Construction Of A Physical Map For Chromosome A9 And Cloning And Characterization Of Major Genes In Brassica Juncea

The genome A is shared among Brassica rapa (A A), B. napus (AACC) and B. juncea (AABB). However, research on chromosomal structural variation of A genome in B. napus (AACC) and B. juncea (AABB) during the allopolyploidization is scarce. The chromosome A9 of Brassica carries a number of genes controlling such major traits as seed coat color, seed size, synthesis of glucosinolates which were interested by current rapeseed-mustard breeders. Research on the A9 chromosome will help dissecting the genes controlling these important traits. In this study, genetic and physical mappings were used to construct BAC contig of chromosome A9 in B. juncea and to investigate the structural variation of chromosome A9 in Brassica. Meanwhile, TT8 and TT4, the genes involved in seed color trait were cloned and characterized. The following are the major accomplishments and results of this study.1. A F2 segregating population of Brassica juncea derived from Sichuan Yellow X Purple-leaf Mustard was used to construct a high-density genetic map by Diversity Arrays Technology (DArT). In this genetic map, the linkage group A9 covered 107.9 cM, contained 99 loci consisting of 207 markers. By aligning these informative markers with the published genomic sequences of B. rapa and B. napus, eight conserved genomic blocks were identified and one inversion covering about 2 Mb was observed when compared to B. rapa genome.2. A bacterial artificial chromosome (BAC) contig composed of 188 BACs was constructed by screening genomic BAC library of B. juncea through PCR walking method. This contig covered 16 410 Kb and 14 401 Kb by anchoring the BAC end sequences (BESs) to B. rapa and B. napus A9 pseudomolecule, respectively. A large inversion covering about 4 Mb was observed when compared to B. rapa genome, which corresponded the inversion identified in A9 linkage map of B. juncea. Eleven BACs harboring seed color gene on chromosome A9 were sequenced, and assembled into a pseudomolecule with size of 1052 711 bp in length in which sixty-five genes including the transcription factor gene BjuA.TT8 were annotated. By comparing the homologs in this region of Brassica, twelve gene(s) insertion/deletion were identified. Synonymous nucleotide substitution rate analysis showed that the divergence time of A genome between B.juncea and B. rapa were later than between B. napus and B. rapa, which may result from a slow genetic improvement of B. juncea.3. Allele analysis of TT8 gene in 192 B.juncea accessions collected from 33 countries or regions found four alleles for BjuA.TT8 and two alleles for BjuB.TT8. Simultaneous mutation of both BjuA. TT8 and BjuB. TT8 was only found in oilseed mustard (B. juncea spp. juncea), which implies that yellow seed trait results from artificial selection after the speciation of B. juncea.4. Y1H (Yeast One Hybrid) assays showed that wild-type Bju. TT8 genes interacted with the promoter of the proanthocyanidins biosythetic genes DFR, ANS and BAN, but mutated Bju.TT8 did not, indicating that Bju.TT8s are responsible for activation of transcription of the genes DFR, ANS and BAN in B. juncea and mutation of Bju. TT8s bring about no expression of the genes DFR, ANS and BAN and inhibition of proanthocyanidins biosythesis in seed coat of Brassica species.5. Homology-based cloning strategy was used to clone the TT4 genes for proanthocyanidins biosynthesis in B juncea. Full length sequences of seven TT4 copies were obtained by screening the B. juncea BAC library and sequencing the positive BAC clones. TT4-1～TT4-4 and TT4-5～TT4-7 were shown by BLAST to be located in A- and B-genome of B. juncea, respectively. RNA-seq analysis showed that only TT4-5 is highly expressed in seed coat of B. juncea and displayed expression differential significantly between yellow-and black-seeded ones.In summary, this study discovered that (1) there is an inversion as well as gene deletion /insertion in chromosome A9 of B. juncea; (2)transcription factor genes BjuA.TT8 and BjuB.TT8 controlling seed color have four and two alleles, respectively; and (3)expression of the genes for proanthocyanidin biosynthesis can not be activated by mutated Bju.TT8s. These results have provided a foundation for elucidating structural variation on chromosomes A9 during alloploidization of Brassica, and uncovered the Bju.TT8-regulated molecular mechanism underlying yellow seed in B. juncea.