Cloning And Functional Analysis Of Grain Length And Weight Related Genes OsLG3a And OsLG3b In Rice

Rice is one of the world's most important cereal and a staple food crop.It is one of the critical problems to improve rice yield for protecting the food security of China and the world.Most of the agronomic traits of rice are complex and generally controlled by multiple genes.However,traditional linkage analysis detects only genetic variation at which any two parents differ.Although thousands of QTLs on grain size have been mapped in rice,only 11 of them have been cloned and functionally validated.The era of deep-sequencing of vast arrays of germplasm has coming and GWAS has been carried out by vast SNPs.Highly-efficient identification of important genes by skillful combination of GWAS and linkage mapping will play an important role in dissection of genetic architecture of complex traits when entering an era of deep-sequencing of vast numbers accessions.But it is still challenging for GWAS to resolve a single gene because of the low rate of LD decay in rice.Herein,we not only developed a novel method to rapidly identify the genes associated with agronomic traits utilizing large amount of sequence data and the linkage mapped QTL information,but also validated the function of two new gene controlling the grain length in rice.We identified 99 QTLs for grain length by GWAS based on?10 million SNPs from 504 cultivated rice,13 of them were validated by four linkage populations and 92 of them were new loci for grain length.We scanned the Ho(observed heterozygosity per locus)index of coupled-parents of crosses mapping the same QTL,based on linkage and association mapping,and identified three genes for grain length.We named this approach as Ho-LAMap.A simulation study of six known genes showed that Ho-LAMap could mine genes rapidly across a wide range of experimental variables using deep-sequencing data.We used Ho-LAMap to clone a new gene,OsLG3a as a positive regulator of grain length,which could improve rice yield without influencing grain quality.Quantitative PCR analysis indicated that higher expression of OsLG3a is correlated with longer grain.Further analysis showed that OsLG3a alleles in the indica and japonica evolved independently,from distinct ancestors and low nucleotide diversity of OsLG3a in indica indicated artificial selection.Phylogenetic analysis showed that OsLG3a might have much potential value for improvement of grain length in japonica breeding.The results demonstrated that Ho-LAMap is a potential approach for gene discovery and OsLG3 is a promising gene to be utilized in genomic assisted breeding for rice cultivar improvement.We also report the molecular cloning and characterization of a novel middle effect QTL for grain length in tropical japonica rice,qGL3-2,which encodes a MADS-box transcription factor(OsLG3b).A candidate region association analysis showed that six SNPs in OsLG3b,which resulted in an alternative splicing at the last exon,were significantly associated with grain length in rice.A premature stop caused by the alternative splicing increased grain length and weight significantly.Quantitative PCR analysis indicated that OsLG3b expression was higher during panicle and seed development stages than other stages in rice.Haplotype analysis and introgression analysis revealed that the long-grain allele might occurred in after javanica domestication and spread to subspecies indica and temperate japonica from continuous natural crossing and artificial selection.Our combined population genetics and network analyses suggest that OsLG3b likely represents a target of human selection for adaptation to tropical region during rice domestication and/or improvement,demonstrating an important role of middle-effect quantitative trait loci in crop breeding.Phylogenetic analysis and pedigree records showed that OsLG3b had been employed by breeders and had much potential value for improvement of grain length in indica breeding.The results demonstrated that the cloning of OsLG3b might aid efforts to elucidate the molecular basis that control grain development and domestication in rice plants,and could facilitate the genetic improvement of rice yield.