2 2 5The Brassica genus contains a diverse range of oilseed and vegetable crops important for human nutrition 1 . Crops of particular agricultural importance include three diploid species, Brassica rapa (AA), Brassica nigra (BB) and Brassica oleracea (CC), and three allopolyploid species, B. napus (AACC), B. juncea (AABB) and Brassica carinata (BBCC). The evolutionary relationships among these Brassica species are described by what is called the 'triangle of U' model 2 , which proposes how the genomes of the three ancestral Brassica species, B. rapa, B. nigra and Brassica oleracae, combined to give rise to the allopolyploid species of this genus. B. juncea formed by hybridization between the diploid ancestors of B. rapa and B. nigra, followed by spontaneous chromosome doubling. Subsequent diversifying selection then gave rise to the vegetable-and oil-use subvarieties of B. juncea. These subvarieties include vegetable and oilseed mustard in China, oilseed crops in India, canola crops in Canada and Australia, and condiment crops in Europe and other regions 3 . Cultivation of B. juncea began in China about 6,000 to 7,000 years ago 4 , and flourished in India from 2,300 BC onward 5 .The genomes of B. rapa, B. oleracea and their allopolyploid offspring B. napus have been published recently [6][7][8] , and are often used to explain genome evolution in angiosperms [6][7][8] . The genomes of all Brassica species underwent a lineage-specific whole-genome triplication 6,7,9 , followed by diploidization that involved substantial genome reshuffling and gene losses 6,10-13 . In general, plant genomes are typically repetitive, polyploid and heterozygous, which complicates genome assembly 14 . The short read lengths of next-generation sequencing hinder assembly through complex regions, and fragmented draft and reference genomes usually lack skewed (G+C)-content sequences and repetitive intergenic sequences. Furthermore, in allopolyploid species, homoeolog expression dominance or bias, and specifically differential homoelog gene expression, has often been detected, for instance in Gossypium [15][16][17] Triticum 18,19 and Arabidopsis 20,21 , but the role of this phenomenon in selection for phenotypic traits remains mechanistically mysterious 22 .We reported here the draft genomes of an allopolyploid, B. juncea var. tumida, constructed by de novo assembly using shotgun reads, single-molecule long reads (PacBio sequencing), genomic (optical) mapping (BioNano sequencing) and genetic mapping, serving to resolve complicated allopolyploid genomes. The multiuse allopolyploid B. juncea genome offers a distinctive model to study the underlying genomic basis for selection in breeding improvement. These findings place this work into the broader context of plant breeding, highlighting The Brassica genus encompasses three diploid and three allopolyploid genomes, but a clear understanding of the evolution of agriculturally important traits via polyploidy is lacking. We assembled an allopolyploid Brassica juncea genome by shotgun and single-m...

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.

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T 1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.

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.