Genetic Dissection Of Oil Content Based On Dynamic QTL And Time-Series Transcriptome During Seed Development In Brassica Napus

Rapeseed is one of the most important oil crops,and rapeseed oil is an important source of high quality edible oils and clean fuels.With the increasing demand for vegetable oil in China in recent years,it has important scientific and practical significance to increase the oil content per unit area of rapeseed.To understanding the genetic mechanism of oil synthesis and metabolism is essential for increasing oil content and improving oil quality.Oil content(OC)is a complex quantitative trait controlled by multiple genes,and is susceptible to environment.At present,there are relatively few studies on the genetic analysis of OC in Brassica napus L.Therefore,dissecting the genetic mechanism of oil accumulation and identified key candidate genes are of great significance for improving OC.In this study,a high-density genetic linkage map was constructed using 300 lines of the KN DH population.Quantitative trait loci(QTL)were identified for OC and protein content(PC)in mature seeds,and the competition mechanism of oil and protein were analyzed.At the same time,the dynamic QTL mapping of OC and fatty acid compositions(FAs)during seed development was carried out.Finally,the genetic mechanism of OC was analyzed in combination with the transcriptome based on two pools constructed with high oil material and low oil material respectively,and some important candidate genes were identified.The main findings are as follows:(1)Construction of high-density genetic linkage maps.The 348 DH lines of the KN population were genotyped using a Brassica 60 K SNP array,and 300 high-quality genotyped DH lines were used to construct a high-density genetic linkage map.The map contains 3207 molecular markers,including 3106 SNP-bins and 101 non-SNP markers.The genetic linkage map covers 3072.7 c M of B.napus genome(the length of the A and C genomes is 1398.6 c M and 1674.1 c M,respectively),and the average genetic distance between adjacent markers is only 0.96 c M.This map is a high-density genetic map based on SNP currently constructed by the largest population in B.napus.(2)QTL mapping and candidate gene analysis for OC and PC in mature seeds.Correlation analysis showed a significant negative correlation between OC and PC.QTL mapping was performed for OC in 12 environments and PC in 11 environments using high-density genetic linkage map.164 and 68 identified QTL were detected for OC and PC,respectively.By meta-analysis,67 consensus QTL for OC and 38 consensus QTL for PC were obtained,of which 6 and 4 major QTL were identified for OC and PC,respectively.Among the six major OC-QTL,cq OC-A2-3,cq OC-A9-10,cq OC-C5-3,cq OC-C5-4 and cq OC-C6-6 were novel and fristly discovered in KN population.Eleven unique QTL which control OC and PC at the same time with opposite additive effects,and the hot-spots of OC and PC-QTL with opposite additive effects on C3 and C5,reveal that the genetic basis of competition mechanism beteem OC and PC includes both pleiotropy and close linkage.BSA analysis was performed to further verify QTL detected by high-density map.38 AGRs overlapped with QTL,which proved the high efficiency and accuracy of high-density map for QTL identification.Based on colinearity relationship between genetic map and physical map,448 acyl lipid-related and 11 storage protein-related candidate genes were identified in the confidence intervals of OC and PC-QTL,respectively.A total metabolic network map involving multiple primary carbon metabolic pathways and an interaction network were constructed based on potential candidate genes underlying OC and PC-QTL,which provided clues for the complex effects of other metabolic pathways on OC.(3)Dynamic QTL analysis for OC and FAs during seed development.The accumulation pattern of OC and FAs during seed development showed that the parent Ken-C8 with low OC entered the rapid oil accumulation stage earlier,and the high oil parent N53-2 lasted longer in the rapid oil accumulation stage,and the oil accumulation pattern of the two parents is obviously affected by the environment.There is a more significant difference in the accumulation pattern of FAs in the two parents.Typically,in Ken-C8,C18:1 continued to grow,while N53-2 had higher erucic acid synthesis capacity,and the growth of C22:1 was accompanied by significant decrease of C18:1.A total of 448 unconditional QTL and 397 conditional QTL were identified by dynamic QTL analysis for OC and 10 FAs.Among them,45 unconditional and 27 conditional QTL for OC.Through co-localization analysis,36 pairs of unconditional and conditional QTL were found to be co-localized;45 unconditional and 33 conditional QTL were co-localized with corresponding traits in mature seeds;32 unconditional and 20 conditional QTL for OC were detected to co-localize with unconditional and conditional QTL for FAs,respectively.The results provided new clues for dissecting oil accumulation patterns and identifying candidate genes in B.napus.(4)Time series transcriptome analysis during seed development baed on high and low oil bulk.The RNA-Seq analysis for extreme lines with high and low OC from KN population in 6 satges during seed development revealed the temporal characteristics of oil accumulation regulation during seed development.Analysis of gene expression correlation showed that materials with low OC entered maturity earlier.Longitudinal comparisons between adjacent stages of seed development revealed that photosynthesis and lipid metabolism related pathways were activated from 17 th day to 24 th day after flowering.In the horizontal comparison between H and L in 6 periods,a total of 1926 differentially expressed genes were identified,and the difference in gene expression between H and L was most significant at 42 th day after flowering.By integrating RNA-Seq,dynamic OC-QTL during seed development and OC-QTL detected in at mature seed,some key differentially expressed candidate genes for further functional analysis were identified: Bna C02g39140 D,Bna A08.SESA4,Bna A03g30740 D,Bna A03.FIB,Bna A06.LCR69,Bna C03.TT4,Bna A03.MFP2,Bna A09.CAC2 and Bna A02.KASI.In conclusion,a high-density genetic linkage map was constructed.Through QTL mapping and dynamic QTL mapping for OC and related traits in mature seeds and during seed development,the mechanism of oil synthesis and regulation was deeply analyzed,and some key candidate genes were identified.The results laid the foundation for revealing the mechanism of oil accumulation and cultivating new varieties with high OC in B.napus.