Data Sheet 2_Integration of GWAS and transcriptome analysis to identify temperature-dependent genes involved in germination of rapeseed (Brassica napus L.).xlsx

Low temperature germination (LTG) is one of crucial agronomic traits for field-grown rapeseed in the Yangtze River Basin, where delayed sowing frequently exposes germination to cold stress. Because of its importance, the genetic basis underlying rapeseed germination under different temperatures has been continuously focused. By long-term field observation, we screened out two cultivars with significantly different LTG performance (JY1621 and JY1605) in field and lab conditions, which therefore were further used for the transcriptome sequencings at three key timepoints under normal and low temperatures. Comparative analysis among multiple groups of differentially expressed genes (DEGs) revealed a set of either early or late temperature response germination (ETRG or LTRG) genes, as well as cold-tolerant (CDT) and temperature-insensitive (TPI) candidate regulators at different germination stages. Furthermore, we performed a genome-wide association study (GWAS) using germination index of 273 rapeseed accessions and identified 24 significant loci associated with germination potential under normal temperatures. Through integrated analysis of transcriptome sequencing and GWAS, we identified a series of candidate genes involved in temperature-dependent germination. Based on the comprehensive analysis, we hypothesized that BnaA3.CYP77A4 and BnaA3.NAC078 could be important candidate genes for LTG due to their expression patterns and haplotype distributions. This study performed the multi-omics analysis on temperature-dependent germination and provided potential genetic loci and candidate genes required for robust germination, which could be further considered for low-temperature germination breeding of rapeseed.

低温发芽（Low temperature germination, LTG）是长江流域大田种植油菜的关键农艺性状之一，该区域因播种延后常使发芽阶段遭受低温胁迫。鉴于其重要性，不同温度下油菜发芽的遗传基础始终是学界关注的重点。通过长期大田观测，我们在大田与实验室条件下筛选出两个低温发芽性状差异显著的油菜品种（JY1621与JY1605），随后以二者为材料，在正常与低温条件下的三个关键时间点开展转录组测序。对多组差异表达基因（differentially expressed genes, DEGs）进行比较分析后，我们鉴定出一批低温发芽早期响应（early temperature response germination, ETRG）与晚期响应（late temperature response germination, LTRG）基因，以及不同发芽阶段的耐寒（cold-tolerant, CDT）与温度不敏感（temperature-insensitive, TPI）候选调控因子。此外，我们以273份油菜种质的发芽指数为表型，开展全基因组关联分析（genome-wide association study, GWAS），鉴定出24个与常温下发芽势显著相关的遗传位点。通过转录组测序与GWAS的整合分析，我们鉴定出一系列参与温度依赖型发芽的候选基因。基于综合分析，我们提出假说：BnaA3.CYP77A4与BnaA3.NAC078可作为低温发芽的关键候选基因，其依据为二者的表达模式与单倍型分布。本研究针对温度依赖型发芽开展多组学分析，为油菜健壮发芽提供了潜在的遗传位点与候选基因，可进一步应用于油菜低温发芽性状的遗传育种改良。