Fine-Mapping And Candidate Gene Analysis Of A Major Seed Number Per Silique QTL-un.A8 In Brassica Napus L.

Rapeseed is one of the main source of edible vegetable oil in China.In recent years,there has been a decline in land area cultivated with rapeseed due to the reduction in cultivated land and the decline in labor force caused by population aging.Therefore,increasing the yield of rapeseed has become the most important problem for rapeseed breeders and researchers.The seed number per silique(SN)is one of the three major components of rapeseed yield,and it is also a product of complex physiological processes such as ovule initiation and ovule/seed development.However,most of the previous studies focused on the SN,while ignoring the research on other related traits in the process of its formation.To explore the genetic mechanism regulating the natural variation of SN and SN-related components,quantitative trait locus(QTL)mapping was conducted using a segregated doubled haploid(DH)population derived by crossing C4-146 and C4-58B,which showed significant differences in SN and aborted ovule number(AON),but no differences were observed in ovule number per ovary(ON).The main results are as follows:1.Construction of genetic map for the DH population:In this study,431 pairs of In Del markers and 46 pairs of SNP markers were designed based on the re-sequencing data of two parents(C4-146 and C4-58B),179 pairs of markers with clear amplified bands and polymorphism between parents were selected to analyze the genotypes of190 families in the DH population.Then,a genetic linkage map consisting of 17 linkage groups and 166 loci covering 684c M of Brassica napus L.genomic region of was constructed.2.Genetic analysis of the SN and its related traits:The SN,ON,silique length(SL)and thousand seed weight(TSW)et al.of parents,reciprocal cross F_1,and their DH populations were measured and analyzed across multiple environments.Under different environments,there is no significant difference in the ON between the parents,but there is a two-fold difference in the SN.The performance of the F₁ reciprocal cross is consistent with the low-SN parent,indicating that the difference in the SN was mainly due to the different degrees of ovule abortion,and the low-SN/more ovules abortion was dominant to the high-SN/few ovules abortions.In addition,high-seeded parents also have longer silique and smaller seed weight.The SL,TSW,and ON of the DH population show normal distribution,but the SN,AON,and percentage of aborted ovules(PAO)show an obvious bimodal distribution,indicating that the SN,AON,and PAO in the DH population was controlled by major genes,while the SL,TSW,and ON were controlled by minor genes.In this population,SN showed the highest broad-sense heritability(0.97),followed by TSW/PAO(0.91),AON(0.85),and SL(0.69),the ON was the lowest(0.66).Trait correlation analysis showed that the SN was significantly negatively correlated with the AON,PAO,and TSW,and significantly positively correlated with the SL,but not with the ON.3.QTL mapping of SN-related traits:a total of 40 SN-related QTLs were identified,and it was integrated into 19 consensus and 3 pleiotropic unique QTLs by meta-analysis in DH population.Among them,a new pleiotropic QTL(un.A8)was detected on the A8 chromosome,which showed the main effect on the SN,AON,and PAO(contribution rate>50%).It also has a greater effect on the TSW and SL.The pleiotropic QTL un.A4 could simultaneously regulate the SN and AON,but the phenotypic variation explained to them was less than 10%.The un.A7 locus could simultaneously control the SN,PAO,TSW,and SL,but the effect was greater on TSW(>10%).In addition,two consistent QTL:cq ON.C6-1 and cq ON.C6-2 controls the ON,among which cq ON.C6-2 was the main QTL(contribution rate>10%).4.Fine mapping of un.A8 locus:The primary mapping interval was narrowed by increasing the marker density.By analyzing the genotypes and phenotypes of 290 lines in the DH population,the un.A8 locus was located between the markers BM1663 and BM1646,spanning 820 kb in the Darmor-bzh genome.Then 16,421 F₂ plants from C4-146×C4-58B were used to further fine map un.A8.After increasing marker density and phenotypic statistics of SN,un.A8 was finally narrowed down to the DNA fragment bounded by BM1668 and BM1672,with an approximate length of 58.5 kb in the ZS11genome.5.Candidate gene analysis of un.A8:the number of corresponding annotated genes in un.A8 candidate interval was different in Darmor-bzh,ZS11,and Shengli,with 6,3,and 4 respectively,of which the corresponding annotated genes in ZS11 and Shengli were included in Darmor-bzh.Besides,expression patterns analysis and comparative sequencing of six annotated genes show that Bna A08g07940D encoding subunit F of photosystem I(PSAF)and Bna A08g07950D encoding zinc transporter 10(ZIP10)were the most likely candidate genes for un.A8 loci.In conclusion,this study identified a major locus un.A8 that affects SN by regulating rapeseed ovule abortion through genetic analysis and QTL mapping and identified the candidate gene through fine mapping.The results of this study lay a solid foundation by revealing the molecular mechanism regulating SN differences in rapeseed and provide theoretical guidance and valuable genetic resources for rapeseed high-yield breeding.