Positional Cloning And Preliminary Function Analysis Of Grain Width Quantitative Trait Loci QGW12 In Rice

Rice is one of the main food crops,and increasing the yield per unit area of rice is an important goal of rice breeding.The yield per unit area of rice is composed of many factors,including the number of plants per mu,the number of ears per plant,the number of grains per ear,and the weight of thousand kernels..These agronomic traits are mainly controlled by quantitative traive loci（QTLs）,in which the increase in grain weight is beneficial to directly increase rice yield,but the composition of grain weight traits is more complicated,mainly due to the degree of grain bank storage size composition.Therefore,increasing the size of the storage capacity（that is,increasing the grain shape traits such as grain length,grain width,and grain thickness）can effectively increase the grain weight of rice and thus increase rice yield.（1）In this experiment,’9311’（genetic background）and common wild rice（insert）were used to construct 199 chromosome fragment substitution lines.Each family examined the agronomic traits such as thousand-grain weight,grain length,grain width and aspect ratio..It was found that the traits of grain width were relatively stable,and one of the strains Q187 had a significant difference in grain width compared with ’9311’ and could be stably inherited.The genotype analysis found that the background of Q187 is more complex,so NIL-Ruf and its sister line NIL-9311 and their descendants were screened with a cleaner background for location research.Using the secondary population,the LOD value was 20.6,and the contribution rate was 46.1%.9311 carried favorable genes that increased grain width.Further,using 2497 single plants and 18 pairs of good polymorphic primers,QTL qGW12 was located in the 24Kb region on chromosome 12,containing a gene,and the key exchange single plant offspring was used for verification.（2）qGW12 encodes a U-Box type E3 ubiquitin ligase,which is more conserved in plants.In NIL-Ruf,there is a single-base mutation in qGW12 that causes asparagine to become serine,and there are also 21 SNPs and two deletions in the promoter.Scanning electron microscopy and paraffin section observations showed that qGW12 had no significant effect on the cell size of spikelet spike hulls and had a greater influence on the number of cells,indicating that qGW12 mainly affected the grain width by affecting the cell number of spikelet spike hulls.By measuring the expression level of qGW12 in spikelets,it was found that the expression level of qGW12 in NIL-Ruf increased compared with NIL-9311,indicating that qGW12 negatively regulates the grain width.The transgenic knockout plants of the japonica rice variety Nipponbare（NIP）found that the qGW12-Cas9 plants had an increased seed phenotype.（3）Through subcellular localization observation,qGW12 protein is localized in the cytoplasm.Quantitative discovery of cell cycle-related genes showed that compared with NIL-9311,the expression levels of genes related to MCM family proteins and E2F family proteins in NIL-Ruf decreased significantly.This indicates that qGW12 may be involved in the replication process of meiotic DNA.