Genetic Analysis And Fine Mapping Of Two Leaf Shape Genes In Rice(Oryza Sativa L.)

Rice?Oryza sativa L.?is a vital food crop in the world with a long history of cultivation.As the main vegetative organ of photosynthesis and respiration,the morphological structure of leaves affects a series of growth and development of the plants.Screening and identification of new leaf shape mutants can lay the foundation for studying leaf development mechanism and constructing ideal leaf morphology,which has important theoretical and application value for improving yield and rice quality in plant type breeding.A novel rice mutant named narrow leaf 12?nal12?was identified from an EMS?ethyl methane sulphonate?mutagenized population of japonica rice Wuyunjing 31 in this study.The phenotypic data of plant were measured,and the histological section and microscopic observation were conducted.The F₂ segregating population was constructed from a cross between nal12 and indica cultivar Taizhongbendi1?TN1?,and 1709narrow-leaf individuals we selected for fine mapping of NAL12 with existing SSR,STS and newly developed molecular markers.The nal12 showed narrow leaves at seedling stage,and exhibited dwarf,increased tillers and thinner stems at maturity stage.According to the anatomy observation of tissue sections,the narrow leaf phenotype of nal12 is caused by the decrease in the number of large veins and small veins compared with wild type.Genetic analysis indicated that the narrow leaf trait was regulated by a single recessive nuclear gene,and NAL12 was finally mapped to the range of 64.7 kb between the LC2-RF37 and LC4R-RF39 marker on chromosome 10.A total of 10 ORFs?open reading frames?were found and there are no narrow-leaf related genes was reported in this region.qRT-PCR analysis revealed that the expression level of auxin synthesis and transport-related genes were affected in nal12.After treatment with exogenous brassinolide?BR?,the leaf inclination angle of wild type?WT?and nal12 were both increased,but nal12 is larger than WT.NAL12 is a new leaf-shape regulatory gene with pleiotropy effect.The study laid foundation for further cloning of the gene NAL12 and enriching its molecular regulatory network.A pair of recombinant inbred lines?RILs?contained 149 lines were derived from a cross between tropical japonica cultivar Jamaica and indica cultivar TN1,and the leaf width and leaf length phenotypic values of flag leaf to third leaf were obtained.Using the specific-locus amplified fragment sequencing technology?SLAF-seq?,we developed SNP markers on the Large-Scale and genotyping it to build a High-density genetic linkage map.Finally,QTLs underlying leaf morphology were detected by Qgene software.The leaf width and leaf length value of parents and RILs showed large differences and continuous variation of normal distribution,which met the requirements of QTL mapping.We successfully constructed a high-density genetic linkage map containing 8,361 SLAF markers evenly distributed on 12 linkage groups,total map distance was 1661.34 cM with an average distance between adjacent markers of 0.20 cM.A total of 32 QTLs were detected,and the major QTL qLW4 was located on chromosome 4,which coincides with the position of the Narrow Leaf 1?NAL1?gene.Sequencing revealed that Jamaica had a copy number variation on the NAL1,which resulted in an increased expression level.The high-density genetic linkage map constructed by SLAF-seq technology provides a powerful support for QTL mapping in important agronomic traits.This study provides a new allele for exploring the regulation of leaf development and leaf shape molecular breeding in rice.