The Genetic Analysis And Gene Mapping Of Narrow And Rolling Leaf Mutant Nrl4 In Rice(Oryza Sativa L.)

As all we konw,rice is one of the most important cereal and staple foods in the world.The leaves which are the major photosynthetic organs of rice are closely related to the plant type and the grain productivity.Leaf characters including length,width and rolling index can affect the plant morphology and the plant type of rice.Moderate RLI(Rolling Leaf Index)of leaves can contribute to keep the rice blades upright,reduce the extinction coefficient,and provide better chance for the large population to accept the light energy,which can finally enhance the photosynthesis of rice plants.Therefore,the new rolling leaf mutant of rice and the information about the molecular mechanism related to the rice rolling leaf development can help us to breed the new rice variety with higher yield by breeding through improving the leaf characters.In present study,a narrow and rolling leaf mutant 4(nrl4)which was used for the genetic mechanism analysis was selected from an established rice mutant library,the gene mapping and the functional analysis related to the mutant trait studied.The main results were summarized as follow:1.From the seedling stage,all leaves of plant in mutant nrl4 were rolled inward and the width of leaf was shortened.The leaf rolling index(LRI)of the flag leaf,the second leaf and the third leaf from the top was 45%,37%and 32%,respectively.The angles from leaves to stem were all decreased which made the plant type more upright.Besides the narrow and rolling leaf,the plants in mutant nrl4 had some different phenotypes compared with those in the wide-type(WT)of plant such as the increasing sightly height(5.71%),the elongating grain length(7.63%)and shortening grain width(7.67%).The 1000-grain weight of mutant nrl4 was increased by 9.20%,and the grain number per panicle and filled grain number per panicle in mutant nrl4 were 16.16%and 14.99%lower than those in wild type,respectively.Furthermore,the seed setting rate of the mutant nrl4 was promoted by 1.40%compared with that of wide-type.2.F1,crossed mutant nrl4 with Zhenong 34,demonstrated flat leaves and there were 921 rice plants with normal traits and 311 rice plants with narrow and rolling leaves in F2,which leaded to the segregation ratio of F2 population was 3:1.The results of genetic analysis revealed that the mutant agronomic trait was controlled by a single recessive gene,which had not been reported until to now,and the mutant nr 14 was a novel narrow and rolling leaf mutant.3.The leaf color of mutant nrl4 was deepened resulting from the increasing chlorophyll contents in the mutant.The content of chlorophyll a,chlorophyll b and total chlorophyll content in the leaf of mutant nrl4 were 1.99,0.61 and 3.91 mg/g,respectively,which were all increased significantly when compared to those in the wild type Zhenong 34.4.When compared with the wild type Zhenong 34,the cellulose content and the hemicellulose content of nrl4 were 22.39 mg/g and 31.17 mg/g respectively,which were lower than the the cellulose content(22.81 mg/g)and the hemicellulose content(31.82 mg/g)in Zhenong 34,although the differences were small.The lignin content of mutant nrl4 was 3.31 mg/g and was decreased by 11.73%compare with that of Zhenong 34,which could reduce the strength of cell wall of mutant nrl4.5.Total RNA extracted from the flag leaves of the mutant nrl4 and WT at the tillering stage was used for real-time qPCR of the genes related to the rolling leaf including nal1,ACL1,OsAG07,rl14,nal7,sill and ROC5.The results showed that the expression levels of genes nal1,ACL1,rl14 and sill were higher in mutant than those in wild type,while the expression levels of OsAGO7,nal7 and ROC5 were decreased in mutant nrl4 which nal7 was dramatically reduced.6.The gene nrl4 was targeted in a confined region of 53 kb flanked by two InDel makers 3M11103 and 3M1115 on the long arm of chromosome 3,where five annotated genes were predicted,including LOC_Os03g19760,LOC_Os03g19770,LOC_Os03g19780,LOC_Os03g19800 and LOC_Os03g19820.Based on the result of sequencing and analysis,there was no mutation occurred in the gene sequence,promoter sequence of 5’-UTR and 3’-UTR sequence of these predicted genes,but strong changes in gene expression pattern of LOC_Os03g19770 according to the real time quantitative PCR results.LOC_Os03g19770 encoded a glycine-rich cell wall protein consisting of cell wall and found in sclerenchyma cells and maintaining the mechanical strength of plants.The absence of sclerenchymatous cells in the abaxial side of leaves might account for the rolled leaf phenotype,which reported before.Therefore,we speculated it as the candidate gene related to the narrow and rolling leaf mutant 4(nrl4).