Background: Extensive mapping efforts are currently underway for the establishment of comparative genomics between the model plant, Arabidopsis thaliana and various Brassica species. Most of these studies have deployed RFLP markers, the use of which is a laborious and time-consuming process. We therefore tested the efficacy of PCR-based Intron Polymorphism (IP) markers to analyze genome-wide synteny between the oilseed crop, Brassica juncea (AABB genome) and A. thaliana and analyzed the arrangement of 24 (previously described) genomic block segments in the A, B and C Brassica genomes to study the evolutionary events contributing to karyotype variations in the three diploid Brassica genomes.

SummaryPolyploidy, the possession of multiple sets of chromosomes, has been a predominant factor in the evolution and success of the angiosperms. Although artificially formed allopolyploids show a high rate of genome rearrangement, the genomes of cultivars and germplasm used for crop breeding were assumed stable and genome structural variation under the artificial selection process of commercial breeding has remained little studied. Here, we show, using a repurposed visualization method based on transcriptome sequence data, that genome structural rearrangement occurs frequently in varieties of three polyploid crops (oilseed rape, mustard rape and bread wheat), meaning that the extent of genome structural variation present in commercial crops is much higher than expected. Exchanges were found to occur most frequently where homoeologous chromosome segments are collinear to telomeres and in material produced as doubled haploids. The new insights into genome structural evolution enable us to reinterpret the results of recent studies and implicate homoeologous exchanges, not deletions, as being responsible for variation controlling important seed quality traits in rapeseed. Having begun to identify the extent of genome structural variation in polyploid crops, we can envisage new strategies for the global challenge of broadening crop genetic diversity and accelerating adaptation, such as the molecular identification and selection of genome deletions or duplications encompassing genes with trait‐controlling dosage effects.

Identification of the candidate gene responsible for the seed coat colour variation in Brassica juncea was undertaken following an earlier study where two independent loci (BjSc1 and BjSc2) were mapped to two linkage groups, LG A9 and B3 (Padmaja et al. in Theor Appl Genet 111:8-14, 2005). The genome search from BRAD data for the presence of flavonoid genes in B. rapa identified three candidate genes namely, DFR, TT1 and TT8 in the LG A9. Quantitative real-time PCR revealed absence of transcript for the late biosynthetic genes (LBGs) and showed significant reduction of transcript in the TT8 from the developing seeds of yellow-seeded line. While mapping of two DFR genes, the BjuA.DFR and BjuB.DFR did not show perfect co-segregation with the seed coat colour loci, that of the two TT8 genes, BjuA.TT8 and BjuB.TT8 showed perfect co-segregation with the seed coat colour phenotype. The BjuA.TT8 allele from the yellow-seeded line revealed the presence of an insertion of 1,279 bp in the exon 7 and did not produce any transcript as revealed by reverse transcriptase PCR. The BjuB.TT8 allele from the yellow-seeded line revealed the presence of an SNP (C→T) in the exon 7 resulting in a stop codon predicting a truncated protein lacking the C-terminal 8 amino acid residues and produced significantly low level of transcript than its wild-type counterpart. Hence, it is hypothesized that the mutations in both the TT8 genes are required for inhibiting the transcription of LBGs in the yellow-seeded mutant of B. juncea.

Molecular mapping and tagging of the erucic acid trait (C22:1) in Brassica juncea was done by a candidate gene approach. Two QTLs underlying the variation of seed erucic acid content were assigned to two linkage groups of a B. juncea map using a doubled haploid (DH) mapping population derived from high x low erucic acid F(1) hybrid. Two consensus primers corresponding to the full-length Fatty Acid Elongase 1 ( FAE1) gene, reported to be involved in the elongation of C18:1 to C22:1, were designed. PCR amplification and subsequent cloning and sequencing identified two FAE1 genes ( FAE1.1 and FAE1.2) in both high and low erucic acid mustard lines. Sequence alignment of corresponding FAE1 genes between high and low erucic acid mustard lines identified four substitution type single nucleotide polymorphisms (SNPs) in FAE1.1 and three in FAE1.2. Using the SNuPE method of SNP genotyping, these two genes were mapped to two independent loci that co-segregated with the two QTLs governing the erucic acid trait. Association of wild ( E1E2) and mutant ( e1e2) haplotypes of two FAE1 genes with erucic acid variation in two segregating populations revealed that the e1e1e2e2 genotype identified low erucic acid individuals (<2%) and E1E1E2E2 identified individuals with highest erucic acid content (>40%). The E1e1E2e2 heterozygote was found to be intermediate in phenotype. The applicability of these SNPs in marker-assisted manipulation of the erucic acid trait was verified by genotyping a set of contrasting germplasm of B. juncea belonging to two distinct gene pools (Indian and east European) and other oil-yielding Brassica 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 BC(4)DH 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.