Positional Cloning of the Gene for Seed Color and Molecular Mechanism of Yellow Seed Formation in Brassica Juncea
|School||Hunan Agricultural University|
|Course||Crop Genetics and Breeding|
|Keywords||Brassica juncea seed coat color positional cloning BAC contig TT8 transcriptome|
Extensive studies have shown that yellow Brassica seeds have many quality advantages over their dark-seeded counterparts, for examples, thinner seed coat, higher oil and protein content, lower fiber content. Therefore, it has been long one of the most important breeding goals to breed yellow-seeded cultivars for rapeseed breeders at home and abroad.Cultivation of oilseed rapa in China mainly includes three species, that is, Brassica juncea, B. napus and B. rapa. Brassica juncea （2n=36, AABB） is derived from interspecific hybridization between its two ancestral diploid species B. rapa （2n=20, AA） and B. nigra （2n=16, BB）. B. juncea has a rich germplasm with superior characters such as yellow seeds, drought resistant, high temperature resistant, early maturing and shattering resistance, In B. juncea many spontaneous yellow-seeded accessions have been found and the yellow-seeded trait is found to be controlled by two duplicated loci in many cases. Our previous studies have mapped one locus, i.e., BjuA.YSC to a0.9-cM region of the chromosome A09. In this study, a large BC8F2population was developed for fine mapping BjuA. YSC, the DNA markers tightly or completely linked to BjuA. YSC used to screen the BAC library constructed using the B. juncea variety Ziyejie, the sequenced BACs annotated to identify the candidate gene for BjuA. YSC, and the seed coats from B. juncea variety Sichuan Yellow and its brown-seeded near-isogenic line （NIL） A used for analysis by DNA chip and RNA-seq to reveal the difference in gene expression between yellow-and black/brown-seed coats. The following major results were obtained.1A total of316markers were used to detect polymorphism between the mapping parents and NILs. The primers of these markers were designed on the basis of the B.rapa genomic sequences and Korean sequenced BACs in the public databases as well as introduced from the publications on mapping in Brassica species. Among these screened markers, thirty one were found to be linked to BjuA. YSC. Nineteen markers were further used for analysis of1042BC8F2individual plants. As a result, a regional genetic map covering4.77cM in length was constructed. BjuA. YSC was defined to a0.30-cM region with the flanking markers Na10AO8, BnGMS351and the three co-segregating markers S88-Ⅳ-2, H032N11-2and S121-Ⅰ-2being found.2The additional forty six primer pairs, which were developed from B.rapa genomic sequences and the sequenced B.juncea BACs or BAC ends, were used for fine mapping in a large segregating population consisting of5434BC8F2individual plants. The twelve were shown to be linked, with the co-segregating markers68N6, S88-IV-2, H032N11-2, and132O01R and the flanking markers S134-16and S102-5each0.04cM away being found. Finally, BjuA. YSC has been delimited to a0.08-cM region on the chromosome A09.3The B. juncea BAC library constructed from the mapping parent Purple-leaf Mustard （Ziyejie） was screened by four-dimensional PCR procedure using the co-segregating and the flanking markers. A1-Mb BAC contig has been constructed around the yellow seed locus of the chromosome A09of were used for annotation. BLAST of B. juncea BAC sequencs against the Arabidopsis genome found66Arabidopsis homologoue genes out of which the Gene24homologous to At4g09820（TRANSPARENT TESTA8, TT8）, a transcription factor gene involved in the flavonoid biosynthetic pathway, was inferred as a candidate gene for BjuA. YSC. The features in sequenced B. juncea BACs and ratio of genetic to physical distance suggest that BjuA. YSC be located in the peri-centromeric region of the chromosome A09.4The two TT8copies, designated as BjuA. TT8and BjuB. TT8, were amplified from both parents of the mapping population using the primers designed from the sequenced B juncea BACs. BjuA.TT8was located on the chromosome A09while BjuB.TT8was speculated to be from the B03chromosome of B. juncea. BjuA. TT8had an additional1275-bp-long insertion in the yellow-seeded parent SY as well as SNP variation although BjuB.TT8had no difference between the yellow and black-seeded parents. Digestion of RT-PCR amplicons by restriction enzymes BanⅠ, BsuRⅠ and SacⅡ showed that insertion in the seventh exon brought about premature termination of BjuA.TT8transcription.5Twenty five genes and1304unigenes including the genes for flavonoid biosynthesis pathway were found to be differentially expressed by DNA array and RNA-seq of the seed coats15days after pollination from the yellow-seeded parent SY and its brown-seeded NIL A, respectively. qRT-PCR confirmed that these genes for flavonoid biosynthesis pathway such as CHS, DFR, LDOX and ANR were not expressed at all or significantly down-regulated in the seed coat of SY, indicating that these genes were involved in seed color formation in B. juncea.Taken together, insertional mutation in the seventh exon in the yellow-seeded parent SY of Brassica juncea brings about premature transcription termination of BjuA.TT8by which the bHLH transcription factor encoded has defect and can not normally participate in formation of the BMW transcription factor complex, which might be caused no expression of the genes for proanthocyanidin biosynthesis and correspondingly no accumulation of proanthocyanidins in the seed coat. As a result, the seed coat becomes transparent, the yellow embryo is visible, and the seed looks yellow.