Excavation And Genetic Improvement Of Grain Shape Genes In Northeast Japonica Super Rice Background

Rice grain shape is closely related to yield and quality traits and has become an important target trait in molecular design breeding for high yield and quality.The key to genetic improvement of grain shape lies in the discovery,innovation and utilization of related variants.So far,remarkable achievements have been made in the research on the genetic basis of grain shape and the mapping and function of grain shape genes in rice.Currently,more and more grain shape related QTLs and cloned genes have been reported,but most of the QTL_S and genes come from mutant materials,which restricts the direct application of breeding to a large extent.With the deepening of related studies,it is difficult to find new genes for natural variation of important agronomic traits,and most of the induced mutants are new functions or new alleles of known genes.On the other hand,the molecular basis of grain morphogenesis and its genetic regulatory network remain unclear.Currently,there is a trend of long-grain breeding in northern japonica rice,but the excavation of grain length genes is relatively weak,which restricts the improvement of grain length.Therefore,it is necessary to mine more grain shape genes,enrich the grain shape regulation network and apply it to the grain shape breeding practice in northeast japonica rice.This research is mainly carried out from the following four aspects:1.The recombinant inbred line population（RIL）constructed by the cross of Shennong 265（SN265）and Huhui 99（R99）was used as the test material.The high-density genetic map constructed by high throughput sequencing contained 3569 Bin markers,the total map distance was 1965.33c M,and the genetic distance of each chromosome was 84.05—217.30 c M.A total of seven major QTL_S regulating grain shape were detected in the three environments.Among them,qGS5 on chromosome 5 was the most important QTL contributing to the grain phenotype,and sequence alignment showed that qGS5 was the cloned grain width gene GW5.The dominant gene qGS9 was located at 15.8-16.6 Mb of chromosome 9 by BSA-seq technique.There were 8 frameshift mutated genes in the region,one of which was the cloned upright panicle gene DEP1.Sequence alignment and gene knockout tests confirmed DEP1 as a candidate gene for qGS9.In japonica Sasanishiki rice,grain length was significantly shortened,plant height and ear length were also significantly reduced after DEP1 was knocked out,suggesting that DEP1 could regulate rice grain length pleiotropy in addition to panicle type upright,grain number per panicle and nitrogen use efficiency.2.The RIL population constructed by Longjing 31（LG31）and Habataki（Haba）hybridizations was used as test material.The high-density genetic map constructed by high-throughput sequencing contained 8366 Bin markers,the total map distance was 2570.11c M,and the genetic distance of each chromosome was 105.35—285.60 c M.A total of 45 major QTLS regulating grain shape were detected in the three environments.Among them,chromosome 5 qGL5-2 was the QTL that contributed the most to grain shape phenotype,which could explain 19.80%,15.80%and 19.38%of grain length variation,respectively,and was named GL5.Further fine mapping targeted GL5 to a 59 kb region with 10 open reading frames（ORF1-10）,which had no reported genes regulating grain shape.Sequencing showed that there was a 1.1kb base deletion at the GL5 site of Haba,which was a structural mutation and could not be detected by second-generation short-read and long-read sequencing.This may be the reason why GL5 has not been cloned.Phenotypic identification showed that the grain length of GL5 knockout mutant increased significantly,suggesting that GL5 was a negative regulator of grain length.The GL5 loci of wild rice,Aus rice and 9 japonica rice varieties were identical to LG31,while 17 out of 21 indica rice varieties were consistent with Haba,suggesting that GL5may be a locus regulating the differentiation of indica japonica subspecies with important agronomic traits3.QTL_S for SLG Grain shape traits were mapped using BSA-seq technique from F₂populations constructed by hybridization of SN265 and Super Large Grain（SLG）.HiFi reads technology was used to assemble high-quality reference genomes of SLGS,and candidate genes were identified by gene annotation and genome sequence alignment analysis.Phenotypic identification showed that the 1000-grain weight of SLG could reach more than 60g,and the epidermal cells of the glume shell extended significantly.Based on the ED and SNP-index association algorithm,the dominant QTL regulating the formation of rice supergrains was located in the 16.4-27.6 Mb region of chromosome 3.By comparing the genomic sequences of SLG and SN265,we found that the cloned plasmid GS3,GL3.1,OsLG3 and OsMADS1 were polymorphic among parents,and four loci of SLG were excellent alleles that increased the length of the plasmid.The analysis of the interaction effect between genes showed that the more alleles the RIL population contained,the higher the phenotype values of grain length and grain weight.The grain size of the polymerized lines with four excellent genes was close to that of the parent SLG,indicating that the super-large phenotype of SLGwas due to the cumulative effect of polymerized multiple excellent genes.4.Based on these results,we propose a strategy to synergically improve rice quality and yield by using CRISPR/Cas9 technology to target G-proteinγ-subunit DEP1 and GS3 to combine the advantages of the upright panicle superior allele dep1 with the long-grain type allele gs3.The results showed that dep1 significantly increased grain number per panicle and yield per plant compared with the wild type,but had negative effects on the appearance quality,texture characteristics and cooking taste quality of rice.gs3 decreased the number of grains per panicle,but had positive effects on appearance quality,texture characteristics,RVA characteristic value and cooking taste quality of rice.Histocytological analysis showed that dep1 inhibited the elongation of the outer epidermal cells of the glume,thus reducing the length to width ratio of grains,and making the arrangement of starch particles more loose and irregular,thus increasing the chalkiness and chalkiness grain rate,while gs3 did the opposite.On this basis,we used CRISPR/Cas9 technique to eliminate GS3 gene in japonica rice variety SN265（with the superior allele dep1）,and investigated the yield and quality characters of the mutant and wild type.The results showed that the 1000-grain weight,yield per plant,appearance quality,cooking taste quality and starch RVA characteristic values of sn265/gs3 mutant were increased.