Gene Mapping And Identification Of Candidate Genes Associated With Yellow Seed Color Minor QTL(BnSCA05) Of Brassica Napus L.

Brassica napus is one of the most important oil crops in the world.Its high oil content and high protein content have always been a general concern of rapeseed breeders.Against the same hereditary background,yellow-seeded rape has such advantages as thin seed coat,high protein and oil content compared with black-seeded rape.Therefore,breeding of yellow-seeded B.napus is a hot spot for research.As the yellow-seeded B.napus belongs to typical quantitative character,susceptible to the impact of a variety of factors such as light,temperature,maturity and harvest time.Its specific genetic mechanism is still unclear.Therefore,this study conducted a QTL analysis of the seed coat color of advanced generation recombinant inbred line in a multiple year and site environment using the composite interval mapping.We located a minor QTL locus associated with the seed coat color character on A05 chromosome,and identified and analyzed the candidate genes within the minor QTL interval combined with the re-sequencing,RNA-Seq,qRT-PCR and other technical methods.A total of four candidate genes related to seed coat color were screened.Finally,over-expression vectors and RNAi expression vectors of these candidate genes were constructed,respectively,and the basic biological functions of the candidate genes were verified through genetic transformation of Arabidopsis thaliana and B.napus.The concrete research results are as follows:1 Minor QTL mapping analysis of the seed coat color of B.napusUsing GH06 as the female parent of yellow-seeded rape,and Zhongyou 821 as the male parent of black-seeded rape,the hybrid offspring was consecutively self-crossed by the “single-seed descent method” to obtain the advanced generation recombinant inbred line population.Then using the advanced generation recombinant inbred line as material,we conducted a QTL analysis of the seed coat color in a multiple year and site environment by means of the composite interval mapping method(CIM).A minor QTL locus associated with a stable seed coat color character was detected on A05 chromosome,and the single QTL could account for 1.38%-5.53% phenotypic variation.By mapping the tightly linked SNP marker unto “Darmor-bzh” reference genome of B.napus,it was found that this minor QTL(BnSCA05)corresponded to the 13Mb-16 Mb interval(< 3Mb)on A05 chromosome.2 Screening and identification of candidate genes associated with seed coat colorAccording to the annotation information of the sequenced “Darmor-bzh” genomeof B.napus,it was found that the interval contained a total of 277 annotated genes.Based on the variation of candidate genes in 24 re-sequenced B.napus samples,we screened 40 differential candidate genes with uniform variable sites in different yellow-seeded or black-seeded B.napus samples.Subsequently,the homologous cloning of variable sites in 40 candidate genes was performed for verification using three pairs of yellow-seeded and black-seeded B.napus near-isogenic lines.The results of genetic homologous alignment showed that 4 genes exhibited regular base mutations in multiple pairs of yellow-seeded and black-seeded B.napus samples.BnSCA05-14 exhibited a fragment deletion in a black-seeded sample(B1),and a base mutation in the other two black-seeded samples(B2,B3),which in turn caused a mutation in the amino acid sequence.BnSCA05-17 exhibited a deletion mutation in the 15 th base sequence of the two black-seeded samples(B1,B2).BnSCA05-18 showed consistent sequence in yellow-seeded sample(Y1,Y2,Y3),whereas it displayed mutations in multiple sites in one black-seeded sample(B2).BnSCA05-03 exhibited mutations in multiple loci in two pairs of samples(Y1,Y2,B1,B2).We listed these 4 genes as candidate genes and conducted a preliminary analysis of their related functions.3 Functional analysis of candidate genesIn this study,we successfully constructed over-expression vectors of candidate genes(BnSCA05-03,BnSCA05-14,BnSCA05-17 and BnSCA05-18),and successfully obtained the candidate gene over-expression of T2 generation A.thaliana plant lines through genetic transformation of A.thaliana by the floral dip method.The phenotypic analysis of the transgenic A.thaliana plants revealed that compared with wild-type A.thaliana,the stem epidermis of OVBnSCA05-14,OVBnSCA05-17 and OVBnSCA05-03 strains turned purple,while the stem epidermis of OVBnSCA05-18 strain was consistent with that of the wild type,indicating that BnSCA05-14,BnSCA05-17 and BnSCA05-03 may affect or participate in the flavonoid metabolic pathway.Using the ultra-deep microscope,phenotypic analysis of T2 generation A.thaliana seeds revealed that the seed epidermis of the transgenic lines OVBnSCA05-14,OVBnSCA05-17 and OVBnSCA05-18 became dark compared with that of the wild type,whereas there was no significant change in the seed phenotype of OVBnSCA05-03 line,indicating that the overexpression of BnSCA05-14,BnSCA05-17,and BnSCA05-18 may affect the color of the seed epidermis by participating in the flavonoid metabolic pathway in the seed epidermis.The specific mechanism still needs to be further explored.4 Analysis of the candidate gene promoterUsing the Getway method,we constructed the deletion expression vectors of the candidate gene promoters,respectively,and analyzed the expression specificity and expression intensity of the candidate gene.The results showed that only the full-length promoter of BnSCA05-03 showed a circular blue band in the transiently transformed tobacco leaves after they were stained by GUS histochemical assay.When the promoter fragment was shortened(-353bp),this phenomenon was absent,indicating that BnSCA05-03 promoter(-543bp--353bp)may have a core element that affects the specific expression of the gene in the tissue.Currently,the identification of the original core of the candidate gene promoter is still in progress.5 Genetic transformation of candidate genes of B.napusTo verify the specific biological functions of candidate genes(BnSCA05-14,BnSCA05-17,BnSCA05-18 and BnSCA05-03)in B.napus,the constructed overexpression vectors of candidate genes and the RNAi expression vector were genetically transformed into B.napus(GH30 and Zhongshuang 11)with stable yellow-seeded and black-seeded character expression by means of hypocotyl dip method.The transgenic plants of B.napus are in the stage of seedling differentiation,and the identification of its transgenic plants and functional analysis of candidate genes will be completed in our follow-up study.