Qtl Mapping And Candidate Genes Analysis For Grain Thickness In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.),one of the most important cereal crops in the world,and more than half of the world’s population feeds on rice.Grain size and shape are characterized by a combination of grain length,grain width and grain thickness,which are quantitative traits.Grain size and shape are not only closely related to rice yield,but also has a great impact on rice quality.Therefore,the comprehensive understanding of the genetic regulation mechanism of grain size and shape is significantly important to the improvement on rice grain yield and quality.Although some QTLs and genes that control grain size and shape have been mapped and cloned,the regulatory network of rice grain size and shape is still unclear.Compared with grain length and grain width genes,there are few grain thickness genes have been reported and cloned in rice.In this study,we conducted a quantitative trait locus(QTL)mapping of grain thickness by a previously constructed high-density genetic maps using 192 recombinant inbred lines.We detected a novel QTL for rice grain thickness and grain width on Chromosome 9,named WTG9.And combined sequence alignment,gene function annotation,expression analysis and haplotype analysis for candidate gene analysis of WTG9.The main results are as follows:1.Base a high density genetic map,we identified a total of 6 QTLs for grain thickness(GT)in three environments,they distributed on chromosomes 4,5,7,9,and each QTL could explain 6.14% ～ 10.21% % of phenotypic variance.Among them,two stable QTLs(q GT5 and q GT7.1)with high heritability reside in the know grain width gene GW5/q SW5 and GL7/GW7.We detected two novels and adjacent QTL for rice grain thickness(q GT9.1 and q GT9.2)on Chromosome 9,they treated as the same QTL and renamed q GT9.2.We found q GT9 in this study overlap with a QTL for grain width(q GW9)on Chromosome 9 of our Previous studys,suggest that this QTL may both control grain width and grain thickness,so we renamed it WTG9(Wide and Thick Grain on Chromosome 9).Coupled with the results of previous studies,WTG9 was narrow down to a 30-kb interval on Chromosome 9 by sequence alignment.Within 30-kb candidate region of WTG9,there are eight predicted genes.Although many nucleotide changes(SNPs and In Dels)were observed among the eight genes in the 30-kb region between parents.According to gene functional annotation,we think that Os09g0535500,which encodes a zinc finger C3HC4-type domain with deubiquitination activity,was predicted to be the best candidate in WTG9.3.By gene expression profiles analysis for eight predicted genes of the mapping region of WTG9,we found that Os09g0535500 was mainly expressed in young panicle and developed grains.Combining gene function annotation and expression profile information,we further verified the expression of candidate genes Os09g0535500 by q RT-PCR.The results showed that the expression of Os09g0535500 was significantly higher in 02428 lines than in YZX lines during the development of the young panicle and grains all stage.4.Based on the 3K Rice SNP information,we performed haplotype analysis for the genomic region and promoter of candidate gene Os09g0535500 through the haplotype module of the RFGB platform.We found major haplotypes based on the SNPs in the genomic region or promoter of Os09g0535500 were significantly correlated with the grain width and thickness.Moreover,we observed different haplotypes unevenly distributed between two domesticated subpopulations(indica and japonica).This suggest that the locus may a target for artificial selection.5.Additionally,based on the key variation site of known grain width genes(GS3,GW5 and GL7),we evaluated the effect of WTG9 on grain width when it carried same genotype of known grain width genes.The result shown that GW5 plays an important role in affecting grain width,and carrying WTG9 wide-grain allele can significantly increase grain width.We also found the parent YZX having rare promoter haplotype in all 3,000 rice accessions(3K rice genome),this suggests that the rare allele of WTG9 carried by YZX is favorable for breeding high quality rice cultivars.In summary,a total of 6 QTLs were identified for rice grain thickness base on a high genetic map,Among them,q GT9 locus has been repeatedly verified in different environments.Using gene annotation and gene expression analysis,we identified a candidate gene,Os09g0535500,which encodes a zinc finger C3HC4-type domain,Further haplotypephenotype association analysis revealed that Os09g0535500 was strongly correlated with grain width and grain thickness.These results lay a foundation for the gene cloning and molecular mechanism of WTG9,and provide new gene resources for breeding for high yield and quality rice cultivars.