Identification Of QTLs And Genes Cloning For Grain-Related Traits In Rice (Oryza Sativa L.)

Rice(Oryza sativa L.)is one of the most important food crops to feed more than half of the world's population,and increasing of rice production and improvement of grain quality are the main objectives of breeding research in rice.Rice grain performance is the key agronomic trait affecting rice production and quality,and the regulation of genetic performance of grain traits is the significant procedure to achieve production and grain-quality improvement.Rice grain-related traits of grain shape,grain weight,and chalkiness are the complex quantitative traits controlled by multiple genes,and easily affected by the environment.In this study,comprehensive investigations of morphology,filling physiology,starch physico-chemical characteristics,and histocytology were performed to dissect the growth and development of rice grain.Using the recombinant inbred lines derived from indica slender-grain germplasm "Yuzhenxiang" and Japonica bold-grain germplasm "02428",high-density genetic map was constructed,and genetic analysis and QTL mapping for grain-related traits were carried out.Combined with gene expression profile,we revealed the gene expression regulation on grain development,and screened the differentially expressed genes(DEGs)used to QTL co-mapping.Subsequently,we conducted cloning and preliminary functional identification of the screened candidate genes.We achieve the construction of genotyping technology system and development of grain-related molecular markers,used in molecular screening of rice germplasm.The main conclusions are as follows:1.Wide range of genetic variation of grain-related traits was detected among tested germplasm.Grain area and perimeter can be usded to reflect adequately the grian shape.Grain circularity has important referential value for evaluation of grain appearance.The grain weight and chalkiness property have the highest coefficient of variation.Indica rice has a wide range of change extent of 13.31~27.22% for AC value and the ASV has highest variation coefficient.RVA values have significant correlation with the grain appearance and physicochemical properties,and AC and GC were the two main indicators affecting RVA profile.The grain shape correlated with the effective grain-filling,active grainfilling period,and incremental grain-filling period,while grain weight was mainly correlated to filling rate related parameters.Significant correlation was detected between PGWC and DEC with active grain-filling period and time of maximum filling rate.Threre was significant difference of enzymatic activity of AGPase,SSS and SBE among different materials,while GBSS was relatively similar.Cytological observations indicated that there were significant differences of hull development,caryopsis cell morphology and structure of starch granule cells of endosperm between YZX and 02428.2.Using the GBS technology,a genetic map with 2,711 Bin marker was constructed based on the mapping population of YZX × 02428 RIL population,with the average genetic distance between markers of 0.86 c M.Survey of population phenotypic traits under 4 environments showed continuous variation pattern separation for various grain traits,most of which were close to the unimodal normal distribution.Environment interaction has greater influence on GW,PL,GL,GW,LWR,and DEC,and the broad-sense heritability ranged from 4.26% to 70.67%.Correlation analysis showed that there was obvious network relationship between GL,GW and chalkiness.Comprehensive QTL analysis under multiple environments was carried out and a number of important QTLs for grain shape(GL,GW,AS,CS,and PL),grain weight and chalkiness characters(PGWC and DEC)were obtained.Among them,major QTLs of GS3,GL7/GW7,gw5/q SW5 and q PGWC-7 were identified repeatly in our study,verifing the reliability of QTL mapping.Fourteen important QTL clusters associated to grain shape or chalkiness were identified with high effect value,which were little sensitive to environment,and ten of which were first reported in this study.3.Gene expression profile sequencing showed that 5,056 significantly DEGs between parents and 668 ones between the two bulk segregant pools(two segregating bulks were made by selecting offspring individuals with extreme value on grain shape and chalkiness).The DEGs between bulk segregant pools mainly enriched in biological process categories of biological control,cellular processes,metabolic processes,stimulating response,and largely enriched in molecular function categories of binding and catalytic activity.These genes involves in the pathways of ribosome,phenylpropanoid biosynthesi,protein processing in endoplasmic reticulum,plant hormone signal transduction,and homologous recombination.Based on BSR analysis,forty-four SNPs highly associated to grain shape and chalkiness in important region with of significant ?SNP-index peak were obtained,which suit for the development of diagnostic molecular markers of grain traits.4.With co-localization of DEGs around the QTL regions,4 major candidate genes were screened: UDP arabinose epimerase of Os UEL-2,bidirectional sugar transporter of Os SWEET3 a,glycosyl hydrolase family gene of Os5bglu20,and non-lysosomal glucosylceramidase of Os NLGase.These genes mainly involved in the pathways of carbohydrate metabolic synthesis.Os UEL-2 was predicted to be associated with grain length;Os SWEET3 a was relevant to grain width or chalkiness;and Os5bglu20 and Os NLGase were associated with chalkiness.Cloning and sequence variation analysis for these andidate genes revealed important SNP mutations.RNA expression analysis show that 4 genes all had higher expression in grain caryopsis,indicating close relation to the grain development regulation.Subcellular localization show that Os UEL-2 protein was localized in the cytoplasm and cell membrane,while Os SWEET3 a exercised its function in the cell membrane.Using genetic transformation,positive transgenic plants of the three candidate genes were obtained and preliminary phenotypic identification was carried out.Os SWEET3 a negatively regulated on grain width probably,and then influenced grain weight.5.With the technology of specific adsorption of nucleic acid by magnetic beads,we developed the high-throughput automated DNA extraction and purification methods.By combination with development of functional molecular markers,a high-efficiency genotyping technology system was achieved.We carried out marker-trait association analysis and breeding application with partial important QTL loci associated to grain shape or chalkiness trait,identifying a number of breeding lines carrying multiple favorable-allele.Overall,this study dissected the genetic performance of rice grain traits;constructed the high-density genetic linkage map based on GBS technology,and identified important QTL loci firstly reported in this study.By combining with gene expression profile,4 candidate genes were primary identified.Moreover,molecular markers for grain triats were developed and applied to breeding practice.These works no only laid the foundation for the genetic and molecular regulation mechanism of giran traits,but also provided the theoretical basis for the molecular breeding utilization in rice.