Genetic Dissection Of Seed Weight Based On QTL Mapping And Time-series Transcriptomics In B.napus

High yield is always the primary goal of crop breeding.Thousand seed weight(TSW)is one of the important components,and increasing seed weight(SW)is of great significance for breeding high yield rapeseed varieties.Therefore,it is very important to study the genetic mechanism of SW in rapeseed.In this study,based on the KN double-haploid(DH)population of Brassica napus(B.napus),QTL mapping for thousand seed weight(TSW)phenotypes in the winter,semi-winter and spring environments was conducted,and differential gene expression analysis based on time-series transcriptome of high-throughput RNA sequencing were performed to reveal the genetic basis for the formation of SW and identify the important candidate genes that have dramatic impact on SW.The main results are as follows:(1)QTL mapping for TSW and identification of potential candidate genes within QTL regions for TSW in B.napus.TSW of rapeseed is a typical quantitative trait,which can be inherited stably in winter,semi-winter and spring ecological environment.There were 116 identified QTL distributing across the 17 chromosomes except for chromosome A08 and C03.After the integration of QTL,we obtained 13,2,3,2 stably expressed QTL in the winter environment,the spring environment,the winter and the semi-winter environment,the winter and the spring environment,respectively.Among them,QTL cq TSW.A4-1and cq TSW.A4-3 were stably identified repeatedly in three environments.In addition,we identified cq TSW.A7-3,cq TSW.C1-1 and cq TSW.C1-3 as three major QTL for TSW,among which cq TSW.C1-1 and cq TSW.C1-3 were major QTLs newly identified.A total of 594 potential candidate genes within the QTL regions were identified,mainly involving in IKU,ubiquitin-protease,plant hormone,embryogenesis,embryogenic cell elongation,biosynthesis of storage protein and TAG,etc.(2)QTL hot spots of TSW and other traits,and epistatic interaction of TSW in B.napus.Hot spots of QTL for seed yield per plant,oil yield content,oil content and TSW were identified on chromosome A07,C01 and C06,respectively,which indicated that TSW was highly correlated with seed yield per plant and oil yield content.Thus,increasing the TSW could efficiently improve the yield and oil yield in B.napus.In addition,epistatic interaction analysis revealed that epistatic interaction pairs that influenced the TSW of B.napus were mainly distributed between A and C sub-genome(54.18%),among which 130 epistatic interaction pairs were in 51 QTL regions of TSW,implying the complexity of TSW regulation is formed by the interaction between the genomes after the fusion of A genome of B.rapa and C genome of B.oleracea.(3)Twenty days after flowering(DAF),e.g.,S3 stage,is the significant period of seed development and material biosynthesis.Seventeen～twenty-six DAF was the important period of storage material biosynthesis and accumulation.Correlation,hierarchical clustering and principal component analysis of gene expression showed that20-26 DAF(S3～S4 stage)was the most important stage of seed development and material synthesis.Longitudinal analysis showed that DEGs and DETFs had the strongest growth regulation and the highest biochemical metabolism enrichment degree at S3/ S2 stage.Horizontal analysis showed the number of DEGs and DETFs were the largest at 20 DAF(S3stage)and 990 differentially expressed genes closely related to seed growth and storage material synthesis were up-regulated.The S3 stage was a critical period for the storage materials biosynthesis and accumulation,especially the seeds with heavy SW had higher anabolic activity at this stage.(4)In a certain period of time,seed development and the increase of storage material biosynthesis and accumulation time can effectively promote the improvement of SW.The material biosynthesis of heavy seed weight seeds still has advantages in the late stage of seed development.From S3 / S2 stage,heavy SW significantly enriched a large number of biological metabolic pathways.Earlier biosynthesis of heavy SW accumulated more storage materials in seeds,and heavy SW had a strong photosynthetic capacity and material biosynthesis process in S5 / S4 stage.Horizontal GO analysis showed that heavy SW had entered the period of rapid cell proliferation and biosynthesis of lipids,proteins,fiber and lignin,etc,from the S2 stage.Horizontal KEGG analysis showed that heavy SW significantly enriched more metabolic pathways in S3 stage,which promoted more storage material synthesis and accumulation.In addition,heavy SW still had advantages in material synthesis and accumulation in S4 and S5 stages.Three gene coexpression modules in heavy SW and seven gene co-expression modules in light SW showed high correlation with specific stages of seed development.Meanwhile,most gene co-expression modules of heavy and light SW were conserved,but individual gene coexpression module showed specificity between between heavy and light SW materials.(5)Identification of key candidate genes using QTL mapping and time-series transcriptome.Integration of time-series transcriptome and QTL mapping results,a total of 31 common key candidate genes related to seed development,the biosynthesis and accumulation of storage materials were identified.These genes main involved in transcriptional regulation(Bna A02g17180),seed coat formation(Bna A01g25630 D,Bna C04g29170 D,Bna C05g31880 D,Bna A02g23180 D,Bna C06g18870 D and Bna C05g31880D),lipid biosynthesis and metabolism(Bna A02g30470 D and Bna A07g26670D),lipids and storage proteins(Bna C01g18950 D,Bna C01g19300 D,Bna C01g19310 D,Bna C01g19330 D,Bna C04g32530 D and Bna C08g11970D),hormone regulation(Bna C06g36410D),ubiquitin(Bna C08g03250D)and seed development(Bna C08g31760D),etc.In conclusion,QTL mapping and time-series transcriptome techniques were used to reveal the regulation mechanism of SW formation in B.napus and identify common potential candidate genes.These results were fully prepared for the next step of gene cloning and functional verification.Meanwhile,it also provided guidance for revealing the genetic mechanism of seed materials biosynthesis and accumulation and cultivating rapeseed new varieties with heavy SW and high yield.