QTL Identification For Rice Grain Shape And Molecular Mechanism Analysis Of OsPPKL3 Involved In Rice Grain Shape Regulation

Rice（Oryza sativa L.）is one of the most important food crops in our country,and increasing and stabilizing its yield is important to our country’s food security.The rice grain yield is mainly determined by three factors:effective number of panicles per unit area,number of filled grains per panicle,and grain weight.The grain weight is determined by grain shape and degree of filling.The grain shape is associated with grain length,width and thickness,which are complex quantitative traits controlled by multiple genes.In addition,grain shape is also closely related to grain appearance quality.Therefore,the research on the identification,localization and regulation mechanism of quantitative trait loci（QTLs）or genes related to grain shape has great significance for the genetic improvement of rice grain yield and appearance quality traits.In this study,a large-grain japonica/geng landrace Suyunuo from Tai Lake Region and a natural population of 241 accessions from all over the world were used as experiment materials.Through the recombination inbred population-based linkage mapping and genome-wide association study（GWAS）,QTLs for rice grain shape were detected and qTGW6/OsGASR7 was cloned by map-based cloning approach;the molecular mechanism of OsPPKL3 in rice grain shape regulation was also studied.The main results are as follows.1.Previously,a large-grain japonica/geng landrace Suyunuo（SYN）from Tai Lake Region,with a thousand-grain weight of about 40 g,was used to cross three relatively smallgrain landraces（with a thousand-grain weight of about 25～28 g）,including Bodao（BD）,Jiangnanwan（JNW）,and Heikezigeng（HKZG）,and three RIL populations were constructed using the method of single seed decent（SSD）:SYN×BD（F2:7）,SYN×HKZG（F2:11）,and SYN×JNW（F2:8）-The correlation analysis of grain length,width and weight in three RIL populations showed that,grain weight in different populations was significantly positively correlated with grain length or grain width.Using the SYN×JNW population and corresponding molecular marker linkage map,QTLs for grain length,width and thousand-grain weight were detected,and 13 QTLs were detected,such as GS3,a major cloned gene for grain length,which was located between the molecular markers RM6959 and RM3646 on chromosome 3;GLW7,a major gene for grain length,which was located between RM3186 and RM346 on chromosome 7;and GS5,a major gene for grain width,which was located between RM1182 and RM3853 on chromosome 5.In addition,two unreported major QTLs for grain shape were detected:one was qGL1 for grain length and weight with a contribution rate of 3.54%and 8.05%respectively,which was located between the molecular markers RM27129 and RM27150 on chromosome 11;the other was qGW5.2 for grain width with a contribution rate of 9.18%,which was located the long arm of chromosome 5.2.Using a RIL population with 124 lines constructed from a cross of Suyunuo and Bodao as experimental material,combined a linkage map（1,939.667 cM）constructed by RAD-Seq,the grain shape/weight-related QTLs were detected through ICIM-ADD algorithm.In three years（2014,2015 and 2017）,a total of five QTLs were detected,of which three were detected in all trials,named qGL3.2,qTGW6 and qGL8.Among them,qTGW6 was located between the molecular markers Chr6₈833151 and Chr6₉125316 on chromosome 6,with a contribution rate of 17.08%,12.41%and 22.66%,respectively,and all the contributions came from Suyunuo.Using the four new developed InDel markers,the interval size of qTGW6 was reduced to 61.56 Kb,and there were four candidate genes in this region.The results of tissue expression analysis showed that,OsGASR7,a GAST（gibberellic acidstimulated transcript）gene family member cloned from the young panicles of Suyunuo and Bodao for sequence analysis,may be a candidate gene for qTGW6.In addition,there were six single nucleotide variation SNP1～6 and one insertion/deletion variation InDel1 between the CDS sequence of OsGASR7BD from Bodao and OsGASR7SYN from Suyunuo.3.Using a natural population composed of 241 rice accessions from all over the world as experimental materials,the grain length,width and thousand-grain weight of rice harvested in Nanjing in 2015 were examined,and genome-wide association analysis was performed by the mixed linear model（MLM）using constructed 700K SNP genetic map.A total of 62 QTLs were detected.In the association analysis of grain length,the-log10（P）-value of SNP6.8909562 was 4.08,and OsGASR7,a candidate gene of qTGW6,was contained within the associated region of SNP-6.8909562.The results of candidate gene association analysis by using rice 3K genome sequencing data showed that,the InDel of 6-bp insertion（AGCAGC）in the exon of OsGASR7 was most related to grain length,which corresponds to the insertion/deletion variation InDel1 between OsGASR7BD and OsGASR7SYN.The transgenic complementation test showed that,introducing OsGASR7SYN from Suyunuo into Bodao and Nipponbare could increase grain length of Bodao and Nipponbare.Altogether,OsGASR7,a member of the GAST gene family,is likely qTGW6.4.The previous study in our laboratory has shown that OsPPKL3 was a highly homologous gene（CDS identity=88%）of OsPPKL1,a major gene for rice grain length and weight.OsPPKL3 was induced by brassinolide（BL）and Brassinazole（BRZ）at rice seedling stage.The bimolecular fluorescence complementation（BiFC）test proved that OsPPKL3 interacted with the rice GSK protein family（the homologous protein of Arabidopsis brassinosteroid signaling pathway regulator BIN2）in tobacco leaf epidermal cells.Using OsPPKL3 as bait protein,the young panicle cDNA library was screened by yeast two-hybrid assay,and OsFBX235,a protein containing F-box domain,was identified.Further analysis revealed that OsFBX235 interacted with the IN+PP2A region of OsPPKL3 in yeast cells.The T-DNA insertion mutant of OsFBX235 exhibits pleiotropic defects including smaller seed size.OsFBX235 was induced by BL and BRZ treatments at rice seedling stage,and osfbx235 mutant was insensitive to BL treatment.Western blot showed that the protein levels of OsBZR1 and GSK2 were reduced in osfbx235 mutant.These results indicate that OsPPKL3 may interact with OsFBX235 in rice and regulate grain size through the BR signaling pathway.