| Rice(Oryza sativa L.)is a major cereal crop in Asia,as well as a major model crop for genetic improvement.Large amounts of high-density genotype data and related high-throughput phenotypic data of rice have been accumulated and associated with many important candidate genes through GWAS approaches.The regulation of gene expression plays an important role in the formation of crucial crop traits and environmental adaptation.Transcriptome sequencing can simultaneously identify exon variants and quantify gene expression.Identification of expression quantitative trait loci(eQTLs)that affect gene expression levels is critical to understanding how genomic variations regulate gene expression levels.With the aid of transcriptome data,candidate genes associated with phenotypes can be identified more efficiently,especially for screening new candidate genes whose functional annotations are not yet clear.In this study,we conducted the construction of eQTLs mapping of rice natural populations based on population transcriptome data of 287 cultivated rice varieties with flag leaves at tassel stage,identified hotspot regions of transcriptional regulation on the rice genome,and discovered key regulators of diterpenoid antitoxin synthesis.Combined with the phenotypic data of the rice population,the population transcriptome data were used for candidate gene screening.The relevant findings are as follows:(1)Based on 287 natural population transcriptome data,177,853 high-quality exonic SNPs were identified in the rice population,and the population structure of the rice population was constructed from these high-quality exonic SNPs.At the same time,we constructed the gene co-expression network and resolved the differences in gene expression between indica and japonica rice based on the expression data of the population.(2)Genome-wide eQTL analysis was performed and 44,354 expression trait loci(eQTLs)regulating 13,201 genes(e Genes)were identified.Based on the distribution of eQTLs on the genome,a total of 17 proximally regulated eQTL(local eQTL)hotspots and 96 distally regulated eQTL(distant eQTL)hotspots were identified.Two co-regulatory gene clusters related to 3-phosphoglycerate and oleuropein sterol synthesis were identified in the local eQTL hotspots.Based on the enrichment analysis of distal eQTL hotspots,an important distal eQTL hotspot HS002 was identified,whose downstream genes were enriched in the diterpenoid antitoxin synthesis pathway.The coexpression analysis,eQTL analysis and linkage mapping together verified that b HLH026 was identified as a key regulator in the regulatory hotspot HS002.Biochemical experiments demonstrated that b HLH026 could bind the promoters of downstream diterpenoid antitoxin synthesis-related genes and activate the expression of downstream genes.Transgenic experiments also showed that b HLH026 could both promote the synthesis of diterpenoid antitoxins and their intermediates and enhance the disease resistance of rice.(3)Genome-wide association analysis(GWAS)combining phenotypic data of 287 rice photoprotective parameters(NPQ)with co-localization analysis of eQTLs identified in the population transcriptome subsequently identified a key gene for NPQ regulation,OsPsbS1.OsbZIP72 was identified by yeast one hybrid library screening and expression correlation analysis as a key gene regulating the expression of OsPsbS1.Based on the results of yeast one hybrid and EMSA experiments,OsbZIP72 was confirmed to bind to the promoter region of OsPsbS1 and a new OsbZIP72 binding element GACAGGTG was identified.Transcriptional activation assays demonstrated that OsbZIP72 could activate the expression of OsPsbS1 and that SAPK1,a phosphokinase that interacts with OsbZIP72,could promote the transcriptional activation ability of OsbZIP72.The transgenic experiment also demonstrated that OsbZIP72 could positively regulate the expression of OsPsbS1 to improve the NPQ value in rice.(4)Genome-wide analysis of 529 rice core germplasm materials for four sugars,non-structural carbohydrate(NSC),starch,sucrose and glucose,detected 152 QTL related to sugar content.Four new sugar-related candidate genes were identified by screening SNPs for nonsynonymous mutations.A whole transcriptome association analysis(TWAS)combined with the population transcriptome data identified 31 candidate genes related to sugar in leaves at the tassel stage.Based on the co-localization of sugar GWAS with eQTL of the population transcriptome,four sn-glycerol 3-phosphate synthesis-related genes were identified on chromosome 1 that might be associated with starch content in tasseled leaves.The results of this study demonstrate that eQTL analysis can greatly help to unravel rice transcriptional regulatory processes and screen the key regulators,while study of population transcriptome also contributes to the precise localization of candidate genes by phenotypic GWAS.Moreover,this study also provides a large amount of high-quality data of exonic SNPs and gene expression variants for the study of rice functional genome.These findings will improve our understanding of the regulatory mechanisms of transcriptomic variations and the complex regulatory network of rice genome,and facilitate the genetic improvement of future cultivated rice varieties. |
