Cloning And Functional Analysis Of Rice Grain Shape Gene GS2.1

High yield,high quality and high efficiency are important goals for rice breeding.Rice grain traits which include grain length,grain width,grain thickness,play an important role in rice yield,quality formation,and efficient seed production.The grain shape directly determines the grain weight,which in turn affects the yield.The grain shape also determines the rice quality indicators such as appearance and commercial traits.The development of sterile and restorer lines with significant difference in grain shape traits and suitable for mechanical separation and processing is the key to the seed production technology of mixed-sowing hybrid rice,which can greatly increase the benefits of hybrid rice seed production.Therefore,the cloning of rice grain-associated genes and the analysis of the molecular regulation mechanism provided the theoretical and technological basis for breeding rice varieties with different grain shape through molecular breeding.In this study,a grain size related QTL GS2.1 gene was isolated with map-based cloning technology.The gene of GS2.1,which was verified through CRISPR/Cas9 gene editing techniques and genetic complementation experiments,is of great significance to analyze the genetic mechanism of grain shape of rice grains and to improve rice varieties with different grain shape.The main results are as follows:?1?The large-grain near-isogenic line BC₃F₄ population,which was constructed with Nipponbare as the recipient parent and the Tedaxian as the donor parent,was used for fine mapping.29 new InDel marker were polymorphic between the recipient parent and the large grain NIL.Finally,the GS2.1 gene was fine mapped in the 160.6 kb physical region between the Indel markers 2M-8-1 and 2M-9 on chromosome 2.?2?According to Nipponbare reference gene sequence,annotation and prediction of the coding region of the GS2.1 fine mapping interval revealed that the GS2.1 region encodes 11structural genes?ORF1-11?.Sequence analysis revealed that there were 27 SNP and 6 InDel difference in the ORF6 promoter region between the NPB and the NIL.14 bp deletion in promoter,and two SNP mutations in the coding region resulting in two amino acid mutations,were found in the large-grained gene.Therefore,ORF6 was used as a candidate gene for GS2.1 for further functional identification.?3?A large-grained genomic DNA complementary vector and a CRISPR/Cas9 gene editing vector were constructed to transform small-grain Nipponbare and large-grain near-isogenic lines,respectively.Currently,Nipponbare background GS2.1 knockout transgenic lines have been obtained.The six homozygous mutants,which showed a smaller grain size,demonstrated that the candidate gene GS2.1 was involved in controlling the grain size.?4?The analysis of transcript expression patterns of GS2.1 gene indicated that the expression of gene in large-grain NIL spikes was significantly higher than that of Nipponbare.