| Brassica napus is one of the most important oil crops,and a main source of edible oil in our country.Yellow seeds of rapeseed show more admiring characteristics than their black counterparts: thinner seed coat,higher oil content and protein content,less fibers and polyphenols,lower pigment and clean oil product;therefore,breeding the yellow-seeded lines and varieties is a desirable method for improving the oil and meal quality of rapeseed,and the research on the yellow seed trait being a hotspot of quality breeding in rapeseed.In the present study,fine mapping of the seed coat color gene D was conducted in yellow-and brown-seeded near-isogenic lines(Y-NIL and B-NIL)developed from the black-seeded B.napus line 94570 and the resynthesized yellow-seeded line No.2127-17;the transcript profile and flavonoid content in seed coat at different developmental stages in yellow-and brown-seeded NILs have also been characterized.Our result laid the foundation for cloning the seed coat color gene and provided further clues for elaborating the molecular mechanism for seed coat coloration in B.napus.The main findings in this study were as follows:1.Fine mapping of the seed coat color gene D in Brassica napusIP,SSR and SNP markers were developed from the reference genome sequences of B.rapa and B.napus.The target gene was mapped to a 1.53 c M interval between the SSR markers H2SSR120 and H2SSR237 on linkage group 9,corresponding to a 140 kb region on chromosome A9 of B.napus ‘Darmor-bzh' reference genome.By combining whole genome re-sequencing with BSA,a candidate region for the seed coat color gene was detected on chromosome A9 covering 27.65 Mb-32.73 Mb,overlapped with the interval by genetic linkage mapping.Furthermore,19 Indel markers were developed covering an 830 kb region and the gene D was located between Bn A9ID60-3 and Bn A9ID61-1.The region between these two markers was about 105 kb on chromosome A9 in B.napus.2.Procyanidin content was significantly different in the seed coats of yellow-and brown-seeded B.napusChenimal staining of procyanidins and flavanols in seeds,embryoes and seed coats at different developmental stages indicated that procyanidins were primarily accumulated in the endothelium and the procyanidin content in the seed coats of yellow-seeded line was much lower than that in black/brown-seeded lines at 21 days after flowering till the mature stage.LC-ESI-MS/MS analysis suggested that the relative contents of some key soluble metabolites in proanthocyanidin biosynthesis pathway in the seed coats of yellow-seeded line were lower than those in the black/brown ones.Accumulation of insoluble procyanidins showed significant difference between yellow-and black/brown-seeded B.napus at 21-49 days after flowering.3.Comparative transcriptome analysis of seed coats at five developmental stages in yellow-and brown-seeded NILsComparing the transcript profile of seed coats in yellow-and brown-seeded NILs,totally 4,974 differentially expressed genes were identified at five developmental stages,involving 3,128 up-regulated and 1,835 down-regulated genes in the seed coats of yellow-seeded line.GO function enrichment analysis indicated that the biological process of response to stimulus was enriched in the up-regulated genes,while secondary metabolism process was enriched in the down-regulated genes,especially for phenylpropanoid and flavonoid metabolism.KEGG enrichment analysis showed that seven pathways were significantly enriched with 181 up-regulated genes,most of which were related to response to stimulus,and the top two pathways were plant-pathogen interaction and plant hormone signal transduction.Phenylpropanoid and flavonoid biosynthesis pathways were the representative pathways enriched in down-regulated genes.4.The differential expression of genes involved in flavonoid biosynthesis may be responsible for the difference of seed coat color in NILsThe down-regulation of twelve structural genes(PAL,C4 H,4CL3,CHS,CHI,F3 H,DFR,ANS,ANR,TT12,AHA10,and TT19)and three transcription factor genes(TT1,TT8,and TT16)in the seed coats of yellow-seeded NIL resulted in that procyanidin biosynthesis was blocked,which leaded to the difference of seed coat color in two NILs.These genes exhibited similar expression patterns in seed coats during seed development,indicating that these genes associated with PA biosynthesis may be regulated by an unreported common regulator.Three transcription factor(TF)genes,including one b HLH gene(Bna A09g42390D)and two MYB-related genes(Bna A09g44970 D and Bna A09g44370D)showed similar developmental expression patterns to the key PA biosynthetic genes and they might thus potentially participate in flavonoid biosynthesis regulation.5.The candidate gene for the seed coat color in the mapping regionBy co-linearity analysis,the seed coat color gene D was located in the 105 kb interval on chromosome A9 and in the 339 kb interval on chr A09_random in B.napus,meanwhile the gene was located in a 545 kb region on chromosome A9 of B.rapa.Totally 130 potential genes were found in these regions by BLASTP search against the A.thaliana genome and 36 candidate genes were choosed according to the A.thaliana gene annotation and the transcriptome analysis,some of which were the transcription factor genes or the genes encoding the transporters that might be involved in the flavonoid biosynthesis. |
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