de genes. En B. carinata, la sinigrina (2propenil glucosinolato) constituye más del 90% del total de los glucosinolatos presentes en las semillas (Getinet 1996b). Aunque en esta especie se ha postulado que los genes responsables de la síntesis de sinigrina se encuentran localizados en el genoma B (Getinet et al. 1997), aún no se ha estudiado el control genético de la síntesis de este glucosinolato en B. carinata. En formas

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resintetazadas de B. napus se ha sugerido que están implicados tres genes en la síntesis de sinigrina (Gland 1985), aunque este glucosinolato es minoritario en esta especie. En general, los estudios genéticos sobre el contenido en glucosinolatos totales realizados en diferentes especies de Brassica indican un número elevado de genes, estimado en seis o siete en B. juncea (Sodhi et al. 2002), o incluso más de siete en B. napus (Hill et al. 2003). Mediante RFLPs (Restriction Fragment Length Polymorphisms) se han mapeado y caracterizado QTLs (Quantitative Trait Loci) asociados al contenido en glucosinolatos alifáticos en semillas de B. napus (Uzunova et al. 1995, Toroser et al. 1995b) y B. juncea (Cheung et al. 1998, Mahmood et al. 2003) (Figura 1.7). Concretamente en B. juncea, se han identificado tres QTL para sinigrina (2-propenil glucosinolato) y dos QTL para gluconapina (3-butenil glucosinolato) (Mahmood et al. 2003). También en esta especie, Ripley y Ronslinsky (2005) han identificado marcadores ISSR (Inter-Simple Sequence Repeat) asociados al contenido en sinigrina. Por otra parte, se han identificado varios genes implicados en algunas de las reacciones que tienen lugar a lo largo de la ruta de biosíntesis de los glucosinolatos alifáticos. Así, en A. thaliana y en diferentes especies del género Brassica se han mapeado los genes GSL- ALK (responsables de las reacciones de alquilación), GSL-OH (responsables de las reacciones de hidroxilación), y GSL-ELONG (responsables de la elongación del aminoácido precursor) (Magrath et al. 1993 y 1994; Parkin et al. 1994; Mithen et al. 1995; Gianmoustaris y Mithen 1996; Hall et al. 2001) (véase Figura 1.7), éstos últimos clonados por Li y Quiros (2002). También en Arabidopsis se han identificado algunos de los genes que intervienen en la etapa final de la biosíntesis de los glucosinolatos alifáticos, como son los genes GSL-OXID, responsables de las reacciones de oxidación y los genes GSL-OPH implicados en las reacciones de desaturación e hidroxilación (Mithen et al. 1995; Wittstock y Halkier 2002).

7. Uso de marcadores moleculares en Brassica En los últimos años, los marcadores moleculares han sido ampliamente utilizados con fines de identificación varietal y para estudios de diversidad genética en las especies cultivadas de Brassica. Sin embargo, los trabajos realizados en B. carinata son muy limitados.

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Tradicionalmente, los marcadores moleculares más empleados para el análisis de las especies de Brassica han sido los RFLPs y RAPDs (Random Amplified Polymorphic DNA). El uso de estos marcadores ha permitido la realización de diferentes estudios de diversidad genética (Song et al. 1990, Hu y Quiros 1991, Rabbani et al. 1998, Axelsson et al. 2000, Yuan et al. 2004), y la construcción de mapas de ligamiento en B. rapa (Song et al. 1991, Chyi et al. 1992, Lagercrantz y Lydiate 1996), B. nigra (Lagercrantz y Lydiate 1995 y 1996, Lagercrantz 1998), B. oleracea (Lagercrantz y Lydiate 1996, Hu et al. 1998, Sebastian et al. 2000, Gao et al. 2007), B. juncea (Sharma et al. 1994 y 2002, Upadhyay et al. 1996, Ramchiary et al. 2007) y B. napus (Ecke et al. 1995, Toroser et al. 1995b, Parkin et al. 1995, Sharpe et al. 1995, Cloutier et al. 1997, Cheung et al. 1997, Butruille et al. 1999, Xu et al. 2001, Sharpe y Lydiate 2003). Sin embargo, el elevado coste y la complejidad de la metodología que conlleva el uso de marcadores RFLPs, y la inespecificidad que presentan los marcadores RAPDs, han determinado el desarrollo y utilización de otros marcadores moleculares con mayores ventajas, como son los microsatélites. Los marcadores microsatélites, también conocidos como SSRs (Simple Sequence Repeats), consisten en motivos cortos de ADN, de 1 a 6 pares de bases (pb), y repetidos en tándem a lo largo del genoma de procariotas y eucariotas (Oliveira et al. 2006). Los microsatélites destacan por presentar un elevado grado de polimorfismo, herencia mendeliana simple, codominancia, elevada especificidad, fácil análisis y alta fiabilidad. El alto nivel de polimorfismo que muestran los microsatélites se ha descrito en soja (Akkaya et al. 1992, Maughan et al. 1995), trigo (Plaschke et al. 1995, Prasad et al. 2000), cebada (Becker y Heun 1995, Pillen et al. 2000, Russell et al. 1997, Struss y Plieske 1998), arroz (Cho et al. 2000), maíz (Chin et al. 1996, Senior et al. 1998), así como en la mayoría de las especies cultivadas de Brassica (Lagercrantz et al. 1993, Kresovich et al. 1995, Swecz-McFadden et al. 1996, Uzunova y Ecke 1999, Plieske y Struss 2001, Suwabe et al. 2002, Tonguç y Griffiths 2004). Las características que muestran los microsatélites, junto con el hecho de que las secuencias flanqueantes generalmente se conservan entre especies de la familia Brassicaceae (Plieske y Struss 2001), los convierte en marcadores ideales para su aplicación directa en la mejora genética de estas especies.

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El principal inconveniente que presentan los marcadores microsatélites radica en la necesidad de conocer previamente su secuencia para poder diseñar los cebadores en sus regiones flanqueantes. En Brassica, se han empleado diversos métodos para el desarrollo de microsatélites. Concretamente en B. napus, Szewc-McFadden et al. (1996) emplearon el análisis de genotecas mediante la hibridación con oligonucleótidos formados por la secuencia microsatélite buscada y posterior secuenciación de los clones positivos. Sin embargo, únicamente consiguieron que el 1% de los clones hibridaran positivamente con la sonda microsatélite y, tras su secuenciación, no detectaron microsatélites en el 15% de los mismos, por lo que concluyeron que el elevado número de falsos positivos obtenidos limitaba la eficiencia de este método. A pesar de estos resultados, otros autores han seguido utilizando este método para el desarrollo de microsatélites a partir de B. rapa (Suwabe et al. 2002) y B. napus (Plieske y Struss 2001, Saal et al. 2001). Recientemente, se ha conseguido incrementar la eficiencia en la obtención de microsatélites (75 a 90% de clones positivos) en B. rapa, B. nigra, B. oleracea y B. napus (Lowe et al. 2004), usando el método descrito por Edwards et al. (1996) basado en la construcción de genotecas enriquecidas para un motivo repetido (ej. CT/GA, TG/AC, GGA/CCT, GAA/CTT, GGC/CCG). Mediante este procedimiento, Lowe et al. (2004) consiguieron desarrollar hasta un total de 398 microsatélites en Brassica, actualmente disponibles en la base de datos BrassicaDB (http://ukcrop.net). Aunque los microsatélites desarrollados en Brassica han sido caracterizados (Lagercrantz et al. 1993, Kresovich et al. 1995, Szwec-McFadden et al. 1996, Uzunova y Ecke 1999, Plieske y Struss 2001, Saal et al. 2001, Lowe et al. 2002 y 2004), y utilizados con éxito en estudios de diversidad genética (Plieske y Struss 2001, Tonguç y Griffiths 2004, Hasan et al. 2006), y en la construcción de mapas de ligamiento en la mayoría de las especies cultivadas de Brassica (Uzunova y Ecke 1999, Saal et al. 2001, Lowe et al. 2004, Ramchiary et al. 2007, Gao et al. 2007), hasta la fecha no se ha descrito la aplicación de estos marcadores en la mejora genética de B. carinata. Los microsatélites son la herramienta más idónea para el estudio de las relaciones filogenéticas, puesto que son transferibles, es decir, los microsatélites diseñados a partir de una

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determinada especie (especie focal) suelen amplificar correctamente en otras especies pertenecientes al mismo género o a distintos géneros dentro de una misma familia (Oliveira et al. 2006). De esta manera, la transferencia de microsatélites desarrollados en especies relacionadas conlleva una importante reducción del esfuerzo, coste y tiempo que supone el diseño de nuevos microsatélites a partir de especies poco estudiadas a nivel genético, y por tanto con un escaso o inexistente número de secuencias microsatélites registradas. El éxito de la tranferencia de los microsatélites entre las especies relacionadas reside principalmente en la conservación de la secuencia del ADN de las regiones flanqueantes. Numerosos estudios demuestran la transferencia de microsatélites entre especies del mismo género, como en Eucalyptus (Byrne et al. 1996, Brondani et al. 1998), Quercus (Steinkellner et al. 1997), Prunus (Dirlewanger et al. 2002), y Brassica (Lagercrantz et al. 1993, Szewc-McFadden et al. 1996, Plieske y Struss 2001, Lowe et al. 2002 y 2004), entre especies o géneros de la misma familia, como en Fabaceae (Gutierrez et al. 2005), Melilaceae (White y Powell 1997), Myrtaceae (Rossetto et al. 2000), Poaceae (Sourdille et al. 2001, Guyomarc´h et al. 2002a y 2002b, Kuleung et al. 2004, Saha et al. 2004) y Brassicaceae (Westman y Kresovich 1998, Plieske y Struss 2001), e incluso entre especies pertenecientes a familias distintas (Decroocq et al. 2003). En el momento de comenzar la presente Tesis, no se tenía conocimiento de la existencia de microsatélites desarrollados en B. carinata, y la información sobre secuencias de esta especie era prácticamente nula.

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8. Objetivos Esta Tesis Doctoral se enmarca en una línea de investigación dirigida al desarrollo de líneas de Brassica carinata con niveles reducidos de glucosinolatos en semillas y al estudio genético de la síntesis de glucosinolatos en esta especie. Los objetivos de la Tesis son: 1. Estudio genético del contenido en glucosinolatos en semillas en una línea de B. carinata con niveles elevados de estos compuestos. 2. Evaluación de la transferibilidad de marcadores microsatélites (SSRs) desarrollados en B. nigra (BB) y B. napus (AACC) a B. carinata (BBCC) para su aplicación en programas de mejora genética de esta especie. 3. Estudio genético comparativo de varias líneas de B. carinata con niveles reducidos de glucosinolatos en semillas y selección de segregantes transgresivos para este carácter. 4. Reducción del contenido en glucosinolatos en semillas de B. carinata mediante hibridación interespecífica.

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9. Referencias Ackman R.G. y F.M. Loew (1977) The effects of high levels of fats rich in erucic acid (from rapeseed oil) or cetoleic and cetelaidic acids (from partially hydrogenated fish oil) in a short-term study in a nonhuman primate species. Fette Seifen Anstrichmitt. 79: 15-24 Akkaya M.S., A.A.Bhagwat y P.B. Cregan (1992) Length polymorphisms of simple sequence repeat DNA in soybean. Genetics 132: 1131-1139 Alemayehu N. y H. Becker (2002) Genotypic diversity and patterns of variation in a germplasm material of Ethiopian mustard (Brassica carinata A. Braun). Genet. Res. Crop Evol. 49: 573-582 Alonso L.C., O. Fernández-Serrano y J. Fernández-Escobar (1991) The outset of a new oilseed crop: th Brassica carinata with low erucic acid content. En: Proc. 8 Int. Rapeseed Conf., Saskatoon, Canadá,

9-11 julio 1991, pp.170-176. Anand L.J., J.P. Sigh y R.S. Malik (1985) B. carinata a potential oilseed crop for rainfed agriculture. Eucarpia Cruciferae Newsl. 10: 76-78 Angus J.F., P.A. Gardner, J.A. Kirkegaard y J.M. Desmachelier (1994) Biofumigation: isothiocyanates released from Brassica roots inhibit growth of take-all fungus. Plant Soil 162: 107-112 Auld D.L., M.K. Heikkinen, D.A. Erickson, J.L. Sernyk y J.E. Romero (1992) Rapessed mutants with reduced levels of polyunsaturated fatty acids and increased levels of oleic acid. Crop Sci. 32: 657662 Axelsson T., C.M. Bowman, A.G. Sharpe, D.J. Lydiate y U. Lagercrantz (2000) Amphidiploid Brassica juncea contains conserved progenitor genomes. Genome 43: 679-688 Barro F., J. Fernández-Escobar, M. De la Vega y A. Martín (2002) Modification of glucosinolate and erucic acid contents in doubled haploid lines of Brassica carinata by UV treatment of isolated microspores. Euphytica 129: 1-6 Becker J. y M. Heun (1995) Barley microsatellites: allele variation and mapping. Plant Mol. Biol. 27: 835-845 Bell J.M. y A. Shires (1982) Composition and digestibility by pigs of hull fractions from rapeseed cultivars with yelow or brown seed coats. Can J. Anim. Sci. 62: 557-565 Bellostas N., J.C. Sørensen y H. Sørensen (2004) Qualitative and quantitative evaluation of glucosinolates in cruciferous plants during their life cycles. Agroindustria 3: 5-10 Bellostas N., J.C. Sørensen y H. Sørensen (2007) Profiling glucosinolates in vegetative and reproductive tissues of four Brassica species of the U-triangle for their bifumigation potential. J. Sci. Food Agric. 87: 1586-1594

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Berggren G. (1962) Reviews on the taxonomy of some species of the genus Brassica, based on their seeds. Svensk Bot. Tidskr. 56: 65-135 Bones A.M. y J.T. Rossiter (1996) The myrosinase-glucosinolate system, its organization and biochemistry. Physiol. Plant. 97: 194-208 Brondani R.P.V., C. Brondani, R. Tarchini y D. Grattapaglia (1998) Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla. Theor. Appl. Genet. 97: 816-827 Brudenell A.J.P., H. Griffiths, J.T. Rossiter y D.A. Baker (1999) The phloem mobility of glucosinolates. J. Exp. Bot. 50: 745-756 Buskov S., B. Serra, E. Rosa, H. Sørensen y J.C. Sørensen (2002) Effects of intact glucosinolates and products produced from glucosinolates in myrosinase-catalyzed hydrolysis on the potato cyst nematode (Globodera rostochiensis cv. Woll). J. Agric. Food Chem. 50: 690-695 Bussy A. (1840) Sur la formation de l´huile essentielle de moutarde. J. Pharm. 27: 464-471 Butruille D.V., R.P. Guries y T.C. Osborn (1999) Linkage analysis of molecular markers and quantitative trait loci in populations of inbred backcross lines of Brassica napus L. Genetics 153: 949-964 Byrne M., M.I. Márquez-García, T. Uren, D.S. Smith y G.F. Moran (1996) Conservation and genetics diversity of microsatellite loci in the genus Eucalyptus. Aust. J. Bot. 44: 331-341 Cardone M., M. Mazzoncini, S. Menini, V. Rocco, A. Senatore, M. Seggiani y S. Vitolo (2003) Brassica carinata as an alternative oil crop for the production of biodiesel in Italy: agronomic evaluation, fuel production by transesterification and characterization. Biomass Bioenerg. 25: 623-636 Chen S., B.L. Petersen, C.E. Olsen, A. Schulz y B.A. Halkier (2001) Long-distance phloem transport of glucosinolates in Arabidopsis. Plant Physiol. 127: 194-201 Cheung W.Y., G. Champagne, N. Hubert y B.S. Landry (1997) Comparison of the genetic maps of Brassica napus and B. oleracea. Theor. Appl. Genet. 94: 569-582 Cheung W.Y., B.S. Landry, P. Raney y G.F.W. Rakow (1998) Molecular mapping of seed quality traits in Brassica juncea L. Czern., and Coss. Acta Hort. 459: 139-147 Chin E.C.L., M.L. Senior, H. Shu y J.S.C. Simith (1996) Maize simple repetitive DNA sequences: abundance and allele variation. Genome 39: 866-873 Cho Y.G., T. Ishii, S. Temnykh, X. Chen, L. Lipovich, S.R. McCouch, W.D. Park, N. Ayres y S. Cartinhour (2000) Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.). Theor. Appl. Genet. 100: 713-722

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Choudhary B.R., P. Joshi y S. Ramarao (2000) Interspecific hybridization between Brassica carinata and Brassica rapa. Plant Breed. 119: 417-420 Chyi Y.S., M.E. Hoenecke y J.L. Sernyk (1992) A genetic linkage map or restriction fragment length polymorphism loci for Brassica rapa (syn. campestris). Genome 35: 746-757 Ciska E., B. Martyniak-Przybyszewska, H. Kozlowska (2000) Content of glucosinolates in cruciferous vegetables grown at the same site for two years under different climatic conditions. J. Agric. Food Chem. 48: 2862-2867 Cloutier S., M. Cappadocia y B.S. Landry (1997) Analysis of RFLP mapping inaccuracy in Brassica napus L. Theor. Appl. Genet. 95: 83-91 Cohen D.B. y P.F. Knowles (1983) Evaluation of Brassica species in California. En: Proc. 6th Int. Rapeseed Conf., Paris, Francia, 17-19 mayo 1983, pp. 282-287 De Haro A., M. Del Río, E. Cartea y A. Ordás (2006) Mejora de la calidad de especies de Brassica. En: G. Yacer, M.J. Díez, J.M. Carrillo y M.L Badenes (eds.). Mejora Genética de la Calidad en Plantas. Universidad Politécnica de Valencia. Valencia, España, pp. 417-447 Decroocq V., M.G. Fave, L. Hagen, L. Bordenave y S. Decroocq (2003) Development and transferability of apricot and grape EST microsatellite markers across taxa. Theor. Appl. Genet. 106: 912-922 Del Río M., R. Font, J.M. Fernández-Martínez, J. Domínguez y A. De Haro (2000) Fields trials of Brassica carinata and B. juncea in polluted soils of the Guadiamar river area. Fresenius Environ. Bull. 9: 328332 Dirlewanger E., P. Cosson, M. Tavaud, M.J. Aranzana, C. Poizat, A. Zanetto, P. Arús y F. Laigret (2002) Development of microsatellite markers in peach ((Prunus persica) Batsch.). Theor. Appl. Genet. 105:127-138 Downey R.K. (1964) A selection of B. campestris L. containing no erucic acid in its seed oil. Can. J. Plant Sci. 44: 295 Downey R.K. y G. Röbbelen (1989) Brassica species. En: R.K. Downey, G. Röbbelen y A. Ashri (eds.), Oil crops of the world. McGraw-Hill, New York, USA, pp. 339-362 Downey R.K. (1990) Brassica oilseed breeding. Achievements and Opportunities. Plant Breed. Abstracts 60: 1165-1170 Ecke W., M. Uzunova y K. Weissleder (1995) Mapping the genome of rapeseed (Brassica napus L.). II. Localization of genes controlling erucic acid synthesis and seed oil content. Theor. Appl. Genet. 91: 972-977

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Saal B., J. Plieske, J. Hu, C.F. Quiros y D. Struss (2001) Microsatellite markers for genome analysis in Brassica. II. Assignment of rapeseed microsatellites to the A and C genomes and genetic mapping in Brassica oleracea L. Theor. Appl. Genet. 102: 695-699 Sacristán M.D. y M. Gerdemann (1986) Different behavior of Brassica juncea and B. carinata as sources of Phoma lingam resistance in experiments of interespecific transfer to B. napus. Plant Breed. 97: 304314 Saha M.C., M.A. RoufMian, I. Eujayl, J.C. Zwonitzer, L. Wang y G.D. May (2004) Tall fescue EST-SSR markers with transferability across several grass species. Theor. Appl. Genet. 109: 783-791 Sasongko N.D. y C. Möllers (2005) Toward increasing erucic acid content in oilseed rape (Brassica napus L.) through the combination with genes for high oleic acid. J. Am. Oil. Chem. Soc. 82: 445-449 Sebastian R.L., E.C. Howell, G.J. King, D.F. Marshall y M.J. Kearsey (2000) An integrated AFLP and RFLP Brassica oleracea linkage map from two morphologically distinct double-haploid mapping populations. Theor. Appl. Genet. 100: 75-81 Senior M.L., J.P. Murphy, M.M. Goodman y C.W. Stuber (1998) Utility of SSRs for determining genetic similarities and relationships in maize using an agarose gel system. Crop Sci. 38: 1088-1098 Sharma A., T. Mohapatra y R.P. Sharma (1994) Molecular mapping and caracter tagging in Brassica juncea. I. Degree, nature and linkage relationship of RFLPs and their association with quantitative traits. J. Plant Biochem. Biotech. 3: 85-89 Sharma R., R.A.K. Aggarwal, R. Kumar, T. Mohapatra y R.P. Sharma (2002) Construction of an RAPD linkage map and localization of QTLs for oleic acid level using recombinant inbreds in mustard (Brassica juncea). Genome 45: 467-472 Sharpe A.G., I.A.P. Parkin, D.J. Keith y D.J. Lydiate (1995) Frequent nonreciprocal translocations in the amphidiploid genome of oilseed rape (Brassica napus). Genome 38: 1112-1121 Sharpe A.G. y D.J. Lydiate (2003) Mapping the mosaic of ancestral genotypes in a cultivar of oilseed rape (Brassica napus) selected via pedigree breeding. Genome 46: 461-468 Singh H., D. Singh y T.P. Tadava (1988) Comparative performance of genotypes of Indian and Ethiopian mustard under semi-arid region of India. Eucarpia Cruciferae Newsl. 13:36-37 Sivaraman, I., N. Arumugam, Y.S. Sodhi, V. Gupta, A. Mukhopadhyay, A.K. Pradhan, P.K. Burma y D. Pental (2004) Development of high oleic and low linolenic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene. Mol. Breed. 13: 365-375

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Slomiski B.A., J. Simbaya, L.D. Campbell, W. Guenter y G. Rakow (1995) Nutritive profile of yelow-seeded canola/rapeseed. En: Proc. 9th Int. Rapeseed Conf., Cambrigde, Reino Unido, 4-7 julio 1995, pp. 203-210 Sodhi Y.S., A. Mukhopadhyay, N. Arumugam, J.K. Verma, V. Gupta y D. Pental (2002) Genetic analysis of total glucosinolate in crosses involving a high glucosinolate Indian variety and low glucosinolate line of Brassica juncea. Plant Breed. 121: 508-511 Song K.M., T.C. Osborn y P.H. Williams (1990) Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs). 3. Genome relationships in Brassica and related genome origin of B. oleracea and B. rapa (syn. campestris). Theor. Appl. Genet. 79: 497-506 Song K.M., J.Y. Suzuki, M.K. Slocum, P.H. Williams y T.C. Obsborn (1991) A linkage map of Brassica rapa (syn. campestris) based on restriction fragment length polymorphism loci. Theor. Appl. Genet. 82: 296-304 Song K.M. y T.C. Osborn (1992) Polyphyletic origins of Brassica napus: new evidence based on originalle and nuclear RFLP analyses. Genome 35: 992-100 Sourdille P., M. Tavaud, G. Charmet y M. Bernard (2001) Transferability of wheat microsatellites to diploid Triticeae species carrying the A, B and D genomes. Theor. Appl. Genet. 103: 346-352 Stefansson B.R., F.W. Hougen y R.K. Downey (1961) Note on the isolation of rape plants with seed oil free from erucic acid. Can. J. Plant Sci. 41: 218-219 Stefansson B.R. y Z.P. Kondra (1975) Tower summer rape. Can. J. Plant Sci. 55: 343-344 Steinkellner H., C. Lexer, E. Turetschek y J. Glössl (1997) Conservation of (GA)n microsatellite loci between Quercus species. Mol. Ecol. 6: 1189-1194 Stoewsand G.S. (1995) Bioactive organosulfur phytochemicals in Brassica oleracea vegetables - A Review. Food Chem. Toxicol. 33 : 537-543 Stoutjesdijk P.A., C. Hurlstone, S.P. Singh y A.G. Green (1999) Genetic manipulation for altered oil qulaity in brassicas. En: N. Wratten y P.A. Salisbury PA (eds). Proc. 10th Int. Rapeseed Congr., 26-29 Sept 1999, Canberra, Australia, CD ROM Stringam G.R., D.I. McGregor y S.H. Pawlowski (1974) Chemical and morphological characteristics th associated with seedcoat in rapeseed. En: Proc. 4 Int. Rapeseed Conf., Giessen, Alemania, 4-8

junio 1974, pp. 99-108 Struss D. y J. Plieske (1998) The use of microsatellite markers for detection of genetic diversity in barley populations. Theor. Appl. Genet. 97: 307-315

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Suwabe K., H. Iketani, T. Nunome, T. Kage y M. Hirai (2002) Isolation and characterization of microsatellites in Brassica rapa L. Theor. Appl. Genet. 93: 534-538 Szewc-McFadden A.K.S., S. Kresovich, S.M. Bliek, S.E. Mitchell y J.R. McFerson (1996) Identification of polymorphic, conserved simple sequence repeats (SSRs) in cultivated Brassica species. Theor. Appl. Genet. 104: 1092-1098 Tani N., M. Ohtsuru y T. Hata (1974) Isolation of myrosinase producing microorganism. Agric. Biol. Chem. 38: 1617-1622 Tapper B.A. y P.F. Reaney (1973) Cyanogenic glycosides and glucosinolates (mustard oil glucosides). En: G.W. Butter y R.W. Bailey (eds.). Chemistry and Biochemistry of Herbago. Academic Press, Londres, Reino Unido, pp. 447-476 Tayo T.O. y D.G. Morgan (1975) Quantitative analices of the growth, development and distribution of flowers and pods in oil seed rape (Brassica napus L.). J. Agr. Sci. 85: 103-110 Tcacenco F.A., B.V. Ford-Lloyd y D. Astley (1985) A numerical study of variation in a germoplasm collection of Brassica carinata and allied species from Ethiopia. Z. Pflanzenzüchtg. 94: 192-200 Textor S., S. Bartram, J. Kroyman, K.L. Falk, A. Hick, J.A. Pickett y J. Gershenzon (2004) Biosynthesis of methionine-derived glucosinolates in A. thaliana: recombinant expression and characterization of methyltioalkylmalate synthase, the condensing enzyme of the chain-elongation. Planta 218: 10261235 Thangstad O.L., A.M. Bones, S. Holtan, L. Moen y J.T. Rossiter (2001) Microautoradiographic localisation of a glucosinolate precursor to specific cell in Brassica napus L. embryos indicates a separate transport pathway into myrosin cells. Planta 213: 207-213 Tonguç M. y P.D. Griffiths (2004) Genetics relationships of Brassica vegetables determined using database derived simple sequence repeats. Euphytica 137: 193-201 Toroser D., C. Wood, H. Griffiths y D.R. Thomas (1995a) Glucosinolate biosynthesis in oilseed rape (Brassica napus L.): studies with

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U.N. (1935) Genomic analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Jpn. J. Bot. 7: 389-452 Uchimiya H. y S.G. Wildman (1978) Evolution of Fraction I protein in relation to origin of amphidiploids Brassica species and other members of Cruciferae. J. Hered. 69: 299-303 Uhl M., B. Laky, E. Lhoste, F. Kassie, M. Kundi y S. Knasmüller (2003) Effects of mustard sprouts and allylisothiocyanate on benzo(a)pyrene-induced DNA damage in human-derived cells: a model study with the single cell gel electrophoresis/Hep G2 Assay. Teratog. Carcinog. Mutag. 1: 273-282 Upadhyay A., T. Mohapatra, R.A. Pai y R.P. Sharma (1996) Molecular mapping and character tagging in mustard (Brassica juncea). II. Association of RFLP markers with seed coat colour and quantitative traits. J. Plant Biochem. Biotech. 5:17-22 Uzunova M., W. Ecke, K. Weiβleder y G. Röbbelen (1995) Mapping the genome of rapeseed (Brassica napus L.). I. Construction of an RFLP linkage map and localization of QTLs for seed glucosinolate content. Theor. Appl. Genet. 90: 194-204 Uzunova M.I. y W. Ecke (1999) Abundance, polymorphism and genetic mapping of microsatellites in oilseed rape (Brassica napus L.). Plant Breed. 118: 323-326 Vaughan J.G. (1977) A multidisciplinary study of the taxonomy and origin of Brassica crops. Bioscience 27: 35-40 Vavilov N.I. (1951) The origin, variation, immunity and breeding of cultivated plants. Ronald Press, New York, USA Velasco L., J.M. Fernández-Martínez y A. De Haro (1996) Identification o fan Ethiopian mustard line with very high levels of glucosinolates. Eucarpia Cruciferae Newsl. 18: 96 Velasco L., J.M. Fernández-Martínez y A. De Haro (1997) Selection of high sum of oil and protein in Ethiopian mustard (Brassica carinata A. Braun). Eucarpia Cruciferae Newsl. 19: 97-98 Velasco L., J.M. Fernández-Martínez y A. De Haro (1998). Increasing erucic acid content in Ethiopian mustard through mutation breeding. Plant Breed. 117: 85-87 Velasco L., J.M. Fernández-Martínez y A. De Haro (1999) Intraspecific breeding for reduced glucosinolate content in Ethiopian mustard (Brassica carinata A. Braun). Euphytica 106: 125-130 Velasco L., A. Nabloussi, A. De Haro y J.M. Fernández-Martínez (2003) Development of high oleic, low linoleic acid Ethiopian mustard (Brassica carinata) germplasm. Theor. Appl. Genet. 107: 823-830 Velasco L., A. Nabloussi, A. De Haro y J.M. Fernández-Martínez (2004) Allelic variation in linolenic acid content to high erucic acid Ethiopian mustard and incorporation of the low linolenic acid trait into zero erucic acid germplasm. Plant Breed. 123: 137-140

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Verma S.C. y H. Rees (1974) Nuclear DNA and the evolution of allotetrapliod Brassicaceae. Heredity 33: 6168 Vilkki J.P. y P.K. Tanhuanpää (1995) Breeding of high oleic acid spring turnip rape in Finland. En: Proc. 9th Int. Rapessed Conf., Cambridge, Reino Unido, 4-7 julio 1995, pp. 386-388 Vles R.O. y J.J. Gottenbos (1989) Nutritional characteristics and food uses of vegetable oils. En: R.K. Downey, G. Röbbelen y A. Ashri (eds.). Oil Crops of the World. McGraw-Hill, New York, USA, pp. 6386 Westman A.L. y S. Kresovich (1998) The potential for cross-taxa simple-sequence repeat (SSR) amplification between Arabidopsis thaliana L. and crop brassicas. Theor. Appl. Genet. 96: 272-281 White G. y W. Powell (1997) Cross-species amplification of SSR loci in the Meliaceae family. Mol. Ecol. 6: 1195-1197 Wittstock U. y B.A. Halkier (2002) Glucosinolate research in the Arabidopsis era. Trends Plant Sci. 7: 263270 Wong R.S.C. y E. Swanson (1991) Genetic modification of canola oil: high oleic acid canola. En: C. Haberstrohn y C.F. Morris (eds). Fat and Cholesterol Reduced Foods: Technologies and Strategies. Portfolio Publ Co, The Woodlands, TX, USA, pp. 153-164 Xu F.S., Y.H. Wang y J. Meng (2001) Mapping boron efficiency gene(s) in Brassica napus using RFLP and AFLP markers. Plant Breed. 120: 319-324 Yodice R. (1990) Nutritional and stability characteristics of high oleic sunflower seed oil. Fat Sci. Technol. 92: 121-126 Young F.V.K., C. Poot, E. Biernoth, N. Krog, N.G.J. Davidson y F.D. Gunstone (1994) Processing of fats and oils. En: F.D. Gunstone, J.L. Harwood y F.B. Padley (eds.). The Lipids Handbook. Chapman & Hall, Londres, Reino Unido, pp. 249-318 Yuan M., Y. Zhou y D. Liu (2004) Genetic diversity among populations and breeding lines from recurrent selection in Brassica napus as revealed by RAPD markers. Plant Breed. 123: 9-12

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CAPÍTULO 2

Inheritance of very high glucosinolate content in Ethiopian mustard seeds

Márquez-Lema A., J.M. Fernández-Martínez, B. Pérez-Vich and L. Velasco Instituto de Agricultura Sostenible (CSIC), Alameda del Obispo s/n. E-14004, Córdoba, España

Inheritance of very high glucosinolate content in Ethiopian mustard seeds Plant Breeding (2008) (en prensa)

Márquez Lema A.

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Abstract Seed meal amendments rich in glucosinolates are of interest for soil pest and disease control. The Ethiopian mustard (Brassica carinata A. Braun) line N2-6215, with very high levels of seed glucosinolates (160 µmol/g), was developed from the line C-101 (116 µmol/g) following mutagenesis. The objective of this research was to study the inheritance of very high seed glucosinolate content. Plants of N2-6215 were reciprocally crossed with plants of the line C-101. The F1, F2, and BC1F1 plant generations were evaluated in two environments and seeds from individual plants were analysed for total glucosinolate content. The very high glucosinolate content in N2-6215 seeds was largely subject to maternal control. No cytoplasmic effects were detected. The trait was found to be oligogenic and determined by at least two or three genes. The estimates of broad-sense heritability were 0.45 and 0.58 in both environments, whereas the estimates of narrow-sense heritability were 0.35 and 0.50. The moderate heritability and oligogenic control of the trait suggest the feasibility of breeding for increased seed glucosinolate content in Ethiopian mustard.

Key words: Brassica carinata − Ethiopian mustard − genetic study − seed glucosinolates − sinigrin

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Introduction Glucosinolates (GSL) are a family of secondary plant metabolites particularly abundant in seeds and green tissues of the Brassicaceae. Glucosinolates and their breakdown products are associated with toxic and antinutritional effects, which limit the value of the seeds and the meal for food and feed uses (Griffiths et al. 1998). Breeding efforts have mainly focused on reducing seed glucosinolate content in the major oil crops of the family, i.e. Brassica napus, B. rapa, and B. juncea (Love et al. 1990a). However, glucosinolates possess beneficial properties as well. They show broad biocidal activity, which encourages their use as an alternative to synthetic pesticides for pest and disease control (Kirkegaard et al. 1998, Lazzeri et al. 2004). Furthermore, glucosinolates possess anti-carcinogenic activity (Hecht 2000). Accordingly, increasing seed and plant glucosinolate content are interesting objectives for novel applications of Brassica plants and seeds. Ethiopian mustard (Brassica carinata A. Braun) germplasm contains a limited variation for seed glucosinolate content and composition. Alemayehu and Becker (2002) reported a range of variation from 90 to 111 µmol/g seed in an evaluation of Ethiopian mustard germplasm conducted over three environments. Other studies reporting wider variation have been based on single environments (Getinet et al. 1996) or genebank seed evaluations (Velasco and Becker 2000). The glucosinolate fraction of B. carinata seeds is mainly made up of sinigrin (2-propenyl), which accounts for > 95% of the total glucosinolates in the seeds (Getinet et al. 1997). No variation for glucosinolate profile has been reported in this species. Genetic studies on total seed glucosinolate content in Brassica spp. have been mainly directed to the study of reduced levels of seed glucosinolates. In crosses involving B. napus lines with a wide range of total glucosinolate content, Rücker and Röbbelen (1994) found high broadsense and narrow-sense heritabilities of the trait (h2b = 0.95; h2n = 0.87). The authors concluded that low glucosinolate content was determined by four or five recessive genes. In the same species, Hill et al. (2003) studied the inheritance of total glucosinolate content in a cross of the type high by low glucosinolate content, reporting also high heritabilities for the trait (h2b = 0.85; h2n = 0.70), which was estimated to be controlled by at least eleven genes. In a similar high by low glucosinolate content cross in B. juncea, Sodhi et al. (2002) concluded the involvement of six or seven genes in

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the genetic control of total glucosinolate content. Sinigrin accumulation in the seeds has been found to be controlled by three genes both in resynthesized B. napus forms (Gland 1985) and in B. juncea (Mahmood et al. 2003). No genetic studies on increased levels of seed glucosinolates have been conducted so far. Breeding for increased levels of seed glucosinolates has been scarce. The Ethiopian mustard line N2-6215, characterized by very high levels of glucosinolates in the seeds (160 µmol/g seed), was developed from the line C-101 (116 µmol/g seed) following chemical mutagenesis (Velasco et al. 1996). The objective of the present research was to study the inheritance of very high glucosinolate content in N2-6215 seeds.

Materials and Methods Plant material and genetic study C-101 is a standard Ethiopian mustard line with average glucosinolate content approximately 116 µmol/g seed. The line N2-6215, with very high glucosinolate content (160 µmol/g), was developed from C-101 following chemical mutagenesis and pedigree selection (Velasco et al. 1996). Seeds of N2-6215 do not differ significantly from those of C-101 for other seed quality traits such as seed oil content, protein content and fatty acid profile (Velasco 1996).The seeds of N2-6215 used for this study corresponded to the F8 generation. Seeds of the parents were sown in December 2002 and the plants were grown in the greenhouse. Plants of N2-6215 were reciprocally crossed with plants of C-101 in the spring of 2003. Microperforated plastic bags were used to prevent uncontrolled pollination. Crossing was done by emasculating immature flower buds of the female parent followed by immediate pollination of their stigmas with fresh pollen from open flowers of the male parent. F1 plants were grown in pots in a mesh cage enclosure in the spring of 2004 together with parent plants. They were self-pollinated to obtain F2 seed and also backcrossed to both parents. The F1, F2 and BC1F1 plant generations were grown in the field together with plants of the parents in the growing seasons 2004/05 and 2006/07. All the plants were bagged before flowering. The plants were harvested and the seeds analysed for total glucosinolate content as described below. The number of plants analysed for each generation and environment are reported in Table 2.1.

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Table 2.1.- Glucosinolate content (µmol/g seed) in the Ethiopian mustard lines C-101, N2-6215, and F1, F2 and backcross plant generations from reciprocal crosses between them, evaluated in the field in 2005 and 2007.

Total glucosinolate content (µmol/g seed) Generations n

Field 2005

n

Field 2007

C-101

23

127 ± 14a,1

84

105 ± 11a

N2-6215

21

170 ± 10b

52

149 ± 10b

C-101 x N2-6215

24

157 ± 8c

32

125 ± 10c

N2-6215 x C-101

24

157 ± 10c

77

127 ± 11c

Parents

F1

Midparent value2

149**

127

F2 C-101 x N2-6215

219

144 ± 14

190

127 ± 17

To C-101

96

138 ± 14

166

127 ± 14

To N2-6215

81

160 ± 13

131

152 ± 15

BC1F1

1

Within the same environment, averages followed by the same superscript letter are not significantly

different (P < 0.01) 2

t-test, H0: mean-midparent value = 0

Analysis of total seed glucosinolate content Total glucosinolate content was measured on intact seeds by near-infrared reflectance spectroscopy (NIRS) with the Pd-glucosinolate complex method (Thies 1982) used as reference method for NIRS calibration (Velasco et al. 1999). Additionally, seeds of the parents were analysed for glucosinolate content and profile by high performance liquid chromatography (HPLC) as described by Velasco and Becker (1998).

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Statistical analyses Maternal and cytoplasmic effects were studied in F1 and F2 seeds, respectively. The minimum number of genes (k) controlling total glucosinolate content was estimated following Wright (1968): 2

k = (P1 - P2)2 / 8(s F - VE) 2

2

where P1 and P2 are the mean values of the two parents, s F is the variance of the F2 plants, and 2

VE the environmental variance. VE was estimated by averaging the variances of the genetically uniform generations P1, P2 and F1. Estimates of broad-sense (h2b) and narrow-sense (h2n) heritabilities were calculated for total glucosinolate content by using the variance of the parents (P1 and P2), as well as the F1, F2, and BC generations to estimate phenotypic (VP), environmental (VE), total genetic (VG), additive genetic (VA), and dominance genetic variances (VD) (Allard 1960), where: VP = VF2 VE = 0.25 (VP1) + 0.25 (VP2) + 0.25 (VF1) + 0.25 (VF1r) VG = VF2 − VE VA = 2 (VF2) − VBC1P1 − VBC1P2 VD = VBC1P1 + VBC1P2 − VF2 − VE h2b = (VA + VD) / VF2 h2n = VA / VF2

Results Plants of the Ethiopian mustard lines C-101 and N2-6215 grown in pots in the greenhouse in 2003 showed average glucosinolate contents of 113 and 183 µmol/g, respectively. Seeds of both lines had a glucosinolate profile dominated by sinigrin, which in both cases represented > 95% of the total seed glucosinolates. F1 seeds from the cross C-101 x N2-6215 averaged 170 µmol/g, whereas those from the reciprocal cross averaged 207 µmol/g. The results indicated a strong maternal effect on total glucosinolate content. At the F1 plant level (F2 seeds analysed), no significant differences between reciprocal crosses were observed in both environments (Table 2.1), indicating absence of cytoplasmic effects on this trait. The mean value of the F1 did not differ significantly from that of the midparent in one

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environment and it was slightly lower in the other environment (Table 2.1), suggesting absence or very small magnitude of dominance effects. The mean values of the F1 and both parents were clearly different in both environments, revealing an important environmental effect on the trait. The glucosinolate content in F2 and BC1F1 plants in both environments is shown in Table 2.1. Estimates of broad-sense heritability (h2b) in 2005 and 2007 field evaluations resulted in values of 0.45 and 0.58, respectively, whereas narrow-sense heritability (h2n) was estimated in 0.35 and 0.50, respectively. The estimate of the number of genes (k) involved in very high glucosinolate content of N2-6215 in 2005 and 2007 field evaluations was 2.5 and 1.6, respectively, indicating that at least two or three genes must be responsible for the trait.

Discussion Genetic studies on seed glucosinolate content in Brassica species have mainly focused on germplasm with reduced levels of these compounds, but to our knowledge this is the first genetic analysis of germplasm exhibiting increased levels of glucosinolates that are consistently expressed across the environments. The trait was found to be subject to strong maternal effects, which is in agreement with previous studies in B. napus (Kondra and Stefansson 1970, Magrath and Mithen 1993). It is noteworthy that F1 seeds had higher glucosinolate content that their corresponding female parent. This could be attributed to micro-environmental or positional effects and/or to a different distribution of assimilates in branches in which flowers were emasculated in comparison to self-pollinated branches, as crossing usually involves the removal of some flower buds from the racemes (Downey et al. 1980). The latter determines a reduced number of pods per raceme, which might influence translocation and accumulation of glucosinolates into maturing seeds. No cytoplasmic effects were identified. The existence of cytoplasmic effects on total seed glucosinolate content in Brassica species is a matter for controversy, as they have been identified in some studies (Kondra and Stefansson 1970, Love et al. 1990b) but not in others (Rücker and Röbbelen 1994). Heritability estimates were moderate, indicating an important environmental effect on the total seed glucosinolate content, which was also observed when comparing the mean values of the non-segregating generations in the two environments in which they were evaluated. Previous

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studies in B. napus have reported higher values of broad- and narrow-sense heritabilities for total seed glucosinolate content (Rücker and Röbbelen 1994, Hill et al. 2003). The mentioned studies included parents with low glucosinolate content (< 10 µmol/g). However, there are no previous studies on the inheritance of very high glucosinolate content in Brassica species. The biochemical and genetic bases of increased glucosinolate content are expected to differ from those leading to a drastic reduction of seed glucosinolate accumulation. No genetic studies on sinigrin accumulation in Ethiopian mustard seeds have been conducted so far. The trait has been studied in a cross involving a resynthesized B. napus form that accumulated approximately 15% of sinigrin in the seeds, even though sinigrin is not present in significant amounts in conventional B. napus plants (Gland 1985). The authors concluded that sinigrin accumulation was determined by three dominant genes. In B. juncea, Mahmood et al. (2003) conducted Quantitative trait loci (QTL) analysis on a doubled-haploid population segregating for both glucosinolate content and profile, identifying three QTL that explained up to 78% of the variation for sinigrin content. Even though the results are not directly comparable, our results also indicated that at least two or three genes are involved in the accumulation of very high glucosinolate (sinigrin) levels in Ethiopian mustard seeds. The line N2-6215 was developed in the course of a mutagenesis program on the line C-101 (Velasco et al. 1996), used as a parent in the present research. Since we concluded that differences in glucosinolate content between C-101 and N2-6215 are produced by at least two or three genes, it is unlikely that all the genes were modified as the result of the mutagenic treatment. One of the advantages of mutagenesis programmes is the management of very large populations, which maximizes the probability of identifying genetic alterations produced by the mutagenic treatment as well as those already existing in the treated population. In sunflower, Pérez-Vich et al. (1999) found that only one of three recessive genes involved in the high palmitic acid content of the mutant CAS-5 was altered with the mutagenic treatment, the other two being already present in the mutagenized population. In the case of the programme in which the line N2-6125 was identified, 1,011 individual M1 plants and 8,331 individual M2 plants were screened for total glucosinolate content (Velasco et al. 1996), which maximized the probability of identifying genetic modifications

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resulting in high seed glucosinolate content, either produced by the mutagenic treatment or already existing in the mutagenized population. There are interesting applications for Brassica germplasm with very high glucosinolate content in the seeds, due to the biological activity of these compounds. For example, seed meal amendments from high glucosinolate Brassica seeds constitute an effective method of control for soilborne pests and pathogens (Lazzeri et al. 2004, Bellostas et al. 2007). There are differences among the different glucosinolates for biological activities. Seeds of the line N2-6215 mainly contain sinigrin, which is one of the most effective glucosinolates for the biological control of nematodes (Lazzeri 1993, Buskov et al. 2002). It is also more effective than other aliphatic glucosinolates against soilborne fungal pathogens (Bellostas et al. 2007). Accordingly, the very high sinigrin content of N2-6215 seeds may be of value for the production of soil amendments with biocidal activity. The trait has a moderate heritability and is controlled by a reduced number of genes, which will facilitate its transfer to appropriate genetic backgrounds.

Acknowledgements The authors thank Alberto Merino for technical assistance. The research was supported by the project MCYT AGL2001-2293 of the Spanish Government.

References Alemayehu N. and H. Becker (2002) Genotypic diversity and patterns of variation in a germplasm material of Ethiopian mustard (Brassica carinata A. Braun). Genet. Resour. Crop Evol. 49: 573-582 Allard R.W. (1960) Principles of plant breeding John Wiley & Sons, New York Bellostas N., J.C. Sørensen and H. Sørensen (2007) Profiling glucosinolates in vegetative and reproductive tissues of four Brassica species of the U-triangle for their biofumigation potential. J. Sci. Food Agric. 87: 1586-1594 Buskov, S., B. Serra, E. Rosa, H. Sørensen and J.C. Sørensen (2002) Effects of intact glucosinolates and products produced from glucosinolates in myrosinase-catalyzed hydrolysis on the potato cyst nematode (Globodera rostochiensis cv. Woll). J. Agric. Food Chem. 50: 690-695

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Downey R.K., A.J. Klassen and G.R. Stringam (1980) Rapeseed and mustard. In: W.R. Fehr and H.H. Hadley (eds.), Hybridization of Crop Plants, 495-509. American Society of Agronomy and Crop science Society of America, Madison, WI Getinet A., G. Rakow and J.P. Raney (1996) Glucosinolate content variation in Brassica carinata A. Braun germplasm grown at Holetta Ethiopia. Cruciferae Newsl. 18: 84-85 Getinet A, G. Rakow, J.P. Raney and R.K. Downey (1997) Glucosinolate content in interspecific crosses of Brassica carinata with B. juncea and B. rapa. Plant Breeding 116: 39-46 Gland A. (1985) Inheritance of content and pattern of glucosinolates in combinations of resynthesized rapeseed x rapeseed cultivars. In: H. Sørensen (ed.), Advances in the Production and Utilization of Cruciferous Crops. 278-285. Martinus Nijhoff/Dr W. Junk Publishers, Dordrecht, The Netherlands Griffiths D.W., A.N.E. Birch and J.R. Hillman (1998) Antinutritional compounds in the Brassicaceae: analysis, biosynthesis, chemistry and dietary effects. J. Hort. Sci. Biotechnol. 73: 1-18 Hecht S.S. (2000) Inhibition of carcinogenesis by isothiocyanates. Drug Metab. Rev. 32, pp. 395-411 Hill J., P. Lethenborg, P.W. Li, M.H. Rahman, H. Sørensen and J.C. Sørensen (2003) Inheritance of progoitrin and total aliphatic glucosinolates in oilseed rape (Brassica napus L.). Euphytica 134: 179-187 Kirkegaard J.A., M. Sarwar and J.N. Matthiessen (1998) Assessing the biofumigation potential of crucifers. Acta Hortic. 459: 105-111 Kondra Z.P. and B.R. Stefansson (1970) Inheritance of major glucosinolates of rapessed (Brassica napus) meal. Can. J. Plant Sci. 50: 643-647 Lazzeri L., R. Tacconi and S. Palmieri (1993) In vitro activity of some glucosinolates and their interaction products toward a population of the nematode Heterodera schachtii. J. Agric. Food Chem. 41: 825-829 Lazzeri L., O. Leoni and L.M. Manici (2004) Biocidal plant dried pellets for biofumigation. Ind. Crops Prod. 20: 59-65 Love H.K., G. Rakow, J.P. Raney and R.K. Downey (1990a) Development of low glucosinolate mustard. Can. J. Plant Sci. 70: 419-424 Love H.K., G. Rakow, J.P. Raney and R.K. Downey (1990b) Genetic control of 2-propenyl and 3-butenyl glucosinolate synthesis in mustard. Can. J. Plant Sci. 70: 425-429 Magrath R., and R. Mithen (1993) Maternal effects on the expression of individual aliphatic glucosinolates in seeds and seedlings of Brassica napus. Plant Breeding 111: 249-252 Mahmood T., U. Ekuere, F. Yeh, A.G. Good and G.R. Stringam (2003) Molecular mapping of seed aliphatic glucosinolates in Brassica juncea. Genome 46: 753-760

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Pérez-Vich B., J. Fernández, R. Garcés and J.M. Fernández-Martínez (1999) Inheritance of high palmitic acid content in the seed oil of sunflower mutant CAS-5. Theor. Appl. Genet. 98: 496-501 Rücker B. and G. Röbbelen (1994) Inheritance of total and individual glucosinolate content in seeds of winter oilseed rape (Brassica napus L.). Plant Breeding 113: 206-216 Sodhi Y.S., A. Mukhopadhyay, N. Arumugam, J.K. Verma, V. Gupta, D. Pental and A.K. Pradhan (2002) Genetic analysis of total glucosinolate in crosses involving a high glucosinolate Indian variety and a low glucosinolate line of Brassica juncea. Plant Breeding 121: 508-511 Thies W. (1982) Complex-formation between glucosinolates and tetrachloropalladate (II) and its utilization in plant breeding. Fette Seifen Anstrichmitt. 84: 388-342 Velasco L. (1996) Utilización de mutagenesis química y análisis por reflectancia en el infrarrojo cercano para la mejora de la calidad de la mostaza etíope. PhD Thesis, Department of Genetics, University of Córdoba, Spain Velasco L. and H.C. Becker (1998) Analysis of total glucosinolate content and individual glucosinolates in Brassica spp. by near-infrared reflectance spectroscopy. Plant Breeding 117: 97-102 Velasco L. and H. C. Becker (2000) Variability for seed glucosinolates in a germplasm collection of the genus Brassica. Genet. Resour. Crop Evol. 47: 231-238 Velasco L., J.M. Fernández-Martínez and A. De Haro (1996) Identification of an Ethiopian mustard line with very high levels of glucosinolates. Cruciferae Newsl. 18: 96 Velasco L., J.M. Fernández-Martínez and A. De Haro (1999) Intraspecific breeding for reduced glucosinolate content in Ethiopian mustard (Brassica carinata A. Braun). Euphytica 106:125−130 Wright S. (1968) Evolution and the Genetics of Populations, Vol. 1. Univ. of Chicago Press, Chicago

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CAPÍÍTULO 3 CAP

Transferability, amplification quality and genome specificity of microsatellites in Brassica carinata and related Brassica species

Márquez-Lema A., L. Velasco and B. Pérez-Vich Instituto de Agricultura Sostenible (CSIC), Alameda del Obispo s/n. E-14004 Córdoba, España

Transferability, amplification quality and genome specificity of microsatellites in Brassica carinata and related Brassica species Breeding Science (2008) (en revisión)

Márquez Lema A.

Capítulo 3

Abstract No information is available on the transferability and amplification quality of microsatellite (SSR) markers of public domain in Brassica carinata A. Braun. The objective of the present research was to study the amplification of a set of 73 SSRs from B. nigra (L.) Koch and B. napus L. in B. carinata, and to compare the results with those obtained in the amplification of the same markers in the other Brassica species of the U triangle. 94.3% of the SSR markers from B. nigra and 97.4% of those from B. napus amplified SSR-specific products in B. carinata. Very high quality amplification in B. carinata was recorded for 52.8% of the specific loci from B. nigra SSRs and 59.3% of the specific loci from B. napus SSRs, compared to 66.7% and 62.8%, respectively in the focal species. Genome specificity and amplification quality of B. nigra and B. napus SSR markers in the six species under study is reported. Polymorphic SSR loci and cluster analysis revealed the diploid/amphidiploid relationships among the six Brassica species of the U triangle. High quality transferable SSR markers provide an efficient and cost-effective platform to advance in molecular research in B. carinata.

Key words: Brassica carinata − B. napus − B. nigra − microsatellites − molecular markers − simple sequence repeats (SSRs) − transferability

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Introduction Brassica carinata A. Braun (Ethiopian mustard) is an amphidiploid species (genome BBCC) originated as a natural cross between B. nigra (L.) Koch (BB) and B. oleracea L. (CC) in northeastern Africa, probably in the Ethiopian plateau, where wild forms of B. nigra co-exist with cultivated forms of B. oleracea since ancient times (Tsunoda 1980). Brassica carinata has great potential as an oilseed crop due to a better agronomic performance than other Brassica oilseed species under high temperature and low rainfall conditions (Warwick et al. 2006). However, breeding research on this species has been very scarce so far. In particular, no research has been conducted to develop molecular tools for trait breeding in B. carinata. Molecular research on B. carinata have focused exclusively on studies of genetic diversity within the species using RAPD (Teklewold and Becker 2006) and AFLP markers (Genet et al. 2005, Warwick et al. 2006). Simple sequence repeat (SSR, microsatellite) markers are widely used in molecular research and breeding in Brassica species because they are numerous, codominant, highly polymorphic and informative, technically simple and reproducible, and relatively inexpensive when primer information is available. Microsatellites have been developed from several Brassica species, including B. napus L., B. nigra (L.) Koch, B. oleracea L. and B. rapa L. (Plieske and Struss 2001, Lowe et al. 2002, Suwabe et al. 2002, Lowe et al. 2004). Several studies have demonstrated a high transferability of microsatellites from the focal species in which they were identified to other Brassica species or even to other related genera. Plieske and Struss (2001) tested the amplification of 81 microsatellite markers identified in B. napus in other Brassica species and found amplification of 62 primer pairs in B. carinata, 61 in B. oleracea, 51 in B. juncea (L.) Czern., 41 in B. rapa and 24 in B. nigra. Additionally, between 24 and 28 primer pairs amplified in related genera such as Sinapis, Diplotaxis, Raphanus, and Eruca. Lowe et al. (2004) studied microsatellite transferability between B. nigra, B. rapa, B. oleracea and B. napus. Except for B. oleracea, the maximum percentage of microsatellites that amplified a product was found in the focal species (from 63.6% in B. oleracea to 85.5% in B. nigra). For the other species, microsatellite amplification ranged from 45.9% (microsatellites from B. napus that amplified in B. nigra) to 72.7% (microsatellites from B. oleracea that amplified in B. napus). The authors concluded that transferability was higher between the A and C genome species (B. oleracea, B. rapa and B. napus)

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than between this group and the B genome species B. nigra. Microsatellite marker development has been recently accomplished in Brassica species by in silico database mining, due to the abundant sequence data already available for some of these species. Sets of 16 and 24 markers have been developed from expressed sequence tags (ESTs) of B. juncea and B. napus, respectively, and 40 SSR markers have been developed from B. oleracea genome shortgun sequences (Burgess et al. 2006, Batley et al. 2007, Hopkins et al. 2007). Hopkins et al. (2007) reported high transferability of the 16 B. juncea EST-SSRs across Brassica species of the U triangle. Microsatellite markers available for B. carinata breeding are very scarce, as no B. carinata SSR markers have been developed and only a few of microsatellite markers of public domain have been transferred to this species. Since a greater number of markers are required for their practical use in B. carinata breeding, the objective of the present research was to evaluate the amplification and to determine the quality of the amplified products of a set of microsatellite primers from B. nigra and B. napus in B. carinata, and to compare the results with those obtained in the amplification of the same primers in the other Brassica species of the U triangle. Additionally, genome specificity of the SSR loci was determined and cluster analysis among the Brassica species of the U triangle was carried out.

Materials and Methods Plant materials Two different germplasm accessions, cultivars or breeding lines of each species were used to evaluate microsatellite amplification in six Brassica species. Accessions of the diploid species B. rapa (BRA 1609, BRA 1722), B. nigra (BRA 189, BRA 191) and B. oleracea (K 9558, BRA 312) were obtained from the genebank of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) of Gatersleben, Germany. The commercial cultivars Iris and Apollo were used for B. napus. The cultivars Zem-1 and Heera were used for B. juncea. The germplasm accessions C-101 and C-49 were used for B. carinata.

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DNA extraction and PCR analysis Total genomic DNA was extracted separately from young leaves of each line following Berry et al. (1995). The final concentration of DNA was estimated by agarose gel electrophoresis and ethidium bromide staining using known concentrations of Low DNA Mass Ladder (Invitrogen, San Diego, USA) as a standard. The working concentration of DNA was adjusted to 100 ng/µl. A set of 73 microsatellite markers (SSRs) randomly selected from those available from B. nigra (B-genome) and B. napus (AC-genome) (Lowe et al. 2004) were used. The PCR reaction mixture (30 µl) contained 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs (Invitrogen, San Diego, CA, USA), 0.3 µM of primers, 0.7 U of Taq DNA polymerase (BioTaqTM DNA Polymerase, Bioline, London, UK), and 50 ng of template DNA. For seventeen SSR markers, PCR amplification was optimized by adjusting reaction mixtures to varying concentrations of MgCl2 (1.5, 2, 2.5 and 3 mM), primers (0.4, 0.8 and 1.2 µM), Taq DNA polymerase (1 unit) and DNA (25, 50 and 75 ng). PCR reactions were run on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) under the same conditions described by Lowe et al. (2004). Amplified products were separated on 3% Metaphor® (BMA, Rockland, ME, USA) agarose gels in 1x TBE buffer with ethidium bromide incorporated in the gel.

Quality of SSRs transferability Amplification fragments were classified as microsatellite-specific products if they produced amplification products with a size similar (within 100 bp) to that of the corresponding focal species (B. nigra or B. napus). Only these fragments were considered to evaluate the quality of SSR transferability. The microsatellite-specific products were classified into four classes based on the signal intensity and ease of scoring: strong signal and easy score (+++); moderate signal but able to score (++); weak signal and difficult to score (+); and no signal (-).

Cluster analysis The SSR profiles generated by the amplification of the Brassica material using the B. napus and B. nigra SSR markers were scored visually. Complex banding patterns were especially observed in amphidiploid species, which made it difficult the assignment of alleles to loci. This

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prohibited the use of allele-scoring techniques. Instead, the absence/presence of each polymorphic amplification product in each line was determined for each primer combination, and data was recorded in a 0/1 matrix, as described by Hasan et al. (2006) for the analysis of B. napus. The binary matrix was used to estimate Dice similarity index (Dice 1945), which is defined as: S = 2Nab / (2Nab + Na + Nb) where Nab is the total number of bands common in genotypes a and b, and Na and Nb are the total number of bands presents in genotypes a and b, respectively. The similarity matrices were subjected to unweighted pair group method of arithmetic averages (UPGMA) clustering method in order to construct the phenetic dendrograms. The NTSYS-pc software (Exeter Software, Setauket, NY, USA) (Rohlf 1998) was employed for carrying out these analyses. The same software was used to calculate the cophenetic correlation (rc) of each phenogram obtained in this study, and Mantel´s test (Mantel 1967) to check the goodness of fit of cluster analysis to the matrix on which it was based. One thousand randomisations were used to test for significance. Additionally, principal coordinate analysis (PCO) based on the Dice genetic similarity matrices were performed with the NTSYS-pc software package.

DNA sequencing PCR products amplified with four microsatellite markers (Ni2-A12, Ni4-A09, Na10-F08, and Na10-H03) were sequenced in one individual per species. PCR reactions were performed as described above, and amplification products were assessed by electroforesis on 2.5% agarose gels in 1x TBE and staining with ethidium bromide. Specific DNA bands were excised from the gel and purified by using the QIAquick Gel Extraction kit (Qiagen, Valencia, CA, USA). Purified PCR products were directly sequenced in both forward and reverse orientations at GATC Biotechnology (Konstanz, Germany) using the corresponding forward and reverse primers.

Results Thirty-three out of 35 (94.3%) primer pairs from B. nigra and 37 out of 38 (97.4%) primer pairs from B. napus amplified microsatellite-specific products in B. carinata. For the other species, the percentage of specific amplification of B. nigra microsatellites was 94.3% for B. juncea, 91.4% for B.

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oleracea, 85.7% for B. rapa, and 74.3% for B. napus. The percentage of specific amplification of B. napus microsatellites was 97.7% for B. juncea, 97.4% for B. rapa and B. oleracea, and 68.4% for B. nigra. Therefore transferability of B. nigra microsatellites was higher in the species carrying the B and/or C genomes, whereas transferability of B. napus microsatellites was higher in the species carrying the A and/or C genomes. Fifty-four out of 73 primer pairs (74.0%) amplified a microsatellite-specific product in the three Brassica genomes. Microsatellite specificity was confirmed by sequencing amplification products obtained with SSR primers. In all cases, sequenced products contained the same repeat motif described by Lowe et al. (2004) for these microsatellites (Table 3.1).

Table 3.1.- Number and type of SSR motifs across the six Brassica species of the U-triangle determined by a sequence analysis .

Motifb

SSR marker

B. rapa

B. nigra

B. oleracea

B. juncea

B. napus

B. carinata

(AA)

(BB)

(CC)

(AABB)

(AACC)

(BBCC)

SSR from B. nigra Ni2-A12

(GA)20

f

f

f

(GA)17

f

(GA)12

Ni4-A09

(GA)25

-

(GA)8

-

(GA)8

-

(GA)8

SSR from B. napus Na10-F08

(GGC)5

(GGC)5

(GGC)2

(GGC)3

(GGC)5

(GGC)6

(GGC)3

Na10-H03

(GGC)6

(GGC)3

-

(GGC)3

(GGC)4

(GGC)6

(GGC)6

a

- No amplified band; f, failed to sequence

b

As described in Lowe et al. (2004)

Very high quality amplification in B. carinata was recorded for 52.8% of the specific loci from B. nigra microsatellites, compared to 66.7% in the focal species and 63.9% in B. juncea. For the other species, the percentage of amplicons of maximum quality was considerably lower (Table 3.2). Similarly, 59.3% of the specific loci from B. napus produced strong amplification in B. carinata, compared to 62.8% in the focal species, 61.6% in B. juncea, 55.8% in B. rapa and much lower percentages in B. nigra and B. oleracea (Table 3.2). The quality of individual microsatellite-specific loci produced by microsatellites from B. nigra and B. napus in the six species under study is reported in ESM 3.1 and ESM 3.2, respectively.

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Eight B. nigra (BB) microsatellite markers revealed genome specific loci, which showed good quality amplification (+++, ++) only in their own species or in species carrying the B genome (ESM 3.1). Eighteen B. napus (AACC) markers produced specific loci with poor quality amplification products (+) or no amplification (-) in B. nigra (BB). From these markers, Na10-B01 amplified good quality (+++, ++) products only in species with the A genome, and Na12-G05, and Na14-G08 only in species with the C genome (EMS 3.2).

Table 3.2.- Percentage of specific loci amplified by SSRs from B. nigra and B. napus in six Brassica spp. a according to the quality of amplification .

Quality of

B. rapa

B. nigra

B. oleracea

B. juncea

B. napus

B. carinata

(AA)

(BB)

(CC)

(AABB)

(AACC)

(BBCC)

+++

20.8

66.7

16.7

63.9

16.7

52.8

++

19.4

13.9

23.6

13.9

20.8

19.4

+

40.3

18.1

43.1

16.7

34.7

19.4

-

19.4

1.4

16.7

5.6

27.8

8.3

amplification SSRs from B. nigra

SSRs from B. napus

a

+++

55.8

37.2

44.2

61.6

62.8

59.3

++

5.8

14.0

23.3

4.7

17.4

12.8

+

22.1

16.3

25.6

19.8

19.8

19.8

-

16.3

32.6

7.0

14.0

0.0

8.1

+++, ++, and + indicate strong, moderate and weak amplification patterns, respectively; – indicates no amplification

The seventy three primer pairs from B. nigra (n = 35) and B. napus (n = 38) used in the analysis of the cultivated Brassica species showed polymorphisms, with a total of 357 alleles identified. Of these, 164 alleles were obtained with SSRs from B. nigra and 193 alleles were obtained with SSRs from B. napus. Cluster analysis was performed using the combined data of the two sets of SSR markers (Fig. 3.1A), and those obtained separately which each set of SSR markers (Fig. 3.1B and Fig. 3.1C). For the three cluster analyses, cophenetic values among Brassica lines were significantly (P < 0.01) correlated (0.90 < rc < 0.94) with the original values. As expected, Brassica species were grouped according to the three diploid genomes (A, B and C), but some differences were observed.

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Fig. 3.1.- Dendrograms for the six Brassica species (B. rapa: BRA 1609, BRA 1722; B. nigra: BRA 189, BRA 191; B. oleracea: K 9558, BRA 312; B. napus: Iris, Apollo; B. juncea: Zem-1, Heera; and B. carinata: C-101 and C-49) based on cluster analysis (UPGMA) of genetic-similarity estimates (Dice similarity index) from: (A), data from the combined set of 73 SSRs from B. nigra and B. napus; (B), data from the set of 35 SSRs from B. nigra; and (C), data from the set of 38 SSRs from B. napus.

(A)

BRA 1609a BRA 1609b BRA 1722a BRA 1722b BRA 312a BRA 312b K 9778a K 9778b IRIS APOLO C-49 C-101 BRA 189a BRA 189b BRA 191a BRA 191b HEERA ZEM-1

A-genome

C-genome

AC-genome BC-genome B-genome

AB-genome 0.00

0.25

0.50

0.75

1.00

Dice coefficient

(B)

A-genome

AB-genome BC-genome

C-genome

AC-genome

B-genome

0.00

0.25

0.50

0.75

BRA 1609a BRA 1609b BRA 1772a BRA 1722b HEERA ZEM-1 C-49 C-101 BRA 312a BRA 312b K 9778a K 9778b IRIS APOLO BRA 189a BRA 189b BRA 191a BRA 191b 1.00

Dice coefficient

A-genome

(C) B-genome

AB-genome

C-genome

AC-genome BC-genome

0.00

0.25

0.50

0.75

Dice coefficient

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BRA 1609a BRA 1609b BRA 1772a BRA 1722b BRA 189a BRA 189b BRA 191a BRA 191b HEERA ZEM-1 BRA 312a BRA 312b K 9778a K 9778b IRIS APOLO C-49 C-101 1.00

Márquez Lema A.

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The dendrogram obtained with 35 B. nigra SSRs (Fig 3.1B) contributed significantly to differentiate the B-genome diploid species B. nigra, while the dendrogram obtained with 38 B. napus SSRs provided a fairly good separation of the species carrying the C-genome (B. oleracea, B. carinata and B. napus) from those carrying the A-genome (Fig. 3.1C). The general dendogram (Fig. 3.1A) constructed using the combined data of the two set of B. napus and B. nigra SSR markers showed the diploid species carrying the A-genome (B. rapa) closer to that with the Cgenome (B. oleracea) rather than to that with the B-genome (B. nigra), differentiating the Nigra lineage and the Rapa/Oleracea lineages. Principal coordinate analysis (PCO) was used to visualize the genetic relationships among the studied Brassica species. The first three axes of the PCO accounted for 68% (37.4%, 16.2%, and 14.4%, respectively) of the variation observed and revealed the diploid/amphidiploid relations described by U (1935) (Fig. 3.2).

Fig. 3.2.- Three dimensional principal coordinate analysis for the six Brassica species under study (B. rapa: BRA 1609, BRA 1722; B. nigra: BRA 189, BRA 191; B. oleracea: K 9558, BRA 312; B. napus: Iris, Apollo; B. juncea: Zem-1, Heera; and B. carinata: C-101 and C-49). The percentages of the total variation represented by the principal coordinates are indicated.

BRA 1772a

B. rapa

BRA 1772b

(AA)

BRA 1609a

BRA 1609b

B. napus

(1 6. 2%

)

3rd Axis (14.4%)

(AACC)

IRIS

2n d

Ax is

APOLO

ZEM-1 HEERA K 9778a BRA 312a

B. juncea

K 9778b

(AABB) C-49

BRA 189a

B. nigra (BB)

BRA 189b

C-101

BRA 191b BRA 191a

B. carinata (BBCC)

1st

Axi s (3 7.4

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%)

BRA 312b

B. oleracea (CC)

Márquez Lema A.

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Discussion In spite of the great potential of SSR markers, they have not been used so far in B. carinata research and breeding. Accordingly, one of the main objectives of this research was to determine the feasibility of transferring high quality SSR markers to B. carinata. The majority of the B. nigra and B. napus SSR markers tested in B. carinata showed specific SSR amplification products of the expected size. The product specificity was confirmed by sequencing a number of amplification products in the six Brassica species tested. These products showed the same SSR motif found in the focal species, although the number of repeats differed from one species to another. Suwabe et al. (2004) compared nucleotide sequences of one B. rapa SSR amplified in the six Brassica species of the U triangle and found also different microsatellite repeat number among species, which has also been described for SSR cross-species amplification in other crops, such as pulse crops (Gutierrez et al. 2005). Although transferability was in general very high, the number of SSRs that amplified high quality products was lower. 59.3% of the B. napus and 52.8% of the B. nigra markers tested showed strong signal intensity and were easy to score in B. carinata. Slightly higher levels of very high quality transferable markers were obtained for the amphidiploid species B. juncea (genome AABB) for the B. nigra (BB), and B. napus (AACC) markers (63.9% and 61.6%, respectively). Lower transferability values (for very high quality products) were obtained in those species carrying a different genome from that of the focal species, with the B. nigra markers showing the lowest values when tested in non-B genome species (average value of 18.1%). Previous studies have reported the transferability of Brassica SSR markers, although information on the quality of the amplified product was not detailed (Plieske and Struss 2001, Lowe et al. 2004). As expected, the reported percentages of SSR that amplified a product within each of the non focal species tested were higher than those obtained in this research for the amplification of very high quality products in non focal species. The possibility of transferring markers showing very high quality products, together with the availability of more than 700 public Brassica SSR (http://www.brassica.info/), would provide an efficient and cost-effective platform for the establishment of molecular strategies for use in B. carinata breeding and genetics.

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Márquez Lema A.

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Genome specificity is a prominent feature of SSR markers. Some of the SSR markers tested in this study revealed genome specific loci, according to the quality of the amplified product. These genome specific loci would be useful for identifying DNA fragments introgressed into another species, and for specific B and C genome identification in the case of B. carinata. Markers used in this study were those described by Lowe et al. (2004), who tested them in the diploid Brassica species B. rapa, B. nigra, and B. oleracea, and in B. napus. However, the results in relation to genome specificity in the present study were different to those reported by Lowe et al. (2004). The use of different PCR conditions to those used by Lowe et al. (2004) for some of the primer pairs (21 out of 73) due to the optimization PCR process, the different criteria used to classify the genome-specific SSR (in our case, we have considered only SSR products with a size similar to that in the focal species, and classified them based on the amplification quality), and the higher number of species tested in this study may have produced such differences. SSR marker loci amplifying specific loci of good quality (+++, ++) in the six Brassica species (a total of 7 SSR from B. nigra, and 9 SSR from B. napus) were identified (ESM 3.1 and 3.2). These loci are probably the result of highly conserved regions, and they are good candidates to be transferred to additional Brassica species. The amplification of a high number of polymorphic loci provided us an opportunity to determine the genetic relationships among the Brassica species of the U triangle. Some differences in cluster analysis were observed when using different sets of SSR markers. Despite a large number of the markers tested were non-genome specific, in general B. nigra SSR markers discriminated the B-genome (Fig. 3.1B), and B. napus markers discriminated the A and the C genomes (Fig. 3.1C). Cluster analysis using the combined set of B. nigra and B. napus SSRs enabled us to separate the Brassica species into their two evolutionary lineages (Rapa/Oleracea group and Nigra group of Brassica) (Fig. 3.1A) (Warwick and Black 1991, Pradhan et al. 1992). The highest similarity levels among species were observed between B. oleracea/B. napus, and B. oleracea/B. carinata, which is in agreement with results reported by Bornet and Branchard (2004) using ISSR fingerprints. Genetic relationships among the six Brassica species obtained by using both the B. nigra and the B. napus sets of SSRs reproduced the diploid/amphidiploid relations described by U (1935).

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Capítulo 3

Acknowledgments The research was supported by the project MYCT AGL2001-2293 of the Spanish Government.

References Bornet B. and M. Branchard (2004) Use of ISSR fingerprints to detect microsatellites and genetic diversity in several related Brassica taxa and Arabidopsis thaliana. Hereditas 140: 245-248 Batley J., C.J. Hopkins, N.O.I. Cogan, M. Hand, E. Jewell, J. Kaur, S. Kaur, X. Li, A. E. Ling, C. Love, H. Mountford, M. Todorovic, M. Vardy, M. Walkiewicz, G.C. Spangenberg and D.Edwards (2007) Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences. Mol. Ecol. Notes 7: 886-889 Berry S.T., A.J. Leon, C.C. Hanfrey, P. Challis, S.R. Burkholz, G.K. Barnes, M. Rufener, M. Lee and P.D.S. Caligari (1995) Molecular markers analysis of Helianthus annuus L. 2. Construction of a RFLP linkage map for cultivated sunflower. Theor. Appl. Genet. 91: 195-199 Burgess B., H. Mountford, C.J. Hopkins, C. Love, A. Ling, G. Spangenberg, D. Edwards and J. Batley (2006) Identification and characterization of simple sequence repeat (SSR) markers derived in silico from Brassica oleracea genome shotgun sequences. Mol. Ecol. Notes 6: 1191-1194 Dice L.R. (1945) Measures of the amount of ecologic association between species. Ecology 26: 297−302. Genet T., C.D. Viljoen and M.T. Labuschagne (2005) Genetic analysis of Ethiopian mustard genotypes using amplified fragment length polymorphism (AFLP) markers. Afr. J. Biotechnol. 4: 891-897 Gutierrez M.V., M.C. Vaz, T. PattoHuguet, J.I. Cubero, M.T. Moreno and A.M. Torres (2005) Crossspecies amplification of Medicago truncatula microsatellites across three major pulse crops. Theor. Appl. Genet. 110: 1210-1217 Hasan M., F. Seyis, A.G. Badani, J. Pons-Kühnemann, W. Friedt, W. Lühs and R.J. Snowdon (2006) Analysis of genetic diversity in the Brassica napus L. gene pool using SSR markers. Genet. Resour. Crop Evol. 53 (4): 793-802 Hopkins C.J., N.O.I. Cogan, M. Hand, E. Jewell, J. Kaur, X. Li, G.A.C. Lim, A.E. Ling, C. Love, H. Mountford, M. Todorovic, M. Vardy, G.C. Spangenberg, D. Edwards and J. Batley (2007) Sixteen new simple sequence repeat markers from Brassica juncea expressed sequences and their cross-species amplification. Mol. Ecol. Notes 7: 697-700 Lowe A.J., A.E. Jones, A.F. Raybould, M. Trick, C.L. Moule, and K.J. Edwards (2002) Transferability and genome specificity of a new set of microsatellite primers among Brassica species of the U triangle. Mol. Ecol. Not. 2: 7-11

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Lowe A.J., C. Moule, M. Trick and K.J. Edwards (2004) Efficient large-scale development of microsatellites for marker and mapping applications in Brassica crop species. Theor. Appl. Genet. 108: 1103−1112. Mantel N.A. (1967) The detection of disease clustering and a generalized regression approach. Cancer Res. 27: 209-220 Plieske J. and D. Struss (2001) Microsatellite markers for genome analysis in Brassica.I. Development in Brassica napus and abundance in Brassicaceae species. Theor. Appl. Genet. 102: 689-694 Pradhan A.K., S. Prakash, A. Mukhopadhyay and D. Pental (1992) Phylogeny of Brassica and allied genera based on variation in chloroplast and mitochondrial DNA patterns: molecular and taxonomic classifications are incongruous. Theor. Appl. Genet. 85: 331-340 Rohlf F.J. (1998) NTSYS-PC. Numerical Taxonomy and Multivariate Analysis System, Version 2.02. Exeter Software, Setauket, New York Suwabe K., H. Iketani, T. Nunome, T. Kage and M. Hirai (2002) Isolation and characterization of microsatellites in Brassica rapa L. Theor. Appl. Genet. 93: 534-538 Teklewold A. and H.C. Becker (2006) Geographic pattern of genetic diversity among 43 Ethiopian mustard Brassica carinata (A. Braun) accessions as revealed by RAPD analysis. Genet. Resour. Crop Evol. 53: 1173-1185 Tsunoda S. (1980) Eco-physiology of wild and cultivated forms in Brassica and allied genera. In: S. Tsunoda, K. Hinata and C. Gómez-Campo (eds.). Brassica crops and wild allies, Japan Scientific Societies Press, Tokyo, pp. 109-120 U.N. (1935) Genomic analysis of Brassica with special reference to the experimental formation of B. napus and its peculiar mode of fertilization. Jpn. J. Bot. 7: 389-452 Warwick S.I. and L.D. Black (1991) Molecular systematics of Brassica and allied genera (Subtribe Brassicinae, Brassiceae) chloroplast genome and cytodeme congruence. Theor. Appl. Genet. 82: 81-92 Warwick S.I., R.K. Gugel, T. McDonald and K.C. Falk (2006) Genetic variation of Ethiopian mustard (Brassica carinata A. Braun) germplasm in western Canada. Genet. Resour. Crop Evol. 53: 297-312

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Capítulo 3

ESM 3.1.- Quality of microsatellite-specific loci generated by B. nigra microsatellites in six Brassica species.

SSR loci a

B. rapa (AA)

Size

B. nigra (BB)

B. oleracea (CC)

B. carinata (BBCC)

B. juncea (AABB)

B. napus (AACC)

(bp) BRA1609 BRA1722 BRA189 BRA 191 BRA312

K9558

C-101

C-49

Heera

Zem-1

Iris

Apollo

Genome amplified (+++ or ++ quality)b

Ni1-A04 a

200-125

-

-

+

+

-

-

-

-

-

-

-

-

Ni2-A02 a

170-125

+

+

+++

+++

+

+

+++

+++

+++

+++

+

+

Btj

Ni2-A08 a

125-75

+

+

+++

+++

+

+

+++

+++

+++

+++

+

+

Btj

Ni2-A10 a

125-90

-

-

+++

+++

+

++

+++

+++

+++

+++

-

-

BC t j

Ni2-A11 a

600-500

+

+

+++

+++

+

+

+

++

+++

+++

+

+

Btj

Ni2-A11 b

425-325

+

-

+

+

+++

++

-

-

+++

+++

+++

+++

Cjn

Ni2-A12 a

140-75

-

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Ni2-B01 a

260-200

+

+

+++

+++

+++

+++

+++

+++

+++

+++

+

+

BC t j

Ni2-B02 a

100-80

+++

+++

+++

+++

+

+

++

++

+++

+++

+++

+++

AB n t j

Ni2-B03 a

500-125

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Ni2-C06 a

200-100

++

+

+++

+++

+

+

+

+

++

++

+

+

AB j

Ni2-C09 a

225-125

+

+

+++

+++

+

-

+++

+++

+++

+++

-

++

Bntj

Ni2-C12 a

350-250

-

+

+

+

++

++

+++

+++

+++

+++

++

++

Ctj

Ni2-D07 a

250-175

+++

+++

+++

+++

+

+

++

++

+++

+++

+

+

AB t j

Ni2-D08 a

150-100

+++

+++

+++

+++

++

++

+++

+++

+++

+++

++

++

ABC n t j

Ni2-D10 a

275-175

++

++

+++

+++

++

++

+++

+++

++

++

++

++

ABC n t j

Ni2-E04 a

225-125

++

++

+++

+++

++

++

+++

++

+++

+++

++

++

ABC n t j Bntj

Ni2-E05 a

180-100

+

+

+++

+++

+

+

+++

+++

+++

+++

++

++

Ni2-F01 a

300-200

+

+

+

+

-

+

+

+

+

+

+

+

Ni2-F02 a

320-220

+++

-

+++

+++

++

-

+

+

+

+

-

-

ABC

Ni2-F07 a

180-140

+

+

+++

+++

+

++

+++

+++

+++

+++

+

+

BC t j

Ni2-G06 a

225-160

++

++

+++

+++

++

+

+

+

+

+

+

+

ABC

Ni3-E06 a

175-150

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Ni3-G04ba

125-75

++

++

++

++

+

+

+++

+++

+++

+++

+

+

AB t j

Ni3-H07 a

260-175

+

+

+++

+++

+

+

+++

+++

+++

+++

-

+

Btj Btj

Ni4-A09 a

140-80

-

-

++

++

-

-

++

++

++

++

-

-

Ni4-A10 a

850-700

+

+

+

+

-

-

+

-

+

+

-

-

Ni4-B06 a

140-100

++

++

+++

+++

++

+

+++

+++

+++

+++

+

+

Ni4-C06 a

150-75

+

+

+++

+++

-

+

+++

+++

+++

+++

-

-

Btj

Ni4-D01 a

300

-

-

++

++

-

+

++

++

++

++

-

-

Btj

Ni4-D10 a

250-150

+++

++

++

+

++

+++

++

++

++

++

++

++

ABC n t j

Ni4-E08 a

150-60

+

+

+++

+++

+

-

+

+

+

+

-

-

B

Ni4-E11 a

~ 50

-

-

-

+

+

+

-

-

-

-

-

-

Ni4-G02 a

160-100

+

+

+++

+++

+

+

+++

+++

+++

+++

+

+

Btj

Ni4-G04 a

> 850

+++

+++

++

+

+++

+++

+++

+

+++

+++

+++

+++

ABC n t j

Ni4-H04 a

125-75

++

++

++

++

++

++

++

++

+

+

++

++

ABC n t

a

ABC t j

a,b = specific loci. Strong, moderate and weak amplification patterns are indicated as +++, ++ and +, respectively; -: no amplification. b A: B. rapa (A-genome); B: B. nigra (B-genome); C: B. oleracea (C-genome); t: B. carinata (BC-genome); j: B. juncea (AB-genome); n: B. napus (AC-genome).

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Márquez Lema A.

Capítulo 3

ESM 3.2.- Quality of microsatellite-specific loci generated by B. napus microsatellites in six Brassica species.

SSR locia

B. rapa (AA)

Size (bp)

B. nigra (BB)

B. oleracea (CC)

BRA1609 BRA1722 BRA189 BRA191 BRA312

K9558

B. carinata (BBCC) C-101

B. juncea (AABB)

C-49

Heera

B. napus (AACC)

Zem-1

Iris

Apollo

Genome amplified (+++ or ++ quality) b

Na10-B01 a

240-180

+++

+++

+

+

+

+

+

+

+++

+

+++

+++

Anj

Na10-B08 a

170-90

+++

+++

-

-

+++

-

+++

+++

+++

+++

+++

+++

AC t n j

Na10-B10 a

150-100

+++

+++

+++

+++

+

-

+++

+++

+++

+++

+++

+++

AB n t j

Na10-C01 a

300-200

++

+

+++

+++

++

++

+++

+++

+

+

++

++

ABC n t

Na10-C06 a

280-200

+++

-

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Na10-D03 a

225-140

++

++

++

++

+++

+++

+++

+++

+

+

+++

+++

ABC n t

Na10-D09 a

360-260

+++

+++

+++

+++

+++

-

-

-

+++

+++

+++

+++

ABC n j

Na10-D09 b

220-140

-

-

+++

+++

+

+++

+++

+++

+++

+++

+++

+++

BC n t j

Na10-E02 a

160-60

+++

+++

+

+

+++

+++

+++

+++

+++

+++

+++

+++

AC n t j

Na10-E08 a

300-200

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Na10-F01 a

360-260

+++

+++

-

-

++

++

+

+

+

+

+

+

AC

Na10-F06 a

180-100

+++

+++

+++

+++

+

+

+

+

++

++

++

++

AB n j

Na10-F06 b

325-225

+

+

+++

+++

+

+

-

-

+++

+++

+

+

Bj

Na10-F08 a

200-150

+++

+++

++

++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Na10-G01 a

220-120

+

+

++

++

+

+

++

++

+

+

++

+

Bnt

Na10-G06 a

250-175

+++

+++

+

+

+++

+++

+++

+++

+++

+++

+++

+++

AC n t j

Na10-H03 a

150-100

+++

+++

-

-

+++

+++

+++

+++

+++

+++

+++

+++

AC n t j

Na10-H06 a

150-75

+++

+++

+

+

+++

+++

+++

+++

+++

+++

+++

+++

AC n t j

Na12-A01 a

130-100

+++

+++

-

-

++

++

+++

+++

+++

+++

++

++

AC n t j

Na12-A01 b

225-160

-

-

-

-

++

++

-

-

-

-

+

+

C

Na12-A07 a

190-150

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Na12-A10 a

150-100

++

++

++

++

++

++

++

++

++

++

++

++

ABC n t j

Na12-B01 a

450-350

+++

-

+

+

++

++

++

++

+++

+++

++

++

AC n t j

Na12-B05 a

225-125

+++

+++

-

-

++

++

++

++

+++

+++

++

++

AC n t j

Na12-C03 a

260-220

+

-

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

BC n t j

Na12-C07 a

700-600

+++

+++

+++

+++

+++

+++

+

+++

+++

+++

+++

+++

ABC n t j

Na12-C07 b

450-420

-

-

+++

+++

+

+

-

+

+

+

+

+

B

Na12-C08 a

350-275

+

+

+

+

++

++

++

++

-

-

++

++

Cnt

Na12-D07 a

250-150

+++

+++

++

++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Na12-D10 a

180-100

-

-

-

-

+

+

+

+

-

-

+

+

Na12-D10 b

475-425

-

-

++

++

+

+

+

+

+

+

+

+

B

Na12-E01 a

250-150

+++

+++

-

-

++

++

+

++

+++

-

+

+

AC t j

+

+

-

-

+

+

+

+

+

-

+++

+++

n

+++

+++

-

-

+++

+++

+++

+++

+++

-

+++

+++

AC n t j

Na12-E06a a 400-250 Na12-F12 a

190-160

Na12-G05 a

250-150

+

+

-

-

+++

+++

+++

+++

+

+

+++

+++

Cnt

Na12-H09 a

225-125

+++

+++

-

-

+++

+++

+++

+++

+++

+++

+++

+++

AC n t j

Na14-A06 a

800-750

+++

+++

+++

+++

+++

+++

+

+++

+++

+++

+++

+++

ABC n t j

Na14-B05 a

150-100

+

+

+++

+++

+

+

+++

+++

+++

+++

+

+

BC t j

Na14-C12a

~ 900

+

-

-

-

-

-

+++

+++

-

-

+++

+++

nt

Na14-E08 a

125-90

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ABC n t j

Na14-E11 a

425-400

+

+

-

-

-

+

+++

+++

+++

+++

+++

+++

ntj

Na14-G02 a

200-150

+++

+++

+++

+++

+

++

+

+++

+++

+++

+++

+++

ABC n t j

Na14-G08 a

300-200

+

+

+

+

++

+++

+++

+++

+

-

+++

+++

Cnt

a

a,b = specific loci. Strong, moderate and weak amplification patterns are indicated as +++, ++ and +, respectively. -: no amplification. b A: B. rapa (A-genome); B: B. nigra (B-genome); C: B. oleracea (C-genome); t: B. carinata (BC-genome); j: B. juncea (AB-genome); n: B. napus (AC-genome).

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CAPÍÍTULO 4 CAP

Transgressive segregation for reduced glucosinolate content in Brassica carinata A. Braun

Márquez-Lema A., J.M. Fernández-Martínez, B. Pérez-Vich and L. Velasco Instituto de Agricultura Sostenible (CSIC), Alameda del Obispo s/n, E-14004 Córdoba, España

Transgressive segregation for reduced glucosinolate content in Brassica carinata A. Braun Plant Breeding (2006) 125: 400-402

Márquez Lema A.

Capítulo 4

Abstract Successful commercial utilization of the meal by-product of Brassica oilseed crops requires the cultivation of cultivars with low glucosinolate (GSL) content in the seeds; however, such cultivars are not yet available in Brassica carinata. The objective of the present research was to search for transgressive segregants with further-reduced GSL content in the progeny of crosses involving four B. carinata lines with reduced GSL content (90 compared with 120 µmol/g seed in standard germplasm). The four lines were crossed following a diallel design and F2 phenotypes (F3 seed bulked) were analysed for GSL content. F2 phenotypes with a transgressive GSL content lower than the parents were identified in all crosses involving the line S2-1241, suggesting that this line carries alleles for reduced GSL content not present in the other lines. F3:4 lines from transgressive F2 phenotypes were evaluated for 2 years, which resulted in the selection of an F3:4 line with an average GSL content of 58 and 46 µmol/g seed, respectively compared to 84 and 62 µmol/g seed, respectively in S2-1241.

Key words: Brassica carinata – Ethiopian mustard – meal quality – mutagenesis – seed glucosinolates – transgressive segregation

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Márquez Lema A.

Capítulo 4

Introduction Seeds and meals from double-zero cultivars of Brassica spp., also known as canola in Canada, are the second most widely-traded vegetable protein sources after soybean (FAOSTAT 2004). Doublezero quality is applied to those cultivars that produce seed oils with < 2% erucic acid and seed meals with < 30 µmol of aliphatic glucosinolates (GSL) per gram of oilfree meal (Downey 1990). Canola-quality cultivars have been developed in B. napus L., B. rapa L., and B. juncea (L.) Czern. (Raymer 2002). Brassica carinata A. Braun is considered as an alternative to current canola crops in regions with semiarid conditions, where this species shows a better agronomic performance (Knowles et al. 1981, Fereres et al. 1983, Rakow 1995). However, canola-quality strains of B. carinata have not been developed yet; although several genetic sources of low erucic acid content that comply with oil quality canola requirements have been developed (Alonso et al. 1991, Getinet et al. 1994, Fernández-Martínez et al. 2001), no germplasm of B. carinata with canola levels of seed GSLs has been developed so far. Sinigrin, also referred to as 2-propenyl or allyl GSL, is the major GSL in B. carinata seeds, accounting for more than 95% of the total GSL content (Getinet et al. 1997). Even though the limit for low-GSL quality in Brassica oilseeds has been set at 30 µmol of aliphatic GSLs per gram of defatted meal, the presence of sinigrin is particularly detrimental when the meal is intended for animal feed. For this reason, there is strong recommendation to reduce sinigrin to levels below 1 µmol/g seed (Potts et al. 1999). Several strategies have been followed for reducing GSL content in B. carinata seeds. The natural variation reported in germplasm collections is low, with an average GSL content of around 100 µmol/g seed (Alemayehu and Becker, 2002). Nevertheless, Velasco et al. (1999) identified variability within the breeding line C-49, with an average GSL content of 115 µmol/g seed, and isolated a line showing a reduced GSL content of 82 µmol/g seed. A second strategy has been mutagenesis. Velasco et al. (1999) applied chemical mutagenesis to seeds of the line C-101, with average GSL content of 120 µmol/g seed, and isolated five mutants with average GSL contents in the M5 generation between 90 and 101 µmol/g seed. Barro et al. (2002) used mutagenesis on isolated microspores and reported the identification of M2 variants with an average GSL content of

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38 µmol/g seed, although the genetic stability of these mutants has not been confirmed so far. A third strategy has been interspecific breeding. Getinet et al. (1997) crossed B. carinata with doublezero strains of B. juncea and B. napus. Total GSL content was reduced (65 µmol/g seed compared to 137 µmol/g seed in the B. carinata parent) in crosses with B. juncea, but not in crosses with B. napus, which led the authors to hypothesize that genes for GSL synthesis are likely to be located on B genome chromosomes of B. carinata. Australian researchers succeeded in developing B. juncea selections with GSL content within the canola standard by intercrossing several lines developed through different strategies such as mutagenesis, resynthesis, and somaclonal variation (Oram and Kirk 1993). The objective of the present research was to develop transgressive segregants with lower GSL content than the parents, from crosses involving three mutants and a breeding line of B. carinata with reduced GSL content.

Materials and Methods Plant materials The Ethiopian mustard line N2-142, with an average GSL content of 82 µmol/g seed, was developed after three generations of pedigree selection from the breeding line C-49, with an average GSL content of 115 µmol/g seed (Velasco et al. 1999). The mutant lines S2-1241 (93 µmol/g seed), N2-7397 (90 µmol/g seed), and N2-9531 (99 µmol/g seed) were developed as M5 lines following chemical mutagenesis of the line C-101, with average GSL content of 121 µmol/g seed and high erucic acid content (Velasco et al. 1999). The four lines had a GSL profile mainly made up of sinigrin, which accounted for more than 93% of the total GSLs.

Crossing and selection scheme Plants of N2-142, S2-1241, N2-7397, and N2-9531 were grown in a field screenhouse in spring 2002 and crossed following a diallel design without reciprocals. Microperforated plastic bags were used to prevent uncontrolled pollination after crosses were made. F1 plants were grown in the greenhouse in winter 2002-2003 and selfed to obtain the F2 generation. F2 plants were grown in the field in 2003, together with plants of the parents and line C-101 used as check, in rows 5-m long

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with 1 m spacing between rows. Three adjacent rows were sown per parent or F2. The inflorescences were covered with microperforated plastic bags before flowering to ensure selfpollination. Twenty-four individual plants were harvested from the parents and the standard check C-101. From 112 to 144 F2 individual plants were harvested from each cross. F3 seed samples were analysed for GSL content as described below. Twelve F2 plants with lower GSL content than the lower limit of the parents were selected and the corresponding F3 plants were grown in the field in 2004 as described above. F4 seed samples were analysed for GSL content. An F3:4 line that exhibited a clear transgressive and stable reduced GSL content in comparison with the parents was grown again in the field in 2005 together with the parents to confirm the transgressive nature of the trait.

Analysis of glucosinolate content Total GSL content was measured on intact seeds by near-infrared reflectance spectroscopy (NIRS), with the palladium-glucosinolate (Pd-GSL) complex method used as reference analysis. In both cases the methodology reported by Velasco et al. (1999) was followed. In all generations, selection was made using NIRS data and all phenotypes selected were further analysed by the reference method. In general, there were no major discrepancies between NIRS and reference method data, with a coefficient of correlation between them of 0.91 in a pooled comparison that included all the samples analysed by the reference method throughout the present research. The results reported in the present manuscript correspond to the analysis by the reference method. Additionally, seeds of the parents and selected F3:4 lines were analysed for GSL content and profile by high performance liquid chromatography (HPLC) as described by Velasco and Becker (1998).

Results and Discussion Plants of the B. carinata check cultivar C-101, grown under the same environment as the F2 generation, averaged 97 µmol/g seed, whereas the lines with reduced GSL content averaged 67 µmol/g seed (N2-142), 64 µmol/g seed (S2-1241), 83 µmol/g seed (N2-7397), and 69 µmol/g seed (N2-9531) (Table 4.1). The ranges of variation of the lines with reduced GSL content did not overlap with the range of variation of the standard check, except for the line N2-7397 that showed a

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range of variation different to the other three lines. Minimum GSL content was 53 µmol/g seed for S2-1241, around 60 µmol/g seed for N2-142 and N2-9531, and above 75 µmol/g seed for N2-7397.

Table 4.1.- Number of individual plants analysed, mean, and range of variation for total seed glucosinolate (GSL) content (µmol/g seed) in the Brassica carinata lines C-101, with standard GSL content, N2-142, S2-1241, N2-7397, and N2-9531, with reduced GSL content, and F2 populations from crosses involving the lines with reduced GSL content, grown at Córdoba, Spain, in 2003.

Parent or cross

n

Mean

Minimum-Maximum

C-101

24

97

89-108

N2-142

24

67

59-75

S2-1241

24

64

53-75

N2-7397

24

83

76-89

N2-9531

24

69

59-79

S2-1241 x N2-142

120

65

44-85

N2-142 x N2-7397

116

80

61-95

N2-142 x N2-9531

112

83

61-107

S2-1241 x N2-7397

138

78

39-116

S2-1241 x N2-9531

144

69

35-96

N2-7397 x N2-9531

120

89

65-115

The analysis of the F2 generation (F3 seeds bulked) revealed the absence of transgressive segregants with lower GSL content than the parents in the crosses N2-142 x N2-7397, N2-142 x N2-9531, and N2-7397 x N2-9531, and the presence of transgressive segregants in the three crosses in which the line S2-1241 was involved, i.e. S2-1241 x N2-142, S2-1241 x N2-7397, and S2-1241 x N2-9531 (Table 4.1). The minimum GSL content in the latter crosses was 44, 39, and 35 µmol/g seed, respectively. GSL content in the three populations showed continuous distributions where no discrete classes could be identified. The progenies of four transgressive F2 plants with lower GSL content than the parents for each of the crosses were sown and the corresponding F3:4 lines were evaluated for GSL content.

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Plants of the line N2-142 grown in the same environment as the F3 plants averaged 85 µmol/g seed, with a range of variation from 71 to 93 µmol/g seed. Plants of the line S2-1241 had an average GSL content of 84 µmol/g seed, with a range of variation from 69 to 99 µmol/g seed. F3:4 lines had average GSL contents from 58 to 81 µmol/g seed. Even though about half the lines included individuals with transgressive GSL contents, only the F3:4 line that averaged 58 µmol/g seed exhibited a range of variation in which practically all the plants had a GSL content below the lower limit of the parents, between 48 and 72 µmol/g seed. This line, which was derived from the cross S2-1241 x N2-142, was further evaluated in the following season to confirm the genetic basis of such a transgressive range of variation. Plants of the parental lines N2-142 and S2-1241 had average GSL contents of 75 and 62 µmol/g seed, respectively, and ranges of variation from 70 to 83 µmol/g seed for N2-142, and from 57 to 70 µmol/g seed for S2-1241. Plants of the putative transgressive line had an average GSL content of 46 µmol/g seed, ranging from 31 to 59 µmol/g seed. This range of variation, determined by the Pd-GSL complex method, was confirmed by HPLC, resulting in a range of variation from 34 to 54 µmol/g seed, mainly in the sinigrin form (> 95% of the total GSLs). Fig. 4.1 shows the histogram of GSL content in the transgressive F3:4 line in comparison to the parent with the lowest GSL content in this environment, S2-1241. All crosses involving the line S2-1241 yielded transgressive segregants in the F2 and F3 generations, which provided a clear indication that this line may carry alleles for reduced GSL content different from those present in the other three lines evaluated. Barro et al. (2002) reported an M2 mutant with reduced levels of seed GSLs similar to those developed in the present research. Nevertheless, the stability of the trait in more advanced generations has not been reported. Total GSL content in B. carinata seeds is a trait strongly influenced by environmental conditions (Getinet et al. 1997). This can be observed also in the present research, where the GSL content of the lines N2-142 and S2-1241 grown as checks in three environments showed a large environmental influence. Accordingly, the confirmation in a second environment of the transgressive levels of GSL content identified in the present research ensures that this is the result of genetic changes rather than environmental effects.

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Fig. 4.1.- Histograms of glucosinolate content (µmol/g seed) in plants of an F3:4 line of Brassica carinata derived from a cross between the lines N2-142 and S2-1241, with reduced GSL content, and plants of the line S2-1241 used as check, grown at Córdoba, Spain in 2005.

16 F

3:4

12

line

S2-1241 x N2-142

8

Number of plants

4

0 12

0

10

20

30

40

50

60

70

80

90 100

40

50

60

70

80

90 100

g-1

seed)

S2-1241 8

4

0 0

10

20

30

Glucosinolate content (µmol

The GSL fraction of B. carinata seeds is made up mainly of sinigrin (2-propenyl GSL), which accounts for more than 95% of the total GSLs in the seeds (Getinet et al. 1997). This was also the case for the four lines used in the present research and the F3:4 transgressive line developed. Sinigrin is particularly detrimental when the meal is intended for animal feed. For this reason, there is strong recommendation to reduce sinigrin to levels as low as possible, even below 1 µmol/g seed (Potts et al. 1999). As there is practically no variation for GSL profile in B. carinata germplasm (Getinet et al. 1997, Velasco and Becker 2000), the development of double-zero strains of B. carinata will require the development of additional genetic sources of reduced sinigrin content. Brassica carinata is a plant species with impressive potential as an oilseed crop for dry areas because of its heat and drought tolerance (Fereres et al. 1983), resistance to seed shattering

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(Knowles et al. 1981) and resistance to pests and diseases (Malik 1981, Gugel et al. 1990). Additionally, germplasm with optimal seed oil quality such as high oleic acid (Velasco et al. 2003) and low linolenic acid content (Velasco et al. 2004) has been developed. However, a major constraint for the development of B. carinata as a new canola crop for semiarid areas is the lack of availability of germplasm with low GSL content. The level of GSL content achieved in the present research is still too high for the efficient utilization of B. carinata meal as animal feed, and further reductions in GSL content are required to make B. carinata an economic alternative to B. napus and other Brassica species with low GSL content.

Acknowledgements The authors thank Alberto Merino for technical assistance. The research was supported by the project MCYT AGL2001-2293 of the Spanish Government.

References Alemayehu N. and H. Becker (2002) Genotypic diversity and patterns of variation in a germplasm material of Ethiopian mustard (Brassica carinata A. Braun). Genet. Resour. Crop Evol. 49: 573-582 Alonso L.C., O. Fernández-Serrano and J. Fernández-Escobar (1991) The outset of a new oilseed crop: th Brassica carinata with low erucic acid. Proc. 8 Int. Rapeseed Congr., Saskatoon, Canada, 9-11 July

1991, 170-176. GCIRC, Paris, France Barro F., J. Fernández-Escobar, M. De la Vega and A. Martín (2002) Modification of GSL and erucic acid contents in doubled haploid lines of Brassica carinata by UV treatment of isolated microspores. Euphytica 129: 1-6 Downey R.K. (1990) Brassica oilseed breeding: achievements and opportunities. Plant Breeding Abstr. 60: 1165-1170. FAOSTAT (2004) FAO Statistical Databases: Agriculture and Food Trade. Food and Agriculture Organization of The United Nations. (www. faostat.fao.org, updated 7 December 2004, accessed 11 October 2005) Fereres E., J. Fernández-Martínez, I. Mínguez and J. Domínguez (1983) Productivity of Brassica juncea th and B. carinata in relation to rapeseed, B. napus. I. Agronomic studies. Proc. 6 Int. Rapeseed Congr.,

Paris, France, 17-19 May 1983, 293-298. GCIRC, Paris, France Fernández-Martínez J.M., M. Del Río, L. Velasco, J. Domínguez and A. De Haro (2001) Registration of zero erucic acid Ethiopian mustard genetic stock 25X-1. Crop Sci. 41: 282

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Getinet A., G. Rakow, J.P. Raney and R.K. Downey (1994) Development of zero erucic acid Ethiopian mustard through an interspecific cross with zero erucic acid Oriental mustard. Can. J. Plant Sci. 74: 793795 Getinet A, G. Rakow, J.P. Raney and R.K. Downey (1997) GSL content in interspecific crosses of Brassica carinata with B. juncea and B. napus. Plant Breeding 116: 39-46 Gugel R.K., G. Séguin-Swartz and G.A. Petrie (1990) Pathogenicity of three isolates of Leptosphaeria maculans on Brassica species and other crucifers. Can. J. Plant Pathol. 12: 75-82 Knowles P.F., T.E. Kearney and D.B. Cohen (1981) Species of rapeseed and mustard as oil crops in California. In: E.H. Pryde (ed.), New Sources of Fats and Oils, 255-268. AOCS Press, Champaign, IL, USA Malik R.S. (1981) Morphological, anatomical and biochemical basis of aphid, Lipaphis erysimi Kalt., resistance in cuciferous species. Sveriges Utsädesf. Tidskr. 91: 25-35 Oram R.N. and J.T.O. Kirk (1993) Induction of mutations for higher seed quality in Indian mustard. In: B.C. th Imrie and J.B. Hacher (eds.), Proc. 10 Australian Plant Breeding Conf., Gold Coast, Australia, 18-23

April 1993, 187-191 Potts D.A., G.W. Rakow and D.R. Males (1999) Canola-quality Brassica juncea, a new oilseed crop for the th Canadian prairies. Proc. 10 Int. Rapeseed Congr., 26-29 Sept. 1999, Canberra, Australia (CD ROM)

Rakow G. (1995) Developments in the breeding of edible oil in other Brassica species. In: Proc. 9th Int. Rapeseed Congr., Cambridge, UK, 4-7 July 1995, 401-406. GCIRC, Paris, France Raymer P.L. (2002) Canola: an emerging oilseed crop. In: J. Janick and A. Whipkey (eds), Trends in New Crops and New Uses, 122-126. ASHS Press, Alexandria, VA, USA Velasco L. and H.C. Becker (1998) Analysis of total glucosinolate content and individual glucosinolates in Brassica spp. by near-infrared reflectance spectroscopy. Plant Breeding 117: 97-102 Velasco L. and H.C. Becker (2000) Variability for seed glucosinolates in a germplasm collection of the genus Brassica. Genet. Resour. Crop Evol. 47: 231-238 Velasco L., J.M. Fernández-Martínez and A. De Haro (1999) Intraspecific breeding for reduced GSL content in Ethiopian mustard (Brassica carinata A. Braun). Euphytica 106:125-130 Velasco L., A. Nabloussi, A. De Haro and J.M. Fernández-Martínez (2003) Development of high oleic, low linolenic acid Ethiopian mustard (Brassica carinata) germplasm. Theor. Appl. Genet. 107: 823-830 Velasco L., A. Nabloussi, A. De Haro and J.M. Fernández-Martínez (2004) Allelic variation in linolenic acid content of high erucic acid Ethiopian mustard and incorporation of the low linolenic acid trait into zero erucic acid germplasm. Plant Breeding 123: 137-140

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CAPÍTULO 5

Development and characterisation of a Brassica carinata inbred line incorporating genes for low glucosinolate content from B. juncea

Márquez-Lema A., J.M. Fernández-Martínez, B. Pérez-Vich and L. Velasco Instituto de Agricultura Sostenible (CSIC), Alameda del Obispo s/n, E-14004 Córdoba, España

Development and characterisation of a Brassica carinata inbred line incorporating genes for low glucosinolate content from B. juncea Euphytica (2008). DOI 10.1007/s10681-008-9678-5

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Abstract The presence of high levels of sinigrin in the seeds represents a serious constraint for the commercial utilisation of Ethiopian mustard (Brassica carinata A. Braun) meal. The objective of this research was the introgression of genes for low glucosinolate content from B. juncea into B. carinata. BC1F1 seed from crosses between double zero B. juncea line Heera and B. carinata line N2-142 was produced. Simultaneous selection for B. carinata phenotype and low glucosinolate content was conducted from BC1F2 to BC1F4 plant generations. Forty-three BC1F4 derived lines were selected and subject to a detailed phenotypic and molecular evaluation to identify lines with low glucosinolate content and genetic proximity to B. carinata. Sixteen phenotypic traits and 80 SSR markers were used. Eight BC1F4 derived lines were very close to N2-142 both at the phenotypic and molecular level. Three of them, with average glucosinolate contents from 52 to 61 micromoles g-1, compared to 35 micromoles g-1 for Heera and 86 micromoles g-1 for N2-142, were selected and evaluated in two additional environments, resulting in average glucosinolate contents from 43 to 56 micromoles g-1, compared to 29 micromoles g-1 for Heera and 84 micromoles g-1 for N2-142. The best line (BCH-1773), with a glucosinolate profile made up of sinigrin (> 95%) and a chromosome number of 2n = 34, was further evaluated in two environments (field and pots in open-air conditions). Average glucosinolate contents over the four environments included in this research were 42, 31, and 74 micromoles g-1 for BCH-1773, Heera, and N2-142, respectively. These are the lowest stable levels of glucosinolates reported so far in B. carinata.

Key words: Brassica carinata – Brassica juncea – sinigrin – interspecific crosses – phenotypic traits – simple sequence repeats (SSR

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Introduction Ethiopian mustard (Brassica carinata A. Braun) is an amphidiploid species (2n = 34, BC genome), derived from the diploid species B. nigra (L.) Koch (2n = 16, B genome) and B. oleracea L. (2n = 18, C genome). Ethiopian mustard has a great potential as an oilseed crop for many regions, especially those with semiarid or mediterranean climates, where it performs better than conventional Brassica oilseed crops such as rapeseed (B. napus L.) or turnip rape (B. rapa L.) (Knowles et al. 1981, Fereres et al. 1983, Warwick et al. 2006). Nowadays, its development as a cash crop is limited by the absence of germplasm with low glucosinolate content in the seeds, already available in other species of the genus such as B. napus, B. rapa, and B. juncea (L.) Czern. (Raymer 2002). Glucosinolates are a family of β-thioglucoside N-hydroxysulfates which, after tissue damage, are hydrolysed in a variety of products with toxic and antinutritive effects, which limits the value of the meal for food and feed uses (Fahey et al. 2001). Current standards define low glucosinolate varieties as those that contain less than 30 micromoles of aliphatic glucosinolates per gram of defatted meal (Potts et al. 1999). However, B. carinata seeds from germplasm accessions typically contain more than 100 micromoles g-1 seed (Getinet et al. 1996, Alemayehu and Becker 2002, Teklewold and Becker 2005). Even though some authors have reported lower seed glucosinolate levels in B. carinata germplasm, such levels were in general unstable and produced only under certain environments (Getinet et al. 1997) or they corresponded to preliminary reports not confirmed so far (Barro et al. 2002). Unlike the other Brassica oil crops, B. carinata glucosinolate profile is mainly made up of sinigrin (2-propenyl), which accounts for more than 90% of the total glucosinolate content (Getinet et al. 1997). This glucosinolate is particularly detrimental in comparison with other glucosinolate homologues when the meal is intended for animal feed, which has led some researchers to recommend its complete removal, even to levels below one micromol g-1 seed (Potts et al. 1999). Success in glucosinolate reduction in B. carinata seeds has been very limited so far. Velasco et al. (1999) conducted pedigree selection within a germplasm accession that showed variation for this trait, developing a line with a significant reduction in seed glucosinolate content, averaging 86 micromoles g-1 over three environments, compared to 122 micromoles g-1 in the

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standard check grown in the same environments. Velasco et al. (1999) also used chemical mutagenesis and reported the isolation of five lines with a similar reduction of seed glucosinolate content, with average levels for this trait from 93 to 101 micromoles g-1, compared to 121 micromoles g-1 in the check. Such a reduction was proved to be consistent over environments. In crosses involving the abovementioned lines with reduced glucosinolate content, Márquez-Lema et al. (2006) isolated a transgressive line with average glucosinolate contents in two environments of 58 and 46 micromoles g-1, respectively compared to 84 and 62 micromoles g-1, respectively in the seeds of the parent with the lowest glucosinolate content. Despite some success in reducing glucosinolate content through intraspecific breeding, the reported levels as still far from the low levels available in the other Brassica oilseed crops. Getinet et al. (1997) studied the feasibility of reducing glucosinolate content in B. carinata seeds through interspecific crosses with low glucosinolate strains of B. napus and B. juncea. Crosses with B. napus (AC genome) resulted in practically no reduction of total glucosinolate content, whereas crosses with B. juncea (AB genome) led to the identification of BC1F3 segregants with a reduced glucosinolate content of up to 66 micromoles g-1, compared to 137 micromoles g-1 in the B. carinata parent and 58 micromoles g-1 in the B. juncea parent. The authors concluded that genes for sinigrin synthesis are located at the B genome of B. carinata (BC genome). Interestingly, the best BC1F3 segregant did not possess sinigrin as the major glucosinolate, but progoitrin (2hydroxy-3-butenyl), which was attributed to the transfer of genes for glucosinolate precursor chain elongation from B. juncea (Getinet et al. 1997). Taking into consideration the abovementioned results, the objective of the present research was the introgression of genes for low glucosinolate content from B. juncea into B. carinata.

Materials and Methods Plant materials The B. carinata line N2-142, with reduced glucosinolate content, was developed by Velasco et al. (1999) by pedigree selection from the germplasm line C-49. The authors reported average glucosinolate content in N2-142 seeds of 86 micromoles g-1, compared to 122 micromoles g-1 in the

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line C-49. Both lines mainly contain sinigrin (2-propenyl glucosinolate) as the major glucosinolate (> 90% of the total glucosinolates). The B. juncea line Heera was used as a donor of genes for low glucosinolate content. The line was developed in India, were it showed a glucosinolate content of 12 micromoles g-1, mainly in the form of gluconapin (3-butenyl glucosinolate) (Sodhi et al. 2002).

Crossing and selection scheme Seeds of N2-142 and Heera were germinated in moistened filter paper in autumn 1997 and the plants were grown in pots in the greenhouse. Plants of N2-142, used as female, were crossed with plants of Heera. As a poor seed set was expected in the cross B. carinata x B. juncea (Getinet et al. 1997), developing siliquae were removed from 12 to 14 days after pollination, surface sterilised and the dissected ovules were cultivated on solid Murashige and Skoog medium at 24ºC with permanent light. Once roots emerged, plantlets were transferred to pots. The F1 plants were grown in the greenhouse and backcrossed to plants of B. carinata line N2-142. BC1F1 to BC1F4 plant generations were grown in the field at the experimental farm of the Institute for Sustainable Agriculture at Córdoba, Spain, from 1999/2000 to 2002/03 growing seasons, respectively, following a plant-to-row scheme and pedigree selection for both seed glucosinolate content and plant type. In all cases, around 70 plants of both parents were grown as checks. A negative selection based on visual observations was conducted from BC1F2 to BC1F4 plant generations in order to eliminate plants or lines resembling morphological characteristics of the B. juncea parent. Additionally, glucosinolate content of seeds from every individual plant was analysed as described below. From an initial population of 2,380 BC1F2 plants, 43 BC1F4 plants were selected based on both mentioned criteria for a detailed characterisation at the phenotypic and molecular level. BC1F5 plants from selected phenotypes were grown in the field in 2003/04 in rows 5-m long with 1 m spacing between rows. Plants of N2-142 and Heera were grown as checks. Border effects were minimised by sowing N2-142 at border rows. After phenotypic and molecular characterisation of the 43 BC1F4 derived lines, three of them were selected and they were grown in two additional environments (field and pots in open-air conditions) in 2004/05 in order to confirm the consistency of their reduced glucosinolate content. The line with the lowest glucosinolate content was additionally evaluated in two environments (field

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and pots in open-air conditions) in 2005/06 together with the parental lines N2-142 and Heera as an additional confirmatory step. The field plot consisted in two replications of three rows 5-m long separated 70 cm. From 36 to 52 individual plants of the central rows were harvested and analysed for glucosinolate content. The unreplicated pot evaluation consisted of 48 plants of each line.

Phenotypic characterization of BC1F4 derived lines A set of phenotypic traits from the IBPGR Brassica and Raphanus descriptors list (IBPGR 1990) was used for selection of BC1F4 derived lines resembling B. carinata phenotype. The list of phenotypic traits as well as their average values and ranges of variation in both parents is shown in Table 5.1. The trait measures were recorded on five plants randomly chosen from each line, with five replicated measures within each plant. Colour traits were evaluated using the RHS Colour Chart (The Royal Horticultural Society 1995).

Molecular characterization of BC1F4 derived lines Young leaves from five randomly selected plants per line were collected from the field experiment of 2003/04 and stored at −80ºC. The leaf tissue from each line was bulked, lyophilised and ground to a fine powder in a laboratory mill. Total genomic DNA was extracted following Berry et al. (1995). DNA samples from each line were screened with a set of 80 SSR markers derived from B. nigra (B genome) and B. napus (AC genome) (Lowe et al. 2004). SSR markers are listed in Table 5.2. For most SSRs, PCR was performed using 30 microl reactions, each containing 50 ng DNA template, 1x PCR buffer, 0.7 U Taq DNA polymerase (BioTaq, Bioline, London, UK), 1.5 mM MgCl2, 0.2 mM of each dNTP, and 0.3 microM of primers. For some SSRs these concentrations had to be slightly adjusted. A Perkin Elmer 9700 thermocycler was programmed for 2 min of initial denaturation at 95ºC, then an amplification profile described by Lowe et al. (2004) was used. This profile included 35 cycles consisting of 94ºC for 30 s, 56ºC for 1 min, 72ºC for 1 min, and a final extension at 72ºC for 10 min, before cooling at 4ºC. PCR products were resolved on 3% Metaphor® (BMA, Rockland, ME, USA) agarose gels in 1x TBE buffer with ethidium bromide incorporated in the gel.

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Table 5.1.- Phenotypic trait for the B. carinata line parent N2-142 and B. juncea parent Heera evaluated in this study in Córdoba, Spain, during 2003/2004.

Parental lines B. carinata line N2-142 Trait

Min

Mean

B. juncea line Heera Max

Min

Mean

Days to flowering (50%)

112

75

Days to maturity (50%)

139

128

Petal colora

2-A

2-A

Max

Petal length (cm)

1.40

1.56

1.68

0.92

1.09

1.28

Petal width (cm)

0.61

0.63

0.79

0.51

0.61

0.76

Leaf colora Leaf blade shape Seedling leaf margin Leaf apex shape

137-B

146-B

Elliptic

Spathulate

Ovate

Dentate

Serrate

Serrate

Intermediate Intermediate-rounded

Lanceolate Crenate

Dentate

Rounded

Acute 1.00

Dentate

Number of leaf divisions

1.00

1.80

3.00

Total siliqua length (cm)

3.76

5.10

5.93

2.97

3.98

5.01

Siliqua width (cm)

0.32

0.44

0.53

0.18

0.31

0.46

Beak length (cm)

0.31

0.48

0.60

0.33

0.64

0.89

Peduncule length (cm)

0.47

0.66

0.87

0.90

1.26

1.91

Seeds per siliqua

4.00

11.04

21.00

0.00

6.28

15.00

1000-seed weight (g)

2.58

3.21

4.08

1.10

2.46

3.78

a

Colour codes according to RHS Colour Chart (The Royal Horticultural Society 1995)

Statistical analyses Statistical analyses of the phenotypic and SSR data were conducted using the programme NTSYS version 2.02 (Rohlf 1998). The means of the phenotypic observations per line were averaged and normalised prior to cluster analysis by dividing by the standard deviation and subtracting the mean for each trait. A matrix of Euclidean distances (Eij) for lines i and j and phenotypic traits k was created using the SIMINT function and the EUCLID coefficient, where

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Eij =

∑ (x k

− x kj )

2

ki

Cluster analysis was computed from the Euclidean distance matrix with the unweighted pair-group method based on arithmetic averages (UPGMA) using SAHN function. SSR bands for each primer combination were recorded as absent (0) or present (1). The binary matrix was used to estimate Dice´s genetic similarity coefficients (Dice, 1945), which are defined as S = 2Nab / (2Nab + Na + Nb) where Nab is the total number of bands common to lines a and b, and Na and Nb are the total number of bands presents in lines a and b, respectively. The similarity matrix obtained was used to generate a dendrogram using the UPGMA clustering procedure. Euclidean distances of individual BC1F4 derived lines to N2-142 based on phenotypic data were computed and compared to the corresponding genetic distances (genetic dissimilarities) based on SSR analysis, defined as one minus the Dice similarity coefficient (Mohammadi and Prasanna 2003), using Mantel test (Mantel 1967).

Analysis of glucosinolate content From BC1F2 to BC1F4 plant generations, total glucosinolate content was measured on intact seeds by near-infrared reflectance spectroscopy (NIRS), with the Pd-GSL complex method used as reference analysis. In both cases, we followed the methodology reported by Velasco et al. (1999). In all generations, a pre-selection was made using NIRS data and all selected phenotypes were further analysed by the reference method. The different evaluations for seed glucosinolate content of selected BC1F4 derived lines were conducted using the Pd-GSL complex method. Additionally, seeds of the parents and the best selected BC1F4 derived line from the field evaluation of 2004/05 were analysed for glucosinolate content and profile by high performance liquid chromatography (HPLC) as described by Velasco and Becker (1998).

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Table 5.2.- List of 80 Brassica SSR markers and their focal species (Lowe et al. 2004), used to analyse B. carinata line N2-142, B. juncea line Heera and 43 BC1F4 derived lines from interspecific crosses between them. SSR Marker

Species

SSR Marker

Species

Na10-B01

B. napus

Ni1-A04

B. nigra

Na10-B08

B. napus

Ni2-A02

B. nigra

Na10-B10

B. napus

Ni2-A08

B. nigra

Na10-C01

B. napus

Ni2-A10

B. nigra

Na10-C06

B. napus

Ni2-A11

B. nigra

Na10-D03

B. napus

Ni2-A12

B. nigra

Na10-D09

B. napus

Ni2-B01

B. nigra

Na10-E02

B. napus

Ni2-B02

B. nigra

Na10-E08

B. napus

Ni2-B03

B. nigra

Na10-F01

B. napus

Ni2-C06

B. nigra

Na10-F06

B. napus

Ni2-C09

B. nigra

Na10-F08

B. napus

Ni2-C12

B. nigra

Na10-G01

B. napus

Ni2-D06

B. nigra

Na10-G06

B. napus

Ni2-D07

B. nigra

Na10-H03

B. napus

Ni2-D08

B. nigra

Na10-H06

B. napus

Ni2-D10

B. nigra

Na12-A01

B. napus

Ni2-E04

B. nigra

Na12-A02

B. napus

Ni2-E05

B. nigra

Na12-A07

B. napus

Ni2-F01

B. nigra

Na12-A10

B. napus

Ni2-F02

B. nigra

Na12-B01

B. napus

Ni2-F07

B. nigra

Na12-B05

B. napus

Ni2-G06

B. nigra

Na12-C03

B. napus

Ni3-A05

B. nigra

Na12-C07

B. napus

Ni3-E06

B. nigra

Na12-C08

B. napus

Ni3-F02

B. nigra

Na12-D07

B. napus

Ni3-G04b

B. nigra

Na12-D10

B. napus

Ni3-H07

B. nigra

Na12-E01

B. napus

Ni4-A09

B. nigra

Na12-E04

B. napus

Ni4-A10

B. nigra

Na12-E06a

B. napus

Ni4-B06

B. nigra

Na12-F12

B. napus

Ni4-C06

B. nigra

Na12-G05

B. napus

Ni4-D01

B. nigra

Na12-H09

B. napus

Ni4-D10

B. nigra

Na14-A06

B. napus

Ni4-E08

B. nigra

Na14-B05

B. napus

Ni4-E11

B. nigra

Na14-C12

B. napus

Ni4-F02

B. nigra

Na14-E08

B. napus

Ni4-G02

B. nigra

Na14-E11

B. napus

Ni4-G04

B. nigra

Na14-G02

B. napus

Ni4-H04

B. nigra

Na14-G08

B. napus

Ni4-H06

B. nigra

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Mitotic analysis Chromosome numbers were counted in root tips of germinating seeds. The root tips were treated with 2 mM 8-hydroxyquinoline at 28ºC in the dark for 2.5 h, fixed and stored in ethanol: acetic acid (3:1) solution, hydrolysed in 1 N HCl at 60ºC for 11 min and stained with Schiff´s reagent at 20ºC in the dark for 15 min. The chromosomes were observed and counted using a confocal laser scanning microscope (LSM5 Pascal, Carl Zeiss Microimaging) using a 63x objective. Images were captured with a Zeiss AxioCam HR color digital camera driven by LSM5 Pascal software release 3.0.

Results Selection from BC1F2 to BC1F4 plant generations From 2,380 BC1F2 plants evaluated for plant characteristics, 807 of them were discarded because they showed traits resembling B. juncea. The remaining 1,573 plants were harvested and their seeds analysed for total glucosinolate content, together with seeds from the parents grown under the same environment. Plants of B. juncea line Heera showed a glucosinolate content from 21 to 36 micromoles g-1 seed, whereas plants of B. carinata line N2-142 showed a range of variation ranging from 68 to 88 micromoles g-1 seed. Total glucosinolate content in BC1F2 plants ranged from 52 to 152 micromoles g-1 seed. The progenies of 62 BC1F2 plants with glucosinolate content from 52 to 82 micromoles g-1 seed were selected and grown in the field. From 2,321 BC1F3 plants evaluated at the phenotypic level, 639 of them were discarded on the basis of phenotypic resemblance to B. juncea and the remaining 1,682 plants were harvested and their seeds analysed for glucosinolate content. The trait ranged from 19 to 37 micromoles g-1 seed in Heera plants, from 67 to 89 micromoles g-1 seed in N2-142 plants, and from 40 to 128 micromoles g-1 seed in BC1F3 plants. The progenies of 50 of them, with glucosinolate content ranging from 40 to 65 micromoles g-1 seed were selected and evaluated in the following generation. Three hundred and fifteen BC1F4 plants were discarded after phenotypic evaluation on the basis of phenotypic resemblance to B. juncea, from a total of 2,425 plants evaluated. Total glucosinolate content in the seeds of the remaining BC1F4 plants ranged from 21 to 95 micromoles

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g-1 seed, compared to ranges of variation from 32 to 46 micromoles g-1 seed for Heera and from 67 to 88 micromoles g-1 seed for N2-142. The progenies of 43 BC1F4 phenotypes, with glucosinolate content from 21 to 48 micromoles g-1 seed were selected for a detailed phenotypic and molecular characterisation.

Phenotypic and molecular characterisation of BC1F4 derived lines The 43 BC1F4 derived lines had average glucosinolate contents ranging from 38 ± 4 micromoles g-1 seed to 84 ± 7 micromoles g-1 seed, compared to average contents of 35 ± 6 micromoles g-1 seed for Heera and 85 ± 6 micromoles g-1 seed for N2-142. The dendrogram from UPGMA cluster analyses based on phenotypic traits is presented in Fig. 5.1. Two major clusters were observed, each of them containing one of the parents. The cluster containing B. juncea parent Heera included 9 BC1F4 derived lines, whereas the other 34 lines were clustered together with B. carinata parent N2-142. Most of the lines within this cluster were separated at a Euclidean distance below 0.20, with the exception of a subcluster formed by four lines separated from the main cluster at a Euclidean distance around 0.28 (Fig. 5.1). 67 out of 80 SSR markers tested on the 43 BC1F4 derived lines and the parental lines were polymorphic and amplified a total of 145 fragments. Cluster analysis revealed a clear separation between B. juncea line Heera and the BC1F4 derived lines, which were closer to B. carinata line N2-142 (Fig. 5.2). This line was clustered together with a group of seven BC1F4 derived lines that included the lines BCH-1744, BCH-1773, BCH-1818, BCH-1944, BCH-1962, BCH-1969, and BCH1813. The comparison between Euclidean distances of individual BC1F4 derived lines to N2-142 based on phenotypic data and the corresponding genetic distances estimated from molecular data revealed a group of eight BC1F4 derived lines at a short distance to N2-142 according to both criteria. This group included the seven abovementioned lines clustered with N2-142 in the dendrogram based on molecular data, and the line BCH-2220 (Fig. 5.3).

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Figure 5.1.- Dendrogram constructed using UPGMA cluster analysis of pairwise Euclidean distances between 43 BC1F4 derived lines (BCH-) and their parent lines B. carinata line N2-142 and B. juncea line Heera based on 16 phenotypic traits. BCH-789 BCH-1715 BCH-1818 BCH-2170 BCH-2276 BCH-2220 BCH-2259 BCH-2257 BCH-2266 BCH-2267 BCH-1813 BCH-2211 BCH-2279 N2-142 BCH-2264 BCH-1944 BCH-2278 BCH-2280 BCH-2177 BCH-2269 BCH-2294 BCH-1962 BCH-1969 BCH-2258 BCH-2225 BCH-1744 BCH-1773 BCH-2305 BCH-2074 BCH-2122 BCH-2281 BCH-2105 BCH-2067 BCH-2068 BCH-2178 BCH-1994 BCH-2028 BCH-2021 BCH-2082 BCH-2060 BCH-2009 BCH-2010 BCH-2013 BCH-2004 HEERA 0.00

0.14

0.28 Euclidean distance

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0.41

0.55

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Figure 5.2.- Dendrogram constructed using UPGMA cluster analysis of pairwise similarities between 43 BC1F4 derived lines (BCH-) and their parent lines B. carinata line N2-142 and B. juncea line Heera based on SSR marker information.

BCH-789 BCH-2220 BCH-1744 BCH-1773 BCH-1818 BCH-1944 BCH-1962 BCH-1969 BCH-1813 N2-142 BCH-1715 BCH-2170 BCH-2177 BCH-2257 BCH-2258 BCH-2259 BCH-2279 BCH-2269 BCH-2281 BCH-2278 BCH-2266 BCH-2280 BCH-2264 BCH-2276 BCH-2267 BCH-2067 BCH-2122 BCH-2225 BCH-2294 BCH-2305 BCH-2211 BCH-1994 BCH-2021 BCH-2028 BCH-2004 BCH-2009 BCH-2074 BCH-2010 BCH-2013 BCH-2082 BCH-2178 BCH-2105 BCH-2060 BCH-2068 HEERA 0.00

0.25

0.50 Dice coefficient

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0.75

1.00

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Figure 5.3.- Scatter plot of Euclidean distances of individual BC1F4 derived lines to Ethiopian mustard line N2142 based on phenotypic data, and the corresponding genetic distances calculated from SSR marker information.

0,40

Molecular distance to N2-142

0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Phenotypic distance to N2-142

Selection and evaluation of lines The eight BC1F4 derived lines closer to N2-142 according to both phenotypic and molecular criteria (Fig. 5.3) had average glucosinolate contents ranging from 52 ± 4 to 84 ± 7 micromoles g-1 seed, compared to 35 ± 6 micromoles g-1 seed in Heera and 85 ± 6 micromoles g-1 seed in N2-142. The three lines with the lowest glucosinolate content, BCH-1969 (52 ± 4 micromoles g-1 seed), BCH1773 (56 ± 9 micromoles g-1 seed), and BCH-1813 (61 ± 12 micromoles g-1 seed) were selected and evaluated in two additional environments in 2004/05. The results are shown in Fig. 5.4. Environmental effects were in the same direction for N2-142 and the three BC1F4 derived lines, i.e. they showed a greater glucosinolate content in the field experiment than in the pot experiment. Conversely, Heera showed the opposite effect. Seeds from plants of BCH-1773 from the field experiment as well as seeds from plants of the parent lines Heera and N2-142 were further analysed for glucosinolate content by HPLC to confirm the values obtained by the Pd-GSL complex method as well as to determine their glucosinolate profiles. The results obtained by HPLC were in line with those obtained with the PdGSL complex method, with average glucosinolate contents of 25 ± 3 micromoles g-1 seed for Heera,

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103 ± 11 micromoles g-1 seed for N2-142, and 54 ± 5 micromoles g-1 seed for BCH-1773. The predominant glucosinolate in the seeds of Heera was gluconapin (3-butenyl), which accounted for more than 92% of the total glucosinolates in the seeds. The predominant glucosinolate in the seeds of both N2-142 and BCH-1773 was sinigrin, which represented more than 95% of the total glucosinolates. Additionally, the somatic number of chromosomes was counted in plants of BCH1773, which indicated that they had the somatic number of chromosomes of B. carinata, i.e. 2n = 34.

Figure 5.4.- Glucosinolate content of B. carinata line N2-142, B. juncea line Heera, and three selected BC1F4 derived lines grown in pots under open-air conditions and in the field in 2004/05 growing season.

-1

Glucosinolate content (micromoles g )

120 Field Pots

100 80 60 40 20 0 BCH-1773 BCH-1813 BCH-1969

N2-142

Heera

The line BCH-1773 was evaluated again together with the parents in the field and in pots in 2005/06 in order to completely discard the possibility that the reduced levels of glucosinolates were the result of environmental effects. Seeds of BCH-1773 averaged 42 ± 7 micromoles g-1 compared to 34 ± 4 and 58 ± 13 micromoles g-1 seed, respectively for Heera and N2-142 in the field experiment. In the pot evaluation, seeds of BCH-1773 averaged 39 ± 6 micromoles g-1 compared to 30 ± 4 and 70 ± 12 micromoles g-1 seed, respectively for Heera and N2-142.

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Discussion Brassica carinata is a promising oilseed crop for dry areas because of its heat and drought tolerance (Fereres et al. 1983), resistance to diseases (Gugel et al. 1990), and availability of germplasm with good seed oil quality (Velasco et al. 2003). But like the other Brassica oilseed crops, the development of B. carinata crop at a commercial scale will require the previous development of low glucosinolate germplasm. The origin of the low glucosinolate trait was the B. napus (genome AC) cultivar ‘Bronowski’, which was found to contain as few as 12 micromoles g-1 seed (Josefsson and Appelqvist 1968). The Bronowski genes for low glucosinolate content have been used for interspecific transfer of the trait into B. rapa (A genome) (Kondra and Stefansson 1970) and B. juncea (AB genome) (Love et al. 1990a). In the three species carrying the Bronowski genes, the low glucosinolate lines contain only traces of sinigrin. This glucosinolate is not present in significant amounts in germplasm of B. napus (excepting some resynthesized lines) and B. rapa (Gland et al. 1981). B. juncea germplasm include accessions with two contrasting glucosinolate profiles, associated with their geographical origin. The accessions from India and Pakistan mainly contain gluconapin, whereas the accessions from other origins mainly contain sinigrin (Vaughan and Gordon 1973). Genes for low glucosinolate content were transferred from B. rapa to a B. juncea line with gluconapin profile (Love et al. 1990a). Therefore all the breeding success in developing low glucosinolate content in Brassica spp. has been based on genes located in the A and C genomes. Sinigrin is the predominant glucosinolate in B. carinata seeds. No significant variation for other glucosinolates has been identified in this species (Velasco and Becker 2000). Getinet et al. (1997) attempted to transfer genes for low glucosinolate content from B. napus and B. juncea to B. carinata, concluding that the genes responsible for sinigrin accumulation were located in the B genome, which was later confirmed by Ripley and Roslinsky (2005). Getinet et al. (1997) obtained BC1F3 plants with reduced sinigrin content in seed meal from crosses between B. juncea and B. carinata that presented a concomitant increase of progoitrin (2-hydroxy-3-butenyl) and gluconapin (3-butenyl), which was attributed to the simultaneous introgression of genes responsible for the synthesis of 2-amino-6-methyl thio-hexanoic acid, which is the precursor for gluconapin (Getinet et al. 1997). Aliphatic glucosinolates are biosynthesized through two routes, one of them producing

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sinigrin and another one producing gluconapin and/or progoitrin as the final products (Gianmoustaris and Mithen 1996). In the present research, reduction of sinigrin content in the line BCH-1773 was not accompanied by a simultaneous increase of the levels of other glucosinolates, as sinigrin accounted for more than 95% of the total glucosinolates. Breeding efforts to reduce total glucosinolate content in B. carinata have had only limited success so far. Getinet et al. (1997) reported an individual BC1F3 plant with 66 micromoles g-1 seed from crosses between B. carinata and B. juncea, compared with 137 micromoles g-1 seed in the B. carinata parent, but the stability of the trait and the degree of recovery of the B. carinata phenotype were not reported. Velasco et al. (1999) reported the development of six lines with a slight reduced glucosinolate content (80 −100 micromoles g-1 seed compared to 120 micromoles g-1 seed in the check). Crosses between these lines produced transgressive segregation from which a line with a glucosinolate content averaged over two environments of 52 micromoles g-1 seed, compared to 73 micromoles g-1 seed in the best parent, was isolated (Márquez-Lema et al. 2006). The present research represents a further advance in the reduction of glucosinolate content in B. carinata. The best BC1F4 derived line BCH-1773, evaluated over four environments, averaged 42 micromoles g-1 seed, compared to 31 micromoles g-1 seed for double zero B. juncea line Heera and 74 micromoles g-1 seed for the B. carinata line N2-142. The new line combined a low glucosinolate content closer to Heera than to N2-142 with plant and molecular characteristics clearly resembling N2-142. The present research was based on a single backcross generation, which is a similar approach to that followed in a previous research to transfer genes for low glucosinolate content from B. juncea to B. carinata germplasm (Getinet et al. 1997). Simultaneous selection for reduced glucosinolate content and phenotypic resemblance to B. carinata conducted from the BC1F2 to the BC1F4 plant generations, together with a detailed phenotypic and molecular evaluation of BC1F4 derived lines ensured the recovery of the B. carinata plant type in selected lines with reduced glucosinolate content. Molecular marker distances and phenotypic trait distances to N2-142 in BC1F4 derived lines were related in a triangular-shaped manner. A similar association was observed by Teklewold and Becker (2006) in the comparison of phenotypic and molecular distances to predict heterosis in Ethiopian mustard. Such a relationship was studied by Burstin and Charcosset (1997) at a

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theoretical and experimental level. They found that low marker distances were associated with low phenotypic distances, whereas high marker distances were associated with a large range of phenotypic distances, which produced a clear tendency towards a triangular relationship between the two distances. According to these authors, the occurrence of polygenic inheritance and linkage disequilibrium could cause such a relationship. It is noteworthy that N2-142 is a line selected for reduced glucosinolate content by Velasco et al. (1999), who reported a glucosinolate content of 82 micromoles g-1 seed compared to 121 micromoles g-1 seed in the line from which it was isolated, i.e. a reduction in glucosinolate content of 32%. In the present research, glucosinolate content in the seeds of N2-142 was additionally reduced by 43%, resulting in the lowest stable glucosinolate levels reported so far in B. carinata. However, further reductions of glucosinolate content in this crop are still required. The task seems arduous, but it will only possible if approached through several strategies. Previous research conducted through intraspecific selection led to the development of a line with stable glucosinolate content of 52 micromoles g-1 seed (Márquez-Lema et al. 2006). Recombination between that genetic source and the line BCH-1773 developed in the present research seems an attractive approach that might result in a further improvement of the trait. Total seed glucosinolate content in Brassica species is a trait affected by environmental conditions (Milford and Evans 1991, Ciska et al. 2000). Accordingly, reports on improved germplasm should take this point into consideration and to include evaluations over several environments. In the present research, the line BCH-1773 was evaluated over four environments, which confirmed the genetic stability of reduced glucosinolate content. The number of genes involved in sinigrin accumulation in B. carinata is unknown. In B. juncea, Love et al. (1990b) concluded that sinigrin and gluconapin accumulation was controlled by multiple additive alleles at two loci, whereas Stringam and Thiagarajah (1995) proposed a model based on recessive alleles at three loci for the absence of sinigrin in B. juncea seeds. This model was confirmed at the molecular level by Mahmood et al. (2003), who identified three QTL affecting sinigrin content. Total seed glucosinolate content in B. juncea was found to be under the control of seven genes (Sodhi et al. 2002). The partial breeding progress achieved so far in lowering glucosinolate content in B. carinata suggests multigene regulation, which determines that the elimination of this antinutritive

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compound from B. carinata seeds will not be an easy task. Nevertheless, the results of the present research represent a new step towards that objective.

Acknowledgements The authors thank Prof. Dr. Heiko C. Becker and Dr. Christian Möllers, Institute of Agronomy and Plant Breeding, Georg-August University of Göttingen, Germany, for facilities given at the initial steps of this research, and Prof. Dr. A. Martín, Institute for Sustainable Agriculture (CSIC), Córdoba, Spain, for his kind assistance in chromosome counting. The research was partly supported by project MCYT AGL2001-2293 of the Spanish Government.

References Alemayehu N. and H Becker (2002) Genotypic diversity and patterns of variation in a germplasm material of Ethiopian mustard (Brassica carinata A. Braun). Genet. Res. Crop Evol. 49: 573-582 Barro F., J. Fernández-Escobar, M. De la Vega and A. Martín (2002) Modification of GSL and erucic acid contents in doubled haploid lines of Brassica carinata by UV treatment of isolated microspores. Euphytica 129: 1-6 Berry S.T., A.J. Leon, C.C. Hanfrey, P. Challis, S.R. Burkholz, G.K. Barnes, M. Rufener, M. Lee and P.D.S. Caligari (1995) Molecular markers analysis of Helianthus annus L. 2. Construction of a RFLP linkage map for cultivated sunflower. Theor. Appl. Genet. 91: 195-199 Burstin J. and A. Charcosset (1997) Relationship between phenotypic and marker distances: theoretical and experimental investigations. Heredity 79: 477-483 Ciska E., B. Martyniak-Przybyszewska and H. Kozlowska (2000) Content of glucosinolates in cruciferous vegetables grown at the same site for two years under different climatic conditions. J. Agric. Food Chem. 48: 2862-2867 Dice L.R. (1945) Measures of the amount of ecologic association between species. Ecology 26:297-302 Fahey J.W., A.T. Zalcmann and P. Talalay (2001) The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochem. 56: 5-51 Fereres E., J. Fernández-Martínez, I. Mínguez and J. Domínguez (1983) Productivity of Brassica juncea and B. carinata in relation to rapeseed, B. napus. I. Agronomic studies. In: Proceedings of the 6 International Rapeseed Congress, Paris, France, 17-19 May 1983. GCIRC, Paris, France, pp. 293-298

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Getinet A, Rakow G, Raney JP (1996) Glucosinolate content variation in Brassica carinata A. Braun germplasm grown at Holetta, Ethiopia. Cruciferae Newsl 18: 84-85 Getinet A., G. Rakow, J.P. Raney and R.K. Downey (1997) Glucosinolate content in interspecific crosses of Brassica carinata with B. juncea and B. napus. Plant Breed. 116: 39-46 Gianmoustaris A. and R. Mithen (1996) Genetics of aliphatic glucosinolates. IV. Side-chain modification in Brassica oleracea. Theor. Appl. Genet. 93: 1006-1010 Gland A., G. Röbbelen and W. Thies (1981) Variation of alkenyl glucosinolates in seeds of Brassica species. Z. Pflanzenzüchtg 87: 96-110 Gugel R.K., G. Séguin-Swartz and G.A. Petrie (1990) Pathogenicity of three isolates of Leptosphaeria maculans on Brassica species and other crucifers. Can. J. Plant Pathol. 12: 75-82 IBPGR (1990) Descriptors for Brassica and Raphanus. International Board for Plant Genetic resources, Rome, Italy Josefsson E. and L.Å. Appelqvist (1968) Glucosinolates in seed of rape and turnip rape as affected by variety and environment. J. Sci. Food Agric. 19: 564-570 Knowles P.F., T.E. Kearney and D.B. Cohen (1981) Species of rapeseed and mustard as oil crops in California. In: Pryde E.H. (ed.). New sources of fats and oils. AOCS Press, Champaign, IL, USA, pp. 255268 Kondra Z.P. and B.R. Stefansson (1970) Inheritance of major glucosinolates of rapeseed (Brassica napus) meal. Can. J. Plant Sci. 5: 643-647 Love H.K., G. Rakow, J.P. Raneya and R.K. Downey (1990a) Development of low glucosinolate mustard. Can J. Plant Sci. 70: 419-424 Love H.K., G. Rakow, J.P. Raney and R.K. Downey (1990b) Genetic control of 2-propenyl and 3-butenyl glucosinolate synthesis in mustard. Can J. Plant Sci. 70: 425-429 Lowe A.J., C. Moule, M. Trick and K.J. Edwards (2004) Efficient large-scale development of microsatellites for marker and mapping applications in Brassica crop species. Theor. Appl. Genet. 108: 1103-1112 Mahmood T., U. Ekuere, F. Yeh, A.G. Good and G.R. Stringam (2003) Molecular mapping of seed aliphatic glucosinolates in Brassica juncea. Genome 46: 753-760 Mantel N.A. (1967) The detection of disease clustering and a generalized regression approach. Cancer Res. 27: 209-220 Márquez-Lema A., J.M. Fernández-Martínez, B. Pérez-Vich and L. Velasco (2006) Transgressive segregation for reduced glucosinolate content in Brassica carinata A. Braun. Plant Breed. 125: 400-402

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Milford G.F.J. and E.J. Evans (1991) Factors causing variation in glucosinolates in oilseed rape. Outlook Agric. 20: 131-137 Mohammadi S.A. and B.M. Prasanna (2003) Analysis of genetic diversity in crop plants-salient statistical tools and considerations. Crop Sci. 43: 1235-1248 Potts D.A., G.W. Rakow and D.R. Males (1999) Canola-quality Brassica juncea, a new oilseed crop for the Canadian prairies. In: Proceedings of the 10th International Rapeseed Congr., Canberra, Australia, 26-29 September 1999 (CD ROM) Raymer P.L. (2002) Canola: an emerging oilseed crop. In: J. Janick and A. Whipkey (eds.) Trends in new crops and new uses. ASHS Press, Alexandria, VA, USA, pp. 122-126 Ripley V.L. and V. Roslinsky (2005) Identification of an ISSR marker for 2-propenyl glucosinolate content in Brassica juncea L. and conversion to a SCAR marker. Mol. Breed. 16: 57-66 Rohlf F.J. (1998) NTSYS-PC. Numerical Taxonomy and Multivariate Analysis System, Version 2.02. Exeter Software, Setauket, New York Sodhi Y.S., A. Mukhopadhyay, N. Arumugam, J.K. Verma, V. Gupta and D. Pental (2002) Genetic analysis of total glucosinolate in crosses involving a high glucosinolate Indian variety and a low glucosinolate line of Brassica juncea. Plant Breed. 121: 508-511 Stringam G.R. and M.R. Thiagarajah (1995) Inheritance of alkenyl glucosinolate in traditional and microspore-derived dubled haploid populations of Brassica juncea (L.) Czern and Coss. In: Proceedings th of the 9 International Rapeseed Congress, Cambridge, UK, 4-7 July 1995, pp. 804-806

Teklewold A. and H.C. Becker (2005) Variation and covariation of seed quality traits in Ethiopian mustard. J. Appl. Bot. Food Qual. 79: 182-188 Teklewold A. and H.C. Becker (2006) Comparison of phenotypic and molecular distances to predict heterosis and F1 performance in Ethiopian mustard (Brassica carinata A. Braun). Theor. Appl. Genet. 112: 752-759 The Royal Horticultural Society (1995) RHS Colour Chart. The Royal Horticultural Society, London Vaughan J.G. and E.I. Gordon (1973) A taxonomic study of Brassica juncea using the techniques of electrophoresis, gas-liquid chromatography and serology. Ann. Bot. 37: 167-184 Velasco L. and H.C. Becker (1998) Analysis of total glucosinolate content and individual glucosinolates in Brassica ssp. By near-infrared reflectance spectroscopy. Plant Breed. 117: 97-102 Velasco L. and H.C. Becker (2000) Variability for seed glucosinolates in a germplasm collection of the genus Brassica. Genet. Res. Crop Evol. 47: 231-238 Velasco L., J.M. Fernández-Martínez and A. De Haro (1999) Intraspecific breeding for reduced GSL content in Ethiopian mustard (Brassica carinata A. Braun). Euphytica 106: 125-130

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Velasco L., A. Nabloussi, A. De Haro and J.M. Fernández-Martínez (2003) Development of high oleic, low linolenic acid Ethiopian mustard (Brassica carinata) germplasm. Theor. Appl. Genet. 107: 823-830 Warwick S.I., R.K. Gugel, T. McDonald and K.C. Falk (2006) Genetic variation of Ethiopian mustard (Brassica carinata A. Braun) germplasm in western Canada. Genet. Res. Crop Evol. 53: 297-312

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CONC CLUSIONES FINALES CON

Conclusiones finales

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Conclusiones finales Las conclusiones que se derivan de los trabajos de investigación realizados en la presente Tesis son las que se exponen a continuación: 1. El control genético del contenido elevado en glucosinolatos totales en semillas de la línea N26215 está determinado por el genotipo materno y no presenta efectos citoplasmáticos. Este carácter presenta una heredabilidad moderada y está controlado por un mínimo de dos o tres genes. 2. Los marcadores SSRs de B. nigra (BB) y B. napus (AACC) utilizados en la presente Tesis mostraron una buena transferibilidad y capacidad discriminatoria para su aplicación en programas de mejora de B. carinata (BBCC). 3. En los cruzamientos realizados entre las líneas de B. carinata con niveles reducidos en glucosinolatos en semillas N2-142, S2-1241, N2-7397 y N2-9531, se obtuvieron segregantes transgresivos con niveles de glucosinolatos inferiores al mínimo encontrado en sus respectivos parentales únicamente en aquellos cruces en los que estuvo implicada la línea S2-1241, lo que sugiere la presencia en esta línea de una alteración genética diferente a la existente en las otras tres líneas empleadas en este estudio. 4. Se ha seleccionado una línea F3:4 de B. carinata con niveles transgresivos y estables de glucosinolatos totales en semillas (52 µmoles g-1), a partir del cruzamiento realizado entre las líneas S2-1241 y N2-142, ambas con niveles reducidos en glucosinolatos (90 µmoles g-1 frente a 121 µmoles g-1 en material estándar). 5. La utilización de descriptores fenotípicos y marcadores SSRs transferidos a B. carinata en el transcurso de esta Tesis ha permitido realizar una selección de tipo de planta B. carinata a partir de una población procedente de cruces entre B. carinata y B. juncea. Asimismo, las distancias fenotípicas y genotípicas obtenidas en este estudio presentaron una relación de forma triangular, coincidiendo con resultados descritos en trabajos anteriores. 6. A partir de cruzamientos interespecíficos realizados entre una línea de B. carinata con niveles reducidos en glucosinolatos totales (N2-142, 90 µmoles g-1 en semillas) y una línea de B. juncea con bajo contenido en glucosinolatos (Heera, 31 µmoles g-1 en semillas), se ha obtenido una línea de B. carinata (BCH-1773) con bajo contenido en glucosinolatos (42 µmoles g-1) y tipo de

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planta B. carinata. La reducción del contenido en sinigrina en esta línea no ha provocado el incremento de otros glucosinolatos (> 95% sinigrina) como había ocurrido en trabajos anteriores.

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ANEX XO ANE

Justificantes

De: [email protected] Fecha: 20/07/2008 19:51:32 Para: [email protected] Asunto: Plant Breeding - Decision on Manuscript ID PLBR-07-OA-525.R1

20-Jul-2008 Dear Dr. Velasco: It is a pleasure to accept your manuscript entitled "Inheritance of very high glucosinolate content in Ethiopian mustard seeds" in its current form for publication in Plant Breeding. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter. We have to mention that we are legally not allowed to publish your manuscript without the signed "Exclusive Licence Form". Therefore we will continue the production process only after receiving the signed "Exclusive Licence Form" from you. You will find this form in the attachment. Please not the correct Fax-nr is: +49 228-73-2045. Thank you for your fine contribution. On behalf of the Editors of Plant Breeding, we look forward to your continued contributions to the Journal. Sincerely, Prof. Jens Léon Editor in Chief, Plant Breeding [email protected]

Reviewer(s)' Comments to Author: Reviewer: 1 Comments to the Author I have read the marked parts of the revised manuscript and the reply of the reviewers. I find this manuscript now acceptable for publication.

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De: [email protected] Fecha: 27/08/2008 9:49:22 Para: [email protected] Asunto: [JSBBS] Initial Manuscript Received: Submission Number: nhu6936263

Dear Angustias Márquez-Lema, Thank you for submitting your manuscript to the Breeding Science. Your manuscript: Submission Number: nhu6936263 was received and processed by the Office as follows. Manuscript Number: BS08-063P Receipt Date: 2008/08/27 It is going through the editorial process. We will contact you when the editorial decision is made. To access the manuscript information, please login at: [Login at:] http://ess.jstage.jst.go.jp/contributor/JSBBS/contribLogin/-char/en Using the Login ID and the password which you have registered during the manuscript submission process. [Manuscript Detail] Submission Number: nhu6936263 Manuscript Number: BS08-063P Initial Manuscript Submission Date: 2008/08/08 Initial Manuscript Receipt Date: 2008/08/27 Manuscript Type: Research Paper Full Title: Transferability, amplification quality and genome specificity of microsatellites in Brassica carinata and related Brassica species. Corresponding Author: Angustias Márquez-Lema

Please contact below if you have any questions. Best regards, Contact: Breeding Science Office *********************************************************** Editorial Office of Breeding Science, Japanese Society of Breeding Email:[email protected] **********************************************************

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