Fine Mapping And Genetic Analysis Of Two Rice Leaf Shape Genes

Leaves are the primary photosynthetic organs,and also the main place where the process of transpiration takes place.Rice leaves contribute to construct ideal plant architecture in rice,as rice leaf shape affects photosynthetic efficiency and regulates yield formation.Previous studies showed that the narrow and rolling leaves can keep erect and reduce mutual shading,thus improving the transmittance.The effective photosynthetic area was increased as light conditions of late growth stage was improved in rice.With the continuous innovation of methods and technologies in modern molecular biology,the discovery of new leaf shape genes for rice breeding and genetic mechanisms of leaf morphology are of great significance to increase rice yield per unit and ensure food security.At present,many researchers have already located and cloned some genes related to rice leaf traits,but the genetic and developmental mechanisms of leaf morphology are still unclear.In this study,we explored new genes related to leaf morphology and defined its function based on the study of the rice germplasm with distinctive leaf traits and the related mutant in rice.It will help us to elucidate the molecular mechanism of leaf growth and development,establishes a theoretical and practical foundation for leaf shape gene application in rice production.The results of our study are as follows:Mapping and functional confirmation of narrow and rolling leaf gene Nrl3(t)in rice.1.The mutant nrl3(t)was identified from an indica variety Zh8015 subjected to 60Co-y irradiation mutagenesis.Narrow and rolling leaf phenotype of nrl3(t)can be inherited stably as well as other pleiotropic phenotypes.To identify whether the leaf mutation was controlled by a single gene,genetic analysis was conducted using the crosses between nrl3(t)and 02428.The results showed that nrl3(t)was a recessive mutant,given the normal leaf shape of F1 plant and individuals of the F2 segregation population.The ratio of normal leaf shape and narrow and rolling leaf individuals fitted into 3:1(?2<?20.05,1=3.84),indicating that the nrl3(t)mutant was controlled by a single recessive nuclear gene.2.The cell structure was observed in the narrow and rolling leaves of the mutant and the normal leaves of the wild type.There was a very significant reduction in the vascular bundle number in nrl3(t)mutant,and this could lead to a narrow blade.The further analysis revealed that the bulliform cells had abnormal development in nrl3(t)mutant compared to the wild type,and this may be the cause of adaxial side rolling in leaf blade.3.The Nrl3(t)gene was located between the two markers C9 and C10E on the chromosome 2.Then the candidate region was delimited to a 40 kb physical distance(Chr2:32793884?32864280).4.The forecast website(Rice Genome Annotation Project)predicted that there are seven open reading frames(ORFs)in the candidate region.Among the seven ORFs,Os02g0776900 is a transcription factor of the GRF family,which is related to the growth and development of the plant.DNA sequences blast found that there was a 4-bp deletion in the second exon of Os02g0776900 genome sequence,which led to translation of encoding sequence premature termination in nrl3(t).Therefore,we constructed Nrl3(t)complementary vector,Nrl3(t)over expression and Nrl3(t)-GFP fusion vectors.The genetic complementation analysis revealed that the phenotype of nrl3(t)was completely restored to normal in the transgenic lines.All the experiments demonstrated that Os02g0776900 was the target gene responsible for the distinctive phenotype of nrl3(t).QTL analysis of rolling leaf trait,fine mapping and near-isogenic line construction of qRL-3-2 in Xieyou9308 recombinant inbred lines1.The 127 recombinant inbred lines population derived from the super hybrid rice Xieyou9308 and their parents were used to map the rolling leaf controlling gene by using composite interval mapping method(CIM).A total of three QTLs were detected on chromosomes 1,3 and 8,and named qRL-1-1,qRL-3-1,qRL-8-1,respectively.The phenotypic variation explained by qRL-1-1,qRL-3-1,qRL-8-1 was 9.34%,8.42%,8.72%,respectively,and the additive effect of three QTLs came from Zhonghui9308.2.We selected a near isogenic line with stable performance in BC4F2 segregation population derived from a cross between recombinant inbred lines plant S57,S130,S198 and recurrent parent Zhonghui9308.Genetic identification showed that this near isogenic line contained qRL-3-2 loci which could explain 48.7%of the phenotypic variation.3.The statistics analysis on agronomic traits found that the near isogenic line which has the same genotype with J5 in BC4F4 segregation population showed more flat leaf blade,longer panicle,and the increases in the number of grains per panicle,1000-grain weight,grain length and grain width,whereas decreased effective panicles.4.The qRL-3-2 was located on the long arm of chromosome 3 between the two markers 24-3 and 28-2.Then the candidate region was delimited to a 167 kb physical distance,which laid a solid foundation for further cloning of this gene.