Fine Mapping And Functional Research Of A BrSCC1 Gene For Seed Coat Color In Brassica Rapa L

Seed coat color is an important agronomic trait in Brassica crops.Compared with brown and black seeds,yellow seeds have higher oil content and less pigments,making them a high quality oil extraction material.In addition,yellow seeds have the advantages of thin seed coats,low fiber,low husk rate and high protein content,making the remaining meal after oil extraction a valuable source of feed.Brassica rapa belongs to Brassica crops,which is a crucial vegetable and oil crop.Breeders have paid much attention to the breeding of yellow seeds in B.rapa.The exploration and identification of genes related to seed coat color have potential application values for Brassica oil crops breeding.In the early stage of this study,a QTL（q SC3.1）was identified on chromosome A03 for controlling seed coat color in B.rapa.In order to further isolate the seed coat color gene,this study used brown seed coat chromosome segment substitution line CSSL-38 containing q SC3.1,yellow seed coat recurrent parent Rc Br as the parents to generate F₂and F_2:3segregating populations,using BSA-seq technology and recombinant analysis,we precisely mapped the candidate gene controlling seed coat color of B.rapa.By integrating gene expression profiling,genome sequence alignment,histochemical staining and metabolomic analysis of seed coats,and functional verification analysis of ectopic expression in Arabidopsis Thaliana,the seed coat color gene Br SCC1 in B.rapa was finally identified.The results are as follows:1.The relative oil content,1000-grain weight,seed coat thickness and husk rate of the two parents were determined.It was found that the oil content of the yellow-seeded parent Rc Br was significantly higher than that of the brown-seeded parent CSSL-38,and the thickness of seed coats and husk ratio were significantly lower than that of the brown-seeded parent CSSL-38,but the 1000-grain weight of the two parents was no significant difference.The Safranin O and Fast Green staining of the paraffin section of the mature seeds from two parents showed that the pigments mainly accumulated in the palisade layer of the seed coat in CSSL-38.2.In order to identify whether proanthocyanidin（PAs）was present in the seed coats of the two parents,the seed coats at different developmental stages were stained with vanillin and DMACA.The results showed that there was no expected staining reaction in the seed coat of both parents at different developmental stages,indicating that brown seed coat does not contain PAs.3.Using UPLC-ESI-MS/MS system for comprehensive targeted metabolome analysis,a total of 829 metabolites were identified in the mature seed coat of Rc Br and CSSL-38,with 164 metabolites showing significant differences between the two parents.Nearly half of the differentially accumulated metabolites belong to phenolic compounds,most of which are up-regulated in CSSL-38,including 15 flavonoids,48phenolic acids,4 lignans,and 9 coumarins.However,no PAs or their oligomers were detected in either parent.These results,combined with the tissue histochemical staining data,suggest that PAs are not related to the formation of brown seed coat in CSSL-38.4.Genetic analysis proved that the yellow seeds of B.rapa was controlled by a single recessive nuclear gene.A 9.2Mb interval located by BSA-seq technology was superimposed with the genome region of q SC3.1,and the 8.4Mb overlapping region associated with the seed coat color gene was identified.The SSR markers were used to screen the recombinants of a small F₂population containing 213 lines,and the seed coat color gene was initially located in the 1.6-Mb interval between markers SC18and SC25.The SSR and Indel markers were further used to screen a large F₂population containing 1665 individuals and a large F_2:3population containing 2475individuals.Finally,The seed coat color gene was located in the 41.1-kb region between Indel055 and Indel057（A03:20,349,640-20,391,718 bp）,which contained nine candidate genes.5.In order to identify seed coat color genes,we first identified the polymorphisms of nine candidate genes CDS regions based on the resequencing data of the two parents,CSSL-38 and Rc Br.A frameshift deletion and 29 non-synonymous SNPs were identified in the CDS regions of six genes among the nine candidate genes.Then,we analyzed the expression levels of these nine genes in the seed coat of the two parents at different developmental stages,and found that only two genes showed significant differences between the two parents.Based on sequence comparison and expression analysis,Bra A03g040800.3C and Bra A03g040840.3C were the most likely candidate genes for controlling seed coat color.6.In order to determine which candidate gene controls seed coat color,the above two genes were ectopic expressed in wild type Arabidopsis thaliana.The results showed that only the Bra A03g040800.3C transgenic lines exhibited a change in seed coat color,which proved that the Bra A03g040800.3C was the seed coat color gene,named Br SCC1,which was positively correlated with pigments accumulation in the seed coats.