A review of Brassica seed color

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1 A review of Brassica seed color Mukhlesur Rahman 1 and Peter B. E. McVetty 2 1 Department of Plant Science, North Dakota State University, Fargo, ND , USA ( md.m.rahman@ndsu.edu); and 2 Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Received 3 June 2010, accepted 22 December Can. J. Plant Sci. Downloaded from by on 05/01/18 Rahman, M. and McVetty, P. B. E A review of Brassica seed color. Can. J. Plant Sci. 91: Canola oil has excellent fatty acid composition and low saturated fat levels, and canola meal has protein with excellent amino acid composition. Canola seed quality can be further improved by the development of higher oil, higher protein and lower fiber content germplasm through the development of yellow seeded lines. While there is no naturally occurring yellow seeded B. napus, yellow seeded mutants that have arisen in nature can be readily indentified in Brassica rapa, B. juncea and B. carinata species. Brassica napus is widely cultivated in Asia, Australia, Europe and North America. Yellow seed in Brassica species is associated with seed that has higher oil and protein content and lower fiber content. Because of these seed quality advantages of yellow seeded lines, plant breeders around the world have been attempting to develop yellow seeded B. napus genotypes using crosses involving naturally occurring yellow seeded Brassica species. Seed color in B. rapa is controlled by two genes. Two duplicate genes are responsible for seed color in B. juncea. In B. carinata, one repressor gene represses the seed color gene resulting in yellow seed, while the absence of the repressor gene results in brown seed. Several yellow seeded B. napus genotypes have been developed and in most cases three genes are reported as being are responsible for seed color. Numerous different molecular markers for seed color genes in B. rapa, B. juncea and B. napus have been developed for use in marker-assisted selection in plant-breeding programs. These molecular markers can also be used to clone the Brassica seed color gene(s) and then create transgenic yellow seeded B. napus genotypes. This review summarizes past and current research on Brassica seed color breeding, genetics and genomics/biotechnology. Key words: Brassica, seed coat color, inheritance, molecular markers Rahman, M. et McVetty, P. B. E Panorama de la couleur des graines de Brassica. Can. J. Plant Sci. 91: L huile de canola se caracte rise par une excellente composition en acides gras, dont une faible teneur en acides gras saturés, tandisque le tourteau du canola renferme desprote inesd une excellente composition en acidesamine s. Il est possible d ame liorer encore plusla qualité desgrainesde canola en cre ant du matériel ge nétique plusriche en huile et en prote ines, et pluspauvre en fibrespar le de veloppement de ligne esa` grainesjaunes. Bien que B. napus ne donne pasnaturellement de graines jaunes, il est facile d identifier des mutants naturels a` grainesjauneschez Brassica rapa, B. juncea et B. carinata.on cultive abondamment B. napus en Asie, en Australie, en Europe et en Ame rique du Nord. Chez lesespe` cesdu genre Brassica, les graines jaunes sont associe esa` une plusforte teneur en huile et en prote inesainsi qu a` une teneur re duite en fibres. Compte tenu de tels avantages, les phytogéne ticiensdu monde entier tentent de cre er desge notypesde B. napus à grainesjaunesen recourant aux espe` cesdu meˆ me genre produisant naturellement des graines jaunes. Deux ge` nesre gulent la couleur de la graine chez B. rapa. Deux ge` nesdoublesla commandent chez B. juncea. Chez B. carinata, un gène re prime celui codant la couleur desgrainespour en engendrer desjaunes, l absence de ce gène re presseur donnant des graines brunes. Plusieurs ge notypesde B. napus a` graine jaune ont e té développe s. Dans la plupart des cas, on attribue la couleur de leursgrainesa` troisgènes. De nombreux marqueurs mole culairesont e té misau point pour la couleur desgraineschez B. rapa, B. juncea et B. napus afin de faciliter la sélection danslesprogrammesd hybridation. On s est e galement servi de cesmarqueurspour cloner le ou lesge` nesre gulant la couleur de la graine chez Brassica et produire desvariétés transge niquesa` grainesjaunesde B. napus. Ce panorama re sume les recherches actuelles et passe essur l amélioration de la couleur de la graine chez Brassica, ainsi que la ge nétique et la ge nomique/biotechnologie de cette culture. Mots clés: Brassica, couleur de la graine, he re dite, marqueursmole culaires Canola/rapeseed is one of the most important oilseed cropscultivated in many partsof the world; it isused as a source of edible oil for human consumption and as a protein-rich meal for livestock feed. It has been reported that the seed in naturally occurring yellow seeded genotypesin B. rapa, B. juncea and B. carinata contains greater oil, higher protein and lower fiber contentsthan the seed of black/brown seeded genotypes in these respective species (Downey et al. 1975). The yellow seeded Brassica genotypes in these species have a thinner and translucent seed coat resulting in a lower hull proportion with a bigger embryo and consequently greater oil and protein contentsin the seeds(stringam et al. 1974). Proanthocyanidinsand tanninsare the major compoundsinvolved in seed coat pigmentation. These are deposited in the seed coat of black/brown seeded Brassica genotypesand reduce the digestibility of Abbreviations: AFLP, amplified fragment length polymorphism; RAPD, random amplification polymorphism DNA; RFLP, restriction fragment length polymorphism; SRAP, s equence related amplified polymorphism Can. J. Plant Sci. (2011) 91: doi: /cjps

2 Can. J. Plant Sci. Downloaded from by on 05/01/ CANADIAN JOURNAL OF PLANT SCIENCE seed meal for poultry and livestock (Bell and Shires 1982; Slominski et al. 1994). The seed coats of black/ brown seeded Brassica genotypescontainsmore fiber and less protein than those of yellow seeded genotypes (Stringam et al. 1974). Although B. napus isby far the most important Crucifer oilseed species, no yellow seeded types occur naturally. Therefore, yellow seeded B. napus lineshave been developed from interspecific crosses with related species, namely, B. rapa, B. oleracea spp. alboglabra, B. juncea and B. carinata (Shirzadegan and Ro bbelen 1985; Liu et al. 1991; Chen et al. 1988; Chen and Heneen 1992; Rashid et al. 1994; Qi et al. 1995; Tang et al. 1997; Meng et al. 1998; Rahman 2001b). In recent years, molecular markers have increasingly been used in plant breeding to select for traits based on genotype rather than phenotype. Thisstrategy can be very effective for traits that are difficult to assess, affected by the environment, and which are controlled by multigene families. Identification of molecular markerstightly linked to the trait of interest could facilitate the selection of desired traits at very early stages of plant development. Several molecular markers associated with the seed coat color trait in B. napus, B. juncea, and B. rapa have been developed by variousresearch groups (Van Deynze et al. 1995; Somerset al. 2001; Liu et al. 2005b; Negi et al. 2000; Mahmood et al. 2005; Padmaja et al. 2005; Rahman et al. 2007). Restriction fragment length polymorphisms (RFLP), random amplification polymorphism DNA (RAPD), amplified fragment length polymorphism (AFLP), and sequence-related amplified polymorphism (SRAP) marker techniques have been used for mapping the seed coat color genes of B. napus. Recently, molecular markerstightly linked with individual genesin the yellow seed coat multigene family of B. napus have been developed and used high efficiency selection for the yellow seeded trait and for map-based gene cloning of yellow seed coat genes (Zhang et al. 2009). BRASSICA SEED ANATOMICAL STRUCTURE ASSOCIATED WITH SEED COAT PIGMENTATION The pigmentation of the seed coat in Brassica species occurs mainly due to deposition of polyphenols, which are polymersof leucocyanidins(leung et al. 1979; Hu 1988) that belong to the group of flavonoids(nørbæk et al. 1999). According to Van Caseele et al. (1982), the seed coat of B. napus hasthree different cell layers, including the palisade layer, several layers of crushed parenchyma cells, and a single aleurone cell layer. Chen and Meng (1984) reported that the palisade and crushed parenchyma layersbelong to the outer integument of the ovary, while the aleurone layer isthe outermost layer of endosperm. Deposition of the seed coat pigments (flavonoids) occurs in the palisade and crushed parenchyma layersof the seed (Vaughan 1970; Stringam et al. 1974). Stringam et al. (1974) reported that the palisade layer is the cell layer of the seed coat that has higher fiber. The palisade layer is about two-thirds thinner in yellow seed coat lines than in black/brown seed coat lines. The reduction of the palisade layer in yellow seeded lines reduces the proportions of polyphenolsand ligninsin the seed (Anjou et al. 1977; Theander et al. 1977; Slominski et al. 1994). Flavonoid Biosynthesis Flavonoidsare the major compoundsinvolved in the Brassica seed coat pigmentation process (Shirley 1998). Seed flavonoids have been classified into different groups that include flavonols, anthocyanins, phlobaphenes, isoflavones, and proanthocyanidins (Lepiniec et al. 2006). Proanthocyanidin, also known as condensed tannin, is deposited only in the seed coat. It is synthesized through a common phenylpropanoid pathway in the flavonoid pathway (Lepiniec et al. 2006; Fig. 1). The aromatic ring (naringenin) isgenerated by chalcone synthase and chalcone isomerase. Oxidation of the aromatic ring yieldsa dihydrokaemperol (dihydroflavonol), which ishydroxylated at the 3? or 5? positions or on both positions by flavonoid 3? hydroxylase and flavonoid 3?5? hydroxylase to produce two other dihydroflavonolsincluding dihydroquercetin and dihydromyricetin, respectively. These dihydroflavonols are converted by dihydroflavonol 4-reductase to produce different leucoanthocyanidins, and then by leucoanthocyanidin dioxygenase to yield delphinidin (purple color), pelargonidin (orange color) and cyanidin (red-magenta color), respectively, which can further be substituted by O-methyltransferase, UDP Flavonoid glucosyl transferase, and rhamnosyl transferase, resulting in decorated anthocyanins. In seeds, leucoanthocyanidins are substituted by leucoanthocyanidin reductase, generating flavan-3-olsthat undergo a condensation reaction to produce proanthocyanidin (Lepiniec et al. 2006; Fig. 1). Leucoanthocyanidin reductase is absent in Arabidopsis seeds and therefore proanthocyanidin is formed by using anthocyanidin asa precursor through one or more enzyme-catalyzed reactionsby anthocyanidin reductase (Abrahamset al. 2002; Achnine et al. 2004). Proanthocyanidinsstart to accumulate at the micropylar part of the embryo at the very early stages post-fertilization (around 1 to 2 d after fertilization), followed by the endothelium and the chalaza end (about 5 to 6 d after fertilization). Oxidation of proanthocyanidin occurs during the seed desiccation period (30 to 40 d after fertilization) leading to brown pigment formation in the mature seed (Lepiniec et al. 2006). Yellow seed coat color occursonly when one or more gene(s) encoding different enzymesare mutated in the flavonoid biosynthetic pathway, so that proanthocyanidin fails to deposit in the seed and thus produces a transparent seed coat that makes the yellow embryo visible, resulting in yellow seed color (Badani et al. 2006).

3 RAHMAN AND MCVETTY * A REVIEW OF BRASSICA SEED COLOR 439 Phenylpropanoid pathway 4-coumaroyl-CoA + 3 malonyl-coa ACC ase Acetyl -CoA Krebs cycle CHS flavones pentahydroxyflavanone F3H tetrahydroxychalcone FS1 FS2 CHI F3 5 H naringenin F3H flavan-4-ols phlobaphenes DFR F3 H eriodictyol flavanones F3H dihydromyricetin F3 5 H dihydrokaempferol F3 H dihydroquercetin dihydroflavonols Can. J. Plant Sci. Downloaded from by on 05/01/18 DFR leucodelphinidin LODX (ANS) myricetin YELLOW SEEDED BRASSICAS Seed oil content in Brassica seed could be increased by the development and use of yellow-seeded lines/cultivars. It has been reported that the yellow-seeded Brassica species produce seeds with higher oil, higher protein and lower fiber content than black/brown seeded types (Downey et al. 1975). Even partially yellow seeded lines produced improved seed quality compared with black/brown seeded types in B. napus (Liu 1992). Rashid et al. (1995) observed 6% higher oil content, similar protein content and 4% lower fiber content in partially yellow seeded strains compared with blackseeded strains of B. napus. Yellow Seeded Brassica: Nutritive Value of the Seed Meal Brassica seed is mainly used as an oil source, and the seed meal is used as feedstuff for poultry and livestock. Rapeseed meal contains about 40% protein with a well-balanced amino acid composition. There are a few anti-nutritional compounds, including glucosinolates, sinapine, tannins, and phytic acid present in rapeseed meal. Glucosinolates have a negative effect on growth and health of animals. Sinapine produces FLS DFR leucodpelargonidin LODX (ANS) FLS kaempferol DFR leucocyanidin delphinidin pelargonidin cyanidin ANR Epi-flavan-3-ols CE LAR flavan-3-ols condensation Condensed tannins (proanthocyanidins) colorless PPO oxidation Oxidized tannins yellow to brown LODX (ANS) FLS quercetin flavonols leucoanthocyanidins 3-OH-anthocyanidins OMT UFGT RT anthocyanins The figure based on ideas from Lepiniec et al. (2006) Fig. 1. Flavonoid biosynthetic pathway. ACCase, acetyl CoA carboxylase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; CE, condensing enzyme; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3?H, Flavonoid 3? hydroxylase; F3?5?H, Flavonoid 3?5? hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; OMT, O-methyltransferase; PPO, polyphenol oxidase; RT, rhamnosyl transferase; UFGT, UDP flavonoid glucosyl transferase. off-flavored trimethylamine in the seed meal that reduces the usability of the meal to the susceptible brown egg laying hens(fenwick and Curtis1980). The black/brown seed coat contains abundant tannins that reduce the digestibility of seed meal especially affecting protein hydrolysis. Embryo-contained phytic acid affects phosphorus binding as well as other essential minerals(uppstro m 1995; Griffithset al. 1998; Mattha us1998; Naczk et al. 1998; Velasco and Mo llers 1998). The seed meal from yellow seeded Brassica lines hashigher protein content and le santi-nutritional compoundsthat make the meal more favorable for use in the poultry or hog industries (Anjou et al. 1977; Slominski et al. 1994). Yellow Seeded Brassicas: Fiber Content in the Seed Meal Higher fiber content in the seed meal is less desirable for monogastric animals. Yellow seeded Brassica lineshave less tannins and less fiber content in the seed meal than black seeded lines, which significantly improves the meal quality (Anjou et al. 1977; Slominski et al. 1994). Slominski et al. (1999) conducted a comparative study on nutritive value of seed meal derived from yellow

4 Can. J. Plant Sci. Downloaded from by on 05/01/ CANADIAN JOURNAL OF PLANT SCIENCE seeded B. rapa, B. juncea and B. napus, and reported that the meal from yellow seeded B. napus had the lowest dietary fiber content (271 g kg 1 dry matter). In contrast, dietary fiber content was highest in the black seeded B. napus (352 g kg 1 dry matter). The yellow seeded B. napus meal generated the highest metabolizable energy when broilerswere fed thismeal. These results indicate that meal from yellow seeded B. napus could be utilized successfully in the poultry industry. INHERITANCE OF SEED COAT COLOR IN DIFFERENT BRASSICA SPECIES In Brassica species, the maternal genotype mainly controlsseed coat color; however, interplay between the maternal parent and the endosperm and/or embryonic genotype may also affect seed coat color (Chen and Heneen 1992; Rahman et al. 2001). Seed Coat Color Inheritance in B. napus Seed coat color inheritance in B. napus hasbeen investigated by several researchers. Shirzadegan (1986) studied seed coat color inheritance and proposed a three-gene model for seed coat color in B. napus. According to hismodel, (i) yellow seed occursonly when all three loci are in the homozygousrecessive condition, (ii) brown seed occurs when a dominant allele ispresent at either the Bl 2 or Bl 3 locusand the Bl 1 locus iseither in homozygousrecessive or in heterozygous condition and (3) black seed occurs when the Bl 1 isin homozygousdominant condition. Van Deynze and Pauls(1994) adopted the Shirzadegan (1986) model with a modification for the genotypesdisplaying black seed coat color where black seed occurs when the Bl 1 locusisin homozygousdominant condition and at least one dominant allele ispresent at any of the other two loci. Henderson and Pauls (1992) confirmed a trigenic inheritance for seed coat color in the F 2 generation of inbred line crosses (16:47:1 segregation) and in DH lines (4:3:1 segregation) from inbred line crosses in B. napus. Baetzel et al. (1999) observed a trigenic inheritance for seed coat color in inbred lines (27:36:1 segregation) and DH lines(1:6:1 segregation) in B. napus and proposed that black seed coat occurred when all genes were in homozygousdominant condition. Lu hset al. (2000) also reported trigenic inheritance for seed coat color in B. napus. Rahman et al. (2001) reported that three or four gene loci were involved in the determination of seed color, and suggested that yellow seeds were formed when all three geneswere homozygousrece sive. Rahman et al. (2001) classified the self-pollinated DH line progeny into five groupsincluding black/dark brown, reddish-brown, partly yellow, yellow-brown and yellow. Four-gene segregation was reported when the first four seed color groups were pooled into one group and the yellow seeded lines were separated from the pool. In contrast, three-gene segregation was observed with pooling of the black/dark brown, reddishbrown and partly yellow seeds into one group, and those with yellow-brown and yellow seeds into a second group. Rahman et al. (2010) observed the seed color segregation in the five different cross-derived populationsand identified two dominant genesthat independently or separately control the black/brown seed coat color trait, and a third seed coat color gene that is responsible for the dark/light yellow seed coat color. The first two genes were dominant over dark/light yellow or yellow seed coat color genes, and the dark/ light yellow seed coat color gene was partially dominant over the yellow seed coat color genes. The occurrence of dark/light yellow seeds in B. napus hasalso been reported by Rahman et al. (2001) and Liu et al. (2005a), but they did not report any genes responsible for this trait. The third seed coat gene is responsible for the dark/light yellow seed coat trait. Study of the inheritance of the third seed coat color gene was complicated because the dark/light yellow seed coat color may appear from yellow seeded plants due to environmental influence asreported by Van Deynze et al. (1993), and Burbulisand Kott (2005). Therefore, Rahman et al. (2010) conducted a progeny test to differentiate between dark/light yellow and yellow seeds. The dark/light yellow seed coat color trait is controlled by one gene and isincompletely dominant over yellow seed coat color genes in yellow sarson B. rapa (Rahman 2001a; Rahman et al. 2007). Apart from the dominant nature of black seed coat color of B. napus, Li et al. (2003) reported that different yellow seed coat color genes existed in different yellow seeded lines, and that these genes showed dominance, partial dominance and recessive allelic interactions. They also obtained a completely dominant yellow seeded line in a segregating population. Liu et al. (2005a) investigated seed color inheritance in F 2,BC 1 and F 1 -derived DH progeniesand reported that seed coat color wascontrolled by maternal genotype and that a single gene locus was responsible for the yellow seed trait. Thisgene waspartially dominant over the black seed trait. Liu et al. (2005b) investigated the seed coat color inheritance in F 2 and BC 1 populationsof two crosses and also in a DH population, and reported that three genes were responsible for seed coat color in B. napus. They identified a dominant yellow seed color gene that had epistatic effects on the other two independently segregating dominant black seeded genes. Seed Coat Color Inheritance in B. rapa Mohammad et al. (1942) and Jo nsson (1975) reported that three independently segregating genes were responsible for seed coat color inheritance where brown seed color wasdominant over the yellow color type, and yellow seed occurred only when all three loci were in a homozygousrecessive condition. Stringam (1980) observed digenic inheritance of seed coat color in B. rapa, and proposed a model for seed color genes Br1 and Br3. According to the model, the presence of dominant allelesat both loci (Br1-Br3-) or the presence of

5 RAHMAN AND MCVETTY * A REVIEW OF BRASSICA SEED COLOR 441 Can. J. Plant Sci. Downloaded from by on 05/01/18 dominant allelesonly at the first locus(br1-br3br3) generated brown seed color; the yellow-brown seed occurred when the second locus contained at least one dominant allele and the first locus contained homozygous recessive alleles (br1br1br3-); the presence of homozygousrece sive allelesat both loci produced yellow seeds (br1br1br3br3). Schwetka (1982), Zaman (1989), Rahman (2001a) and Rahman et al. (2007) supported this model for seed coat color inheritance in B. rapa. Ahmed and Zuberi (1971), Hawk (1982), Chen and Heneen (1992) observed a single gene inheritance pattern in B. rapa, where the brown seed color was dominant over the yellow seed type. In contrast, Ahmed and Zuberi (1971) and Chen and Heneen (1992) observed single locus inheritance for seed coat color in B. rapa. Rahman (2001a) explained thisapparent anomaly by suggesting that the second locus for yellow-brown seed color gene was in homozygous recessive (br3br3) condition and as a result the first gene (Br1) segregated as a single gene. Seed Coat Color Inheritance in B. juncea In B. juncea two independent dominant geneswith duplicate effects are responsible for the black or brown color trait (Anand et al. 1985; Chauhan and Kumar 1987; Negi et al. 2000; Mahmood et al. 2005). According to these authors, yellow seed occurs when both gene loci are in the homozygous recessive condition, and black or brown seed occurs when at least a dominant allele is present at either of the two loci. These seed coat color genes may also be present in the genomes of B. rapa (AA) and B. nigra (BB), respectively (Negi et al. 2000). Seed Coat Color Inheritance in B. carinata In Brassica species, seed coat color is controlled by the maternal parent where black/brown seed color is dominant over yellow seed color. However, contrasting seed color inheritance in B. carinata wasreported by Getinet et al. (1987), Getinet and Rakow (1997) and Rahman and Tahir (2010). Getinet et al. (1987) studied the seed coat color inheritance in B. carinata and later Getinet and Rakow (1997) confirmed the earlier published result. Getinet and Rakow (1997) observed an interaction between brown seed coat color gene and a dominant repressor (Rp) gene. According to Getinet and Rakow (1997), a dominant repressor (Rp) gene inhibits the function of seed coat color gene and resulted in translucent seed i.e., yellow seed in B. carinata. The absence of the repressor gene will allow the seed coat color gene to be functioned for the synthesis of seed coat pigments resulted brown seed in B. carinata. Recently, Rahman and Tahir (2010) studied the inheritance of seed color gene in genetically diverse B. carinata germplasm and confirmed the report of Getinet and Rakow (1997). According to these studies, no recessive gene for seed color has yet been identified in B. carinata. BREEDING FOR YELLOW SEEDED B. NAPUS Naturally occurring seed coat colors in all Brassica species are black/brown with yellow seeded mutants identified only in B. rapa, B. juncea and B. carinata. There isno naturally occurring yellow seeded B. napus germplasm available. Therefore, development of yellow seeded B. napus hasbeen a primary breeding objective for canola/rapeseed breeders globally for many years. The first yellow seeded B. napus cultivar HUA-yellow No. 1 wasregistered in China in 1990 (Liu et al. 1991). This yellow seeded line was developed from a cross of yellow seeded B. rapa (ssp. chinensis) with black seeded B. napus, followed by backcrosses to B. napus and pedigree selection (Liu 1983). However, the seed coat color wasfrequently affected by changesin the environment resulting in black seed and/or black spots on the seed coat. Shirzadegan and Ro bbelen (1985) developed yellow seeded B. napus (winter type) from a cross of a black seeded winter type B. napus variety Quinta with a resynthesized yellow-brown seeded B. napus line derived from a cross of light yellow-brown seeded B. oleracea ssp. alboglabra and yellow seeded B. rapa. Zaman (1987) developed partially yellow-seeded B. napus from interspecific crosses of yellow seeded B. carinata with yellow seeded B. rapa followed by a backcross to B. napus, and from a cross of black seeded B. napus with yellow seeded B. rapa. He also attempted to develop a yellow seeded CC genome species from a cross of yellow seeded B. carinata (BBCC) and black seeded B. alboglabra (CC), which could be crossed with yellow seeded B. rapa to develop yellow seeded B. napus. However, this attempt did not generate a yellow seeded CC genome species. Chen et al. (1988) resynthesized B. napus from interspecific crosses between yellow seeded B. rapa and yellow or brown seeded B. alboglabra, with yellow seeded types identified from a segregating population. Chen and Heneen (1992) developed yellow seeded B. napus lines from crosses of resynthesized B. napus linescontaining yellow seed coat color genes(chen et al. 1988) with yellow-brown seeded B. napus, or with yellow seeded B. carinata. However, the yellow seeded lines did not breed true in the following generations. Rashid et al. (1994) obtained yellow seeded B. napus through interspecific crosses of black seeded B. napus with two yellow seeded mustard species, B. juncea and B. carinata. To eliminate the B genome chromosomes from both hybrids, the F 1 generationswere backcro sed to B. napus. Further crosses were made between backcross F 2 plantsof the (B. napus B. juncea)b. napus cross and backcross F 2 plantsof the (B. napus B. carinata)b. napus cross. The F 2 intercross population wasgrown in the field and 91 yellow seeded plants were selected from 4858 plants. Qi et al. (1995) obtained yellow seeded B. napus from a cross of yellow seeded B. carinata with a partially yellow seeded B. napus line that carried the yellow seed coat color gene from B. rapa. Tang et al. (1997) developed 16 yellow seeded B. napus lines from various interspecific crosses using naturally

6 Can. J. Plant Sci. Downloaded from by on 05/01/ CANADIAN JOURNAL OF PLANT SCIENCE occurring yellow seeded species, such as (B. rapa B. oleracea) B. napus, B. napus B. juncea, and B. napus B. rapa, intervarietal crosses of B. napus, and from irradiated progeniesof B. napus. Meng et al. (1998) developed yellow seeded B. napus from interspecific cross of yellow seeded B. rapa and yellow seeded B. carinata. Trigenomic hexaploid Brassica (2n 54, AABBCC) wasgenerated from the F 1, which was crossed with partial yellow or brown seeded varieties of B. napus. Most of the F 1 seeds were self-fertile and generated brown seed color. In the F 2 population, 73 out of 2590 open-pollinated plantsproduced yellow seed coat color. Stable yellow seeded lines were developed from two successive self-pollinated generations of the selected lines. Rahman (2001b) developed yellow seeded B. napus from interspecific crosses between yellow seeded B. rapa var. Yellow Sarson (AA), black-seeded B. alboglabra (CC), yellow-seeded B. carinata (BBCC) and resynthesized black-seeded B. napus (AACC) derived from a black-seeded B. alboglabra and yellow seeded B. rapa cross. Three approaches were taken to develop yellow seeded B. napus and yellow seeded CC genome species. In the first approach, yellow seeded B. rapa was crossed with yellow seeded B. carinata, and the trigenomic hybrid (ABC) was crossed with resynthesized black seeded B. napus. The hybrid wasselfpollinated for number of generationsand yielded yellow seeded B. napus lines. In the second approach, brown seeded trigenomic hexaploid Brassica (AABBCC) was developed from the trigenomic hybrid (ABC) of a cross of yellow seeded B. rapa and yellow seeded B. carinata. The hexaploid Brassica was crossed with the resynthesized brown seeded B. napus. The new hybrid wasselfpollinated for several generations but failed to generate any yellow seeded B. napus lines. To develop yellow seeded CC genome species in the third approach, a cross wasmade between black seeded B. alboglabra and yellow seeded B. carinata and the F 1 wasself-pollinated for several generations. A yellowish-brown seeded B. alboglabra (CC) line wasdeveloped, which was crossed with yellow seeded B. rapa, but failed to generate any resynthesized yellow seeded B. napus lines. Burbulisand Kott (2005) identified canola type yellow seeded B. napus in the DH progeniesof a crossbetween two black seeded and six yellow seeded lines. MOLECULAR MARKERS FOR SEED COAT COLOR GENES IN BRASSICA The creation of genetic variation and subsequent selection of desirable genotypes is a key issue in crop improvement and plant breeding. Traditionally, selection of plant materialswith desirable traitsiscarried out using phenotypic selection. Phenotypic traits are rather limited in number and only a limited number of qualitative and/or quantitative genes are responsible for most of the phenotypic variation. Furthermore the expression of genes is often influenced by the environment. In contrast, the development of molecular markers linked to a trait of interest using different molecular marker techniquesallowsthe monitoring of similarity/dissimilarity among different genotypes at the very early stages of plant development, independent of environmental effects. This knowledge can be used directly in a marker-assisted selection program in plant breeding to identify desirable genotypes in segregating populations(f 2 or backcross). Marker-assisted selection can speed breeding progress significantly. The use of molecular markers makes it possible to transfer a trait from one genotype to another with high efficiency, even traitsthat are highly influenced by the environment (Rahman et al. 2010). Molecular Markers for Seed Color Genes in B. napus Only a few researchers have developed molecular markersfor seed coat color genesin B. napus. Van Deynze et al. (1995) developed RFLP markersfor two of the three seed coat color genes in B. napus. Somers et al. (2001) identified eight RAPD markersco-segregating with the major yellow seed coat color gene (pigment1) in B. napus that differentiated over 72% of individualsfor seed coat color variation. Liu et al. (2005b) reported that a single gene locus in the yellow seeded lines was partially dominant over black seeded lines, and two RAPD and eight AFLP markers were developed linked to the seed coat color genes in B. napus. Two of the 10 markerswere very close (3.9 cm and 2.4 cm) to the seed coat color gene, which allowed selection for yellow seeded individuals at an accuracy of 99.91%. However, these RAPD and AFLP markers were not adapted for larger scale marker assisted selection. Therefore, Liu et al. (2006) converted these RAPD and AFLP markersinto reliable SCAR and cleaved amplified polymorphic sequence markers from the sequence information of the most closely linked four AFLP and two RAPD markersfor seed coat color breeding in B. napus. Fu et al. (2007) developed QTLs for seed color genes in B. napus. Two recombinant inbred line populations were used to screen with SSR, RAPD, and SRAP markers. Out of 19 QTLs, sequence of the flanking marker of one major QTL wasanchored very close to Arabidopsis TT10 seed coat color gene. Zhang et al. (2010) used two mapping populations to identify QTL for seed color genes in B. napus. Three putative QTLswere identified in the linkage group N18, N5, and N3 from the first population. In the second population, another three QTLswere identified in the linkage group N9, N18, and N8. The QTL N18 was common in both populations, whereas the remaining QTLswere different from each other, indicating that the seed coat color genes are different in two populations. Rahman et al. (2010) developed three different SRAP markersfor tagging the three different seed coat color genesin the gene family of B. napus. The first SRAP marker wastightly linked to the black/brown seed coat color trait of B. napus and converted into SCAR

7 RAHMAN AND MCVETTY * A REVIEW OF BRASSICA SEED COLOR 443 Can. J. Plant Sci. Downloaded from by on 05/01/18 marker. The second SRAP was also tightly linked to the black/brown seed coat color trait and converted into SNP marker. The third SRAP seed coat color gene marker was closely linked to the dark/light seed coat color trait. Molecular Markers for Seed Color Genes in B. rapa The development of molecular markersfor seed coat color genesin B. rapa hasalso been reported. Rahman et al. (2007) developed a very close (0.47 cm) dominant SRAP marker for the major seed coat color gene (Bl 1 )in B. rapa. For easy use in marker-assisted selection in plant breeding programs, the dominant SRAP marker wasconverted into co-dominant SCAR and SNP markers using chromosome walking technology. Sequence analysis of the SRAP marker showed the same sequence as the second seed coat color gene marker in B. napus (Rahman et al. 2010), indicating the presence of the same gene in B. rapa and in B. napus. Molecular Markers for Seed Color Genes in B. alboglabra Chen et al. (1997) developed a RAPD marker linked to the seed coat color gene located at the terminal region of chromosome 1 of B. alboglabra using B. rapaalboglabra addition lines. Heneen and Jørgensen (2001) used a brown seeded B. rapaalboglabra monosomic additional line that carried chromosome 4 of B. alboglabra and reported that the embryo of a brown seeded additional line contained a gene for dark seed coat color. They developed a RAPD marker for the seed coat color trait linked to chromosome 4 of B. alboglabra. Molecular Markers for Seed Color Genes in B. juncea Negi et al. (2000) developed three AFLP markerstightly linked to the brown seed coat color trait in B. juncea. These AFLP markers were not suitable for large scale application in marker assisted selection in plant breeding. Therefore, PCR-walking technology wasapplied to convert the dominant AFLP marker into simple codominant SCAR markers, which distinguished the yellow and brown seeded B. juncea linesaswell asthe homozygousand heterozygousbrown-seeded individuals. Mahmood et al. (2003) constructed an RFLPbased genetic map of B. juncea and described the linkage groups of A- and B-genomes. On the basis of this genetic map, Mahmood et al. (2005) later identified two QTL linked with two seed coat color genes located on both linkage group specific to A- and B-genome of B. juncea. Padmaja et al. (2005) developed three microsatellite markers(ra2-a11, Na10-A08 and Ni4-F11) strongly associated with seed coat color genes. Two (Ra2-A11, Na10-A08) of the three markerswere placed on linkage group-1 and the other marker (Ni4-F11) wasplaced on linkage group-2. The markersna10-a08 and Ni4-F11 co-segregated without any recombination with the seed coat color genes BjSC1 and BjSC2 in B. juncea, respectively. ENVIRONMENTAL INFLUENCE IN SEED COAT COLORATION In general, warmer temperaturessignificantly reduce color deposition in the seed coat of Brassica species. Van Deynze et al. (1993) conducted systematic research on the influence of temperature on seed coat color in yellow seeded and black seeded lines of B. napus. The experiment wasconducted in growth cabinetsat 16/128C, 18/ 148C, 20/168C, 22/188C and 24/208C day/night temperatureswith a 16-h photoperiod (350 mmol m 2 s 2 ). Yellow seeded lines had improved yellow seed color with increased temperatures and the reduction of color was linearly correlated with increased temperatures. For example, two genotypesproduced black or dark brown seed coat color at 168C, while the genotypesproduced yellow seed coat color at 248C. These results indicate that high temperature might block the function of genes or enzymes involved in the biosynthesis of pigments in seed. Marles et al. (2003) reported that yellow seeded B. carinata produced yellow seed at 25/208C day/night temperature, but that seed coat color turned light brown at 18/158C. Burbulisand Kott (2005) conducted an experiment with 11 yellow to brown-yellow seeded lines grown at three different temperature conditions, 20/ 168C and 28/248C day/night temperaturesin growth chambers, and an outside plot with day time temperaturesabove 308C from bolting to the seed maturity stage. The lines showed an increasing yellowness with higher temperatures, and were darker with cooler growing conditions. It hasbeen cited earlier that proanthocyanidinsare the final compound in the seed coat coloration process, and dihydroflavonol 4-reductase is one of the enzymes responsible for the biosynthesis of proanthocyanidins from dihydromyricetin (Fig. 1). Dihydroflavonol 4- reductase transcripts were absent or less abundant in the yellow seeded lines of B. carinata when they were grown at warm temperatures(25/208c). Cooler (18/ 158C) growing temperaturesincreased the dihydroflavonol 4-reductase expression resulting in the deposition of pigments in the seed coat of yellow-seeded lines, indicating that temperature hasa direct affect on biosynthesis of some transcripts responsible for seed coat pigmentation in plants(marleset al. 2003). SEED COAT COLOR GENE IDENTIFICATION AND CLONING Yellow seed B. napus could be developed using gene transformation techniques if the seed coat color genes are identified in Brassica species. The first and only seed coat color gene in Brassica to be identified, cloned and functionally characterized wasreported by Zhang et al. (2009). Thisismajor advance in Brassica seed coat color research since there are more than 20 genes controlling seed coat color identified and characterized

8 Can. J. Plant Sci. Downloaded from by on 05/01/ CANADIAN JOURNAL OF PLANT SCIENCE in Arabidopsis. Zhang et al. (2009) developed a fine genetic map and identified a SNP marker, which was located inside a Brassica ortholog of TRANSPARENT TESTA GLABRA 1 (TTG1) gene in Arabidopsis. This gene wasfound to have a complete linkage with the leaf hairiness gene of Brassica rapa. The functional analysis of the TTG1 gene in Brassica wasconducted by transforming the gene homolog from the black seeded and the yellow seeded parents into an Arabidopsis ttg1 mutant. The gene homolog from the black seeded parent could recover the seed coat color in the Arabidopsis ttg1 mutant, while the yellow seeded gene homolog failed to recover the seed coat color. This also demonstrated that the TTG1 ortholog of Brassica shared the same gene function in Arabidopsis. CONCLUSIONS Yellow seed coat color in Brassica species is desirable because oil content and protein content are higher and fiber content islower in yellow seeded Brassica lines compared with black/brown seeded Brassica lines. The development of yellow seeded B. napus lineshasbeen an objective for canola/rapeseed breeders for many decades because of the seed quality advantages of yellow seeded Brassica lines. Multi-genic inheritance, environmental influencesand combined maternal and embryonic control make the development of yellow seeded genotypes in B. napus very challenging. Molecular markersfor all yellow seed coat color genes in B. napus and the more recent gene cloning of a yellow seeded gene should greatly facilitate the rapid development of new yellow seeded B. napus canola/rapeseed cultivars. Abrahams, S., Tanner, G. J., Larkin, P. J. and Ashton, A. R Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis. 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