Identification Of QTL For Grain-releted Traits In Rice Through Linkage Analyses And GWAS And Comprehensive Analyses Of Oryza SPL Gene Family

Rice(Oryza sativa L.)is one of the three major food crops supporting more than 50%of whole population worldwide.The growing global population and the deterioration of the environment pose new challenges to the breeding of high-yielding rice.Rice yield is a complex quantitative agronomic trait.Grain size(grain length,width and thickness)and grain weight are closely related to rice yield.Linkage analysis and association analysis are the mainstream methods of QTL mapping,which have developed rapidly in recent years.Bin map method improves the accuracy and computational power of linkage analysis,while multi-locus genome-wide association analysis has solved the problems of high false-negative rate and inadequate detecting of micro-effective QTLs in single-locus association analysis.Multi-locus genome-wide association analysis is a reasonable method for strictly closed-pollinated crops,such as rice.In order to mine the QTLs related to rice grain traits,genetic map was constructed with the high-generation recombinant inbred lines derived from the cross of indica and japonica,then the QTLs related with rice grain traits were detected by ICIMapping.At the same time,GWAS was performed in 533 rice core collections with five multi-locus genome-wide association analysis methods and identified candidate genes associated with grain width and grain weight.Comparing the results of genetic linkage analysis and association analysis,three co-localization regions were obtained,two of which were coincident with published genes,while qGW-5-1 was a new QTL associated with rice grain size and weight.Finally,SPL gene family which related to rice yield was comprehensively analyzed in the Oryza genus.The main results of this study are shown below:(1)RIL population was constructed through the cross of indica Luohui 9 and japonica RPY.We re-sequenced 270 F11 progenies and constructed a high-density genetic map with 4,758 bin marker and an average marker interval of 0.5 cM.The 1000-grain weight traits of F11 were counted in Lingshui,Hainan,2016 and in Ezhou,Hubei,2017.and four grain-size related traits(grain length,grain width,grain length to width ratio,and grain thickness)were tested in Ezhou,Hubei,2018.The ICIM(Inclusive Composite Interval Mapping)method was used to detect QTL related to target traits.At the threshold of LOD greater than or equal to 2.5,8 and 11 QTLs were identified for grain size and grain weight.Among them,qGW-5-1 and qGLWR-5-1 were detected in the same region.Through further analysis,we suggested LOC Os05g09020(WRKY67),belongs to a member of WRKY gene family,as a candidate gene related with rice grain size and weight.(2)Genome-wide association studies were performed for yield-related traits(grain length,grain width,grain thickness,1000-grain weight and yield per plant)in 533 rice accessions using SL-GWAS(MLM)and five ML-GWAS(mrMLM,FASTmrEMMA,pLARmEB,pKWmEB,ISIS EM-BLASSO)methods.For MLM result,p-value(1.65×10-6)were selected for the suggestive genome-wide significance level,and 161,136,160,189,and 171 SNP markers were detected to be associated with the above five traits,respectively.For ML-GWAS,152,106,12,111 and 64 QTNs were detected associated with the above five traits with LOD greater than or equal to 3.0,respectively.Then,two QTNs,simutanously detected by both SL-GWAS and ML-GWAS,were chosen for further analysis.A total of 12 candidate genes located in the LD block of qKGW7-1 and qGW-9-2.Based on gene expression analysis and previous studies,we suggested that two candidate genes LOC_Os07g31450 and LOC_Os09g0280 were related with grain width and 1000-grain weight,separately.(3)The SPL gene family of two cultivated rice and four wild rice in the Oryza genus AA genome were comprehensively analyzed.A total of 105 genes belonging to SPL family of Oryza were identified,which could be divided into two subfamilies ? and ?,of which ? was further divided into ?A,?B and ?C.Among them,? and ?A have similar structural characteristics,with a large number of exons and no miR156 regulation elements significantly different from other subfamilies.In addition,a total of 19 OsSPL homologous groups were identified,all of which were under negative selection pressure.The structure of one specie in the same group was similar,and there were significant differences among different groups,including gene copy number variation,intron length variation,and whether regulated by miR156.The analysis of cis-acting elements showed that SPL genes were mostly related to photoperiod and phytohormone regulation.And transcriptome analysis indicated that the expression pattern of target gene regulated by miR156 was tissue-specific.The results of linkage analysis and GWAS to mine the QTLs related to grain size and grain weight and the structural characteristics and microevolution of SPL gene family in Oryza AA genome species will help us to further understand the genetic mechanism of rice grain size and grain weight traits,and provide an important basis for molecular improvement of grain traits in rice.