QTL Dissection Of Rice Grain Shape,and Cloning And Functional Anylasis Of GL3.3,a Gene Conferring Grain Length In Rice

Rice is one of the most important crops worldwide.To improve rice yield and grain quality is the main task of rice breeders.Grain shape,a trait which both influences grain yield and grain quality,has become the focus of more and more researchers.Grain shape,which includes grain length,grain width,grain thickness and length-to-width ratio will directly influence grain weight and eventually influence rice yield.Besides,grain shape is an important component of rice appearance quality and will also affect the milling quality.Hence,studying the genetic and molecular basis of grain shape will be beneficial for the breeding of rice varieties with both high yield and good quality.Our work is composed of two parts.Firstly,we carried out QTL detection for grain shape using an BC₃F₁ population derived from Jin23B and Qingguai and detected 10 grain shape related QTLs,three of which were detected continuously in two years,and QTL effect validation revealed that these three QTLs were novels QTLs that have not been cloned yet.Secondly,we cloned a grain length QTL,qGL3.3,which was located at the long arm end of chromosome 3,through positional cloning and studied the function of this gene.The main results are shown below:1.We measured the grain shape related traits of BC₃F₂ and BC₃F_2:3 backcross population and carried out QTL detection of these tratits.Totally ten QTLs for grain shape were detected in the backcross population.Three QTLs namely qGW1 located on chromosome 1,qGS3 located on chromosome 3 and qGS7 located n chromosome7 were detected continuously in two years.We developed near-isogenic lines for each of these QTLs and validated the effects of these QTLs,and confirmed that these were genes regulating grain shape on these sites.2.We detected a grain length QTL qGL3.3,which was located on the long arm of chromosome 3,using a recombinant inbred line derived from the cross between Zhenshan97B?ZS97?and Nanyangzhan?NYZ?.The allele from NYZ increased grain length by 0.556mm and explained 11.51%of the phenotypic variance in the RILpopulation.We developped near isogenic lines?NIL?of qGL3.3 and validated the effect of this QTL.3.We screended recombinants and performed progeny tests for qGL3.3 and further mapped qGL3.3 to a 15kb region.In this region,there are two annotated genes,LOC_Os03g62500 and LOC_Os03g62510.Comparing the nucleotides sequence of this region in two parents,we found a G to A variation between ZS97B and NYZ in LOC_Os03g62500,which would cause the disruption of splicing and led to a smaller coding product.We further put this gene as the candidate gene for qGL3.3.4.Knocking out of this gene in ZS97 will increase gain length and over expression of NYZ allele will also enhance grain length.Complementation of ZS97allele in NYZ will recover the short grain phenotype.These results indicated that LOC_Os03g62500 was exactly GL3.3 and it was a negative regulator of grain length.The G to A mutation in NYZ is the functional mutation.5.Real-time PCR and GUS staining of GL3.3 revealed that GL3.3 was expressed in multiple organs.Subcellular localization analysis showed that GL3.3 was located on the nuclus.Scanning electron microscope analysis of the outer surface of the lemma showed that GL3.3 might positively regulate cell division and negatively regulate cell elongation.6.Analyzing the GL3.3 sequence in diverse Oryza sativa accessions revealed that the G to A mutation in NYZ was a rare mutation and only existed in tropical japonica.The polymorphism of the nucleotides flanking GL3.3 showed that GL3.3might have been selected during breeding process.We also found that the tropical japonica with NYZ allele all carried a gs3 allele.And further analysis revealed a long-distance linkage disequibruim between GL3.3 and GS3.Besides,we also noticed that the effect of GL3.3 was much bigger in the background of NYZ than that in ZS97.By analyzing the grain length of four different genotype combinations composed of GL3.3 and GS3 in RIL population,we found that there was an epistatical interaction between GL3.3 and GS3.7.We investigated the yield related traits in NILs and found that the grain length and grain weight are significantly increased in NIL-NYZ.No obvious difference was observed for grain width,plant height and panicle length,and a slight decrease of grain number per panicle was observed,but no difference was observed for grain yield.These results show that GL3.3 can be utilized to improve grain shape without influencing grain yield.In conclusion,we detected a few novel grain shape QTLs through linkage analysis and successfully cloned a grain length gene,GL3.3.GL3.3 negatively regulates grain length and grain weight and does not participate in BR pathway.GL3.3 is epistatically interacted with rice major grain length gene,GS3,and explained the formation of extra-long grains.The gene cloned in this research can be used to improve grain shape without affecting yield and can also provide theoretical basis for the breeding of super-long grain varieties.