Integrating GWAS And Transcriptomics To Identify Candidate Genes Conferring Heat Tolerance At Seedling Stage In Rice

Rice（Oryza sativa L.）is one of the most important food crops in the world.Global warming seriously affects the growth,development and yield of rice.Identifying new loci and favorable alleles associated with heat tolerance is crucial to developing rice heat-tolerant varieties.In this study,we evaluated the heat tolerance at the seedling stage using some 3K rice accessions,and the main research results were as follows.1.In this experiment,620 rice accessions from the 3K Rice Genome Project（3K RG）was used,including 173 Geng,411 Xian,19 admix,7 Aus,9 Basmati and 1 unknown accessions.13-day-old seedlings were exposed to 45℃ for 3 d in a phytotron and then returned to normal conditions（28℃）for 7 d of recovery.The survival rate（SR）and leaf score of heat tolerance（SHT）of rice seedling stage were evaluated,and the results showed that the heat tolerance of GJ-trp and XI-3 was significantly higher than that of other subgroups.A total of 31 SNPs significantly related to heat-tolerant were detected by genome-wide association analysis（GWAS）combining 2802578 single nucleotide polymorphisms（SNPs）.According to LD block analysis,31 SNPS were divided into 6loci.Four loci qHT3.1,qHT3.3,qHT6,qHT7 were identified by SR,and two locus qHT1,qHT3.2 were identified by SHT.Among them,qHT1,qHT3.2 and qHT3.3 are the heat tolerant locus qHTB1,qHTB3-3 and TT2 reported by predecessors,respectively.Among the six detected loci,qHT7 harbored the strongest association signal and the most associated SNPs.By comparing the transcriptomes of two representative accessions with contrasting heat tolerance,LOC＿Os07g48710（Os VQ30）was selected as a promising candidate gene in qHT7 due to the significant difference in its expression level between the two accessions,which was consistent with the results of qRT.2.After high temperature stress,although there was no statistically significant difference in the relative MDA content between heat tolerant material（F64）and sensitive material（PK）,the relative H₂O₂ content of F64 after 72 h of heat stress was significantly lower than that of PK.And,the relative activity of the antioxidant enzymes SOD and POD were significantly higher in F64 than in PK.Moreover,in GO enrichment analysis,F64 and PK heat stress showed significant differences in cell redox homeostasis after 24h,with F64 significantly up-regulated and PK significantly down-regulated.These results suggested that F64 suffered less damage to cell membranes under heat stress than PK,possibly due to more effective active detoxification by ROS scavenging regulation in F64.3.Haplotype 4（Hap4）of LOC＿Os07g48710 was determined as the favorable haplotype for heat tolerance via the gene-based haplotype analysis.The heat-tolerant haplotype LOC＿Os07g48710^Hap4 was highly enriched in the tropical Geng/Japonica accessions,and its frequency has decreased significantly during the improvement process of rice varieties.Based on the GWAS and transcriptomics integrated results,a hypothetical model modulated by qHT7 in response to heat stress was proposed.Our results provide valuable candidate genes for improving rice heat tolerance through molecular breeding.