Mapping and tagging of seed coat colour and the identification of microsatellite markers for marker-assisted manipulation of the trait in Brassica juncea

Microsatellite marker technology in combination with three doubled haploid mapping populations of Brassica juncea were used to map and tag two independent loci controlling seed coat colour in B. juncea. One of the populations, derived from a cross between a brown-seeded Indian cultivar, Varuna, and a Canadian yellow-seeded line, Heera, segregated for two genes coding for seed coat colour; the other two populations segregated for one gene each. Microsatellite markers were obtained from related Brassica species. Three microsatellite markers (Ra2-A11, Na10-A08 and Ni4-F11) showing strong association with seed coat colour were identified through bulk segregant analysis. Subsequent mapping placed Ra2-A11 and Na10-A08 on linkage group (LG) 1 at an interval of 0.6 cM from each other and marker Ni4-F11 on LG 2 of the linkage map of B. juncea published previously (Pradhan et al., Theor Appl Genet 106:607–614, 2003). The two seed coat colour genes were placed with markers Ra2-A11 and Na10-A08 on LG 1 and Ni4-F11 on LG 2 based on marker genotyping data derived from the two mapping populations segregating for one gene each. One of the genes (BjSC1) co-segregated with marker Na10-A08 in LG 1 and the other gene (BjSC2) with Ni4-F11 in LG 2, without any recombination in the respective mapping populations of 130 and 103 segregating plants. The identified microsatellite markers were studied for their length polymorphism in a number of yellow-seeded eastern European and brown-seeded Indian germplasm of B. juncea and were found to be useful for the diversification of yellow seed coat colour from a variety of sources into Indian germplasm.     

A Large Insertion in bHLH Transcription Factor BrTT8 Resulting in Yellow Seed Coat in Brassica rapa

Yellow seed is a desirable quality trait of the Brassica oilseed species. Previously, several seed coat color genes have been mapped in the Brassica species, but the molecular mechanism is still unknown. In the present investigation, map-based cloning method was used to identify a seed coat color gene, located on A9 in B. rapa. Blast analysis with the Arabidopsis genome showed that there were 22 Arabidopsis genes in this region including at4g09820 to at4g10620. Functional complementation test exhibited a phenotype reversion in the Arabidopsis thaliana tt8-1 mutant and yellow-seeded plant. These results suggested that the candidate gene was a homolog of TRANSPARENT TESTA8 (TT8) locus. BrTT8 regulated the accumulation of proanthocyanidins (PAs) in the seed coat. Sequence analysis of two alleles revealed a large insertion of a new class of transposable elements, Helitron in yellow sarson. In addition, no mRNA expression of BrTT8 was detected in the yellow-seeded line. It indicated that the natural transposon might have caused the loss in function of BrTT8. BrTT8 encodes a basic/helix-loop-helix (bHLH) protein that shares a high degree of similarity with other bHLH proteins in the Brassica. Further expression analysis also revealed that BrTT8 was involved in controlling the late biosynthetic genes (LBGs) of the flavonoid pathway. Our present findings provided with further studies could assist in understanding the molecular mechanism involved in seed coat color formation in Brassica species, which is an important oil yielding quality trait.     

Identification of AFLP fragments linked to seed coat colour in Brassica juncea and conversion to a SCAR marker for rapid selection

A Brassica juncea mapping population was generated and scored for seed coat colour. A combination of bulked segregant analysis and AFLP methodology was employed to identify markers linked to seed coat colour in B. juncea. AFLP analysis using 16 primer combinations revealed seven AFLP markers polymorphic between the parents and the bulks. Individual plants from the segregating population were analysed, and three AFLP markers were identified as being tightly linked to the seed coat colour trait and specific for brown-seeded individuals. Since AFLP markers are not adapted for large-scale application in plant breeding, our objective was to develop a fast, cheap and reliable PCR-based assay. Towards this goal, we employed PCR-walking technology to isolate sequences adjacent to the linked AFLP marker. Based on the sequence information of the cloned flanking sequence of marker AFLP8, primers were designed. Amplification using the locus-specific primers generated bands at 0.5 kb and 1.2 kb with the yellow-seeded parent and a 1.1-kb band with the brown-seeded parent. Thus, the dominant AFLP marker (AFLP8) was converted into a simple codominant SCAR (Sequence Characterized Amplified Region) marker and designated as SCM08. Scoring of this marker in a segregating population easily distinguished yellow- and brown-seeded B. juncea and also differentiated between homozygous (BB) and heterozygous (Bb) brown-seeded individuals. Thus, this marker will be useful for the development of yellow seed B. juncea cultivars and facilitate the map-based cloning of genes responsible for seed coat colour trait. Received: 2 October 1999 / Accepted: 11 November 1999     

Fine mapping of loci involved with glucosinolate biosynthesis in oilseed mustard (Brassica juncea) using genomic information from allied species

Fine mapping of six seed glucosinolate QTL (J2Gsl1, J3Gsl2, J9Gsl3, J16Gsl4, J17Gsl5 and J3Gsl6) (Ramchiary et al. in Theor Appl Genet 116:77–85, 2007a) was undertaken by the candidate gene approach. Based on the DNA sequences from Arabidopsis and Brassica oleracea for the different genes involved in the aliphatic glucosinolate biosynthesis, candidate genes were amplified and sequenced from high to low glucosinolate Brassica juncea lines Varuna and Heera, respectively. Of the 20 paralogues identified, 17 paralogues belonging to six gene families were mapped to 12 of the 18 linkage groups of B. juncea genome. Co-mapping of candidate genes with glucosinolate QTL revealed that the candidate gene BjuA.GSL-ELONG.a mapped to the QTL interval of J2Gsl1, BjuA.GSL-ELONG.c, BjuA.GSL-ELONG.d and BjuA.Myb28.a mapped to the QTL interval of J3Gsl2, BjuA.GSL-ALK.a mapped to the QTL interval of J3Gsl6 and BjuB.Myb28.a mapped to the QTL interval of J17Gsl5. The QTL J9Gsl3 and J16Gsl4 did not correspond to any of the mapped candidate genes. The functionality and contribution of different candidate genes/QTL was assessed by allelic variation study using phenotypic data of 785 BC₄DH lines. It was observed that BjuA.Myb28.a and J9Gsl3 contributed significantly to the base level glucosinolate production while J16Gsl4, probably GSL-PRO, BjuA.GSL-ELONG.a and BjuA.GSL-ELONG.c contributed to the C3, C4 and C5 elongation pathways, respectively. Three A genome QTL: J2Gsl1harbouring BjuA.GSL-ELONG.a, J3Gsl2 harbouring both BjuA.GSL-ELONG.c and BjuA.Myb28.a and J9Gsl3, possibly the ‘Bronowski genes’, were identified as most important loci for breeding low glucosinolate B. juncea. We observed two-step genetic control of seed glucosinolate in B. juncea mainly effected by these three A genome QTL. This study, therefore, provides clues to the genetic mechanism of ‘Bronowski genes’ controlling the glucosinolate trait and also provides efficient markers for marker-assisted introgression of low glucosinolate trait in B. juncea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

茎瘤芥异戊烯基转移酶家族基因全基因组鉴定及表达分析北大核心CSCD

该研究以茎瘤芥栽培品种‘永安小叶’为实验材料,在全基因组水平对茎瘤芥基因组中异戊烯基转移酶(IPT)家族基因成员进行鉴定;通过荧光定量PCR检测各基因在不同组织、盐胁迫和根肿菌胁迫条件下的表达模式。结果显示:(1)在茎瘤芥基因组中共鉴定到27个IPT家族基因,分布在14条染色体上,它们在系统进化树中可聚类为7个分支。(2)大部分IPT家族基因主要在茎瘤芥的根和茎中表达,在叶片、花和种荚中表达量相对较低。BjuB006281在茎中的表达水平最高,BjuA027211、BjuB010173、BjuB010174和BjuA001839在根中的表达水平较高。(3)大部分的IPT基因表达受盐胁迫抑制,BjuB006281、BjuA036403、BjuB010173、BjuB026254在盐胁迫12~48 h显著下调表达;BjuB022918和BjuB007352则在盐胁迫24~48 h显著下调表达。(4)大部分茎瘤芥IPT基因在12 h受到根肿菌侵染的显著诱导,其中BjuB006281、BjuA014415、BjuB022918在侵染后12 h的表达水平为0 h对照的15倍以上。该研究鉴定出多个响应盐胁迫和根肿菌胁迫的IPT基因,为进一步研究他们的基因功能奠定了基础。     

Identification of a major gene and RAPD markers for yellow seed coat colour in Brassica napus.

The development of yellow-seeded Brassica napus for improving the canola-meal quality characteristics of lower fibre content and higher protein content has been restricted because no yellow-seeded forms of B. napus exist, and their conventional development requires interspecific introgression of yellow seed coat colour genes from related species. A doubled-haploid (DH) population derived from the F1 generation of the cross 'Apollo' (black-seeded) x YN90-1016 (yellow-seeded) B. napus was analysed via bulked segregant analysis to identify molecular markers associated with the yellow-seed trait in B. napus for future implementation in marker-assisted breeding. A single major gene (pigment 1) flanked by eight RAPD markers was identified co-segregating with the yellow seed coat colour trait in the population. This gene explained over 72% of the phenotypic variation in seed coat colour. Further analysis of the yellow-seeded portion of this DH population revealed two additional genes favouring 'Apollo' alleles, explaining 11 and 8.5%, respectively, of the yellow seed coat colour variation. The data suggested that there is a dominant, epistatic interaction between the pigment I locus and the two additional genes. The potential of the markers to be implemented in plant breeding for the yellow-seed trait in B. napus is discussed.     

Genetic analysis of agronomic and quality traits in mustard (Brassica juncea)

To develop an efficient mustard (Brassica juncea) breeding programme, a better knowledge of the genetic control and relationships of the main selected characters is needed. Thus, doubled haploid (DH) lines were evaluated over 2 years in the field. Days to flowering, plant height, thousand-seed weight, fatty acid composition, seed oil content, sinigrin, gluconapin and total glucosinolate contents were determined in the DH population. The influence of seed coat colour was estimated. Results showed significant differences between yellow and brown seeds only for oil and fatty acid contents. Molecular analysis revealed that seed coat colour is associated with two Mendelian trait loci: Bjc1 [on linkage group (LG) 3] and Bjc2 (on LG6). The quantitative trait loci associated with characters—detected by composite interval mapping—were not co-localised and revealed a genetic independence. The results obtained in this study show that the most important agronomic and quality traits of brown mustard could be bred independently. Correlation between the studied traits is also discussed.     

A linkage map of chickpea (Cicer arietinum L.) based on populations from Kabuli × Desi crosses: location of genes for resistance to fusarium wilt race 0

Two recombinant inbred line (RIL) populations derived from intraspecific crosses with a common parental line (JG62) were employed to develop a chickpea genetic map. Molecular markers, flower colour, double podding, seed coat thickness and resistance to fusarium wilt race 0 (FOC-0) were included in the study. Joint segregation analysis involved a total of 160 markers and 159 RILs. Ten linkage groups (LGs) were obtained that included morphological markers and 134 molecular markers (3 ISSRs, 13 STMSs and 118 RAPDs). Flower colour (B/b) and seed coat thickness (Tt/tt) appeared to be linked to STMS (GAA47). The single-/double-podding locus was located on LG9 jointly with two RAPD markers and STMS TA80. LG3 included a gene for resistance to FOC-0 (Foc0₁/foc0₁) flanked by RAPD marker OPJ20₆₀₀ and STMS marker TR59. The association of this LG with FOC-0 resistance was confirmed by QTL analysis in the CA2139 × JG62 RIL population where two genes were involved in the resistance reaction. The STMS markers enabled comparison of LGs with preceding maps.     

Inheritance of seed color and molecular markers linked to the seed color gene in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Seed color inheritance in Brassica juncea was studied in F₁, F₂ and BC₁ populations. Seed color was found under the control of the maternal genotype, and the brown-seeded trait was dominant over the yellow-seeded trait. Segregation analysis revealed that one pair of major genes controlled the seed coat color. To develop markers linked to the seed color gene, AFLP (amplified fragments length polymorphism) combined with BSA (bulk segregant analysis) technology was used to screen the parents and bulks selected randomly from an F₂ population (Wuqi yellow mustard × Wugong mustard) consisting of 346 individuals. From a survey of 512 AFLP primer combinations, 15 AFLP markers located on either side of the gene were identified, and the average distance between markers was 2.59 cM. P11MG15 was a cosegregated marker, and the closest markers (P03MC08, P16MC02 and P11MG01) were at a distance of 0.3, 0.3 and 0.7 cM from the target gene, respectively. In order to utilize the markers for breeding of yellow-seeded varieties, four AFLP markers, P11MG01, P15MG15, P09MC12 and P16MC02 were successfully converted into SCAR (sequence characterized amplified region) markers. The seed color trait controlled by the single gene together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded varieties. The markers found in the present study could accelerate the step of map-based cloning of the target gene.     

Gene Silencing of BnTT10 Family Genes Causes Retarded Pigmentation and Lignin Reduction in the Seed Coat of Brassica napus

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus.     

Allelic Variation of BnaC.TT2.a and Its Association with Seed Coat Color and Fatty Acids in Rapeseed (Brassica napus L.)

Efficient molecular markers for the selection of rapeseed genetic materials with high seed oil content and ideal fatty acid (FA) composition are preferred by rapeseed breeders. Recently, we reported the molecular mechanism of TRANSPARENT TESTA 2 (TT2) in inhibiting seed FA biosynthesis in Arabidopsis. However, evidence showing the association of rapeseed TT2 homologs and seed FA production are still insufficient. In this study, we collected 83 rapeseed (Brassica napus L.) landraces from different geographical backgrounds to conduct association mapping of BnaC.TT2.a in relation to seed coat color and FA biosynthesis. Population background was corrected by 84 pairs of SSR markers that were uniformly distributed among the linkage groups of the Tapidor-Ningyou-7 DH population. A single copy of BnaC.TT2.a for single nucleotide polymorphism (SNP) assay was cloned by a pair of previously reported specific primers. From the analysis of BnaC.TT2.a allelic variations using GLM+Q model, four SNPs on intron 1 of BnaC.TT2.a that were associated with seed FA were discovered. Moreover, an InDel at position 738 on exon 3 of BnaC.TT2.a indicated a change of protein function that was significantly associated with seed coat color, linoleic acid (C18:2), and total FA content. These findings revealed the role of BnaC.TT2.a in regulating the seed color formation and seed FA biosynthesis in rapeseed, thereby suggesting effective molecular markers for rapeseed breeding.     

Inheritance of seed colour and identification of RAPD and AFLP markers linked to the seed colour gene in rapeseed (Brassica napus L.)

In China Polima cytoplasmic male sterility (cms) is currently the most important hybrid system used for the breeding of hybrids. In an effort to develop yellow-seeded Polima cms restorer lines, we used yellow-seeded, doubled haploid (DH) line No.2127-17 as the gene source in crosses with two elite black-seeded Polima cms R lines, Hui5148-2 and 99Yu42, which originated from our breeding programme. The inheritance of seed colour was investigated in the F₂, BC₁ and F₁-derived DH progenies of the two crosses. Seed colour was found to be under the control of the maternal genotype and the yellow seed trait to be partially dominant over the black seed trait. Segregation analysis revealed a single gene locus for the partial dominance of yellow seed colour. Of 810 randomly amplified polymorphic DNA (RAPD) primers, 240 (29.6%) revealed polymorphisms between the parents. Of the 240 RAPD primers and 512 amplified fragment length polymorphism (AFLP) primer pairs, four RAPDs and 16 AFLP pairs showed polymorphisms between the bulks, with two RAPD and eight AFLP markers being identified in the vicinity of the seed-coat colour gene locus using a DH progeny population—derived from the cross Hui5148-2×No.2127-17—of 127 individuals in combination with the bulked segregant analysis strategy. Seven of these latter ten markers were linked to the allele for yellow seed, whereas the other three were linked to the allele for black seed. The seed-coat colour gene locus was bracketed by two tightly linked markers, EA02MG08 (2.4 cM) and S1129 (3.9 cM). The partial dominance and single gene control of the yellow seed-coat colour trait together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded hybrid varieties.