Mapping And Candidate Gene Analysis Of Yellow-Green Leaf Gene YGL-15 In Rice (Oryza Sativa L.)

Leaf color mutants of rice are mainly caused by gene mutations of photosynthetic pigment metabolism and chloroplast development.Variation of leaf color would lead to changes in photosynthesis,directly or indirectly affecting rice growth and yield.In this study,a yellow-green leaf mutant,named ygl-15（yellow-green leaf-15）,was identified from the hybrid progeny of indica rice lines.To clarify the mechanism of leaf color variation of the ygl-15 mutant,phenotype identification,physiological and biochemical analysis,gene mapping,and analysis of the candidate genes were performed in this study.The main research results are as follows:The ygl-15 mutant showed yellow green leaves phenotype from the seedling stage to the maturity stage.Compared with the wild type,chlorophyll and carotenoid contents of the ygl-15 mutant were significantly reduced at the seedling stage.Finally,agronomic traits of the ygl-15 mutant were affected.Among them,compared with the wild type,plant height,panicle length,total grain number per panicle,total grain number per plant and filled grain number per plant,were decreased in ygl-15.But there was no obvious change for effective panicle number,seed-setting rate and thousand-grain weight.Genetic analysis showed that the mutant phenotype of ygl-15 was controlled by a single nuclear recessive gene.The F₂population of the ygl-15 mutant crossed with Zhonghua 11 were used as the primary population,.The ygl-15 gene was located within a1.25 Mb region between SSR markers chr03mm2399 and chr03mm2612 on the third chromosome of rice,and was closely linked with the polymorphic molecular marker chr03mm2581.New polymorphic molecular markers were developed in the preliminary localization interval.The F₂ population of the ygl-15 mutant crossed with Zhonghua 11were used as the first fine population.The ygl-15 gene was located within a 78.9 kb region between new markers M08124 and M08175,and was closely linked with the new polymorphic molecular marker M08139.In order to further narrow the localization range of the ygl-15 gene,the F₂ population of the ygl-15 mutant crossed with Nipponbare were used as the second fine population.And new polymorphic molecular markers were developed in the preliminary localization interval.Finally,the ygl-15 gene was located within a 26.6 kb region between new markers M08143 and M08165.According to NCBI Blast analysis,and gene annotation on RAP-DB websites,there are three genes in this interval.By sequencing and comparing sequences of WT and ygl-15,point mutations were found on the exon of the second candidate gene resulting in amino acid variation.As a result,the second candidate gene was identified as the candidate gene YGL-15.Gene expression analysis revealed that,the expression level were up-regulated for genes related to chlorophyll synthesis and photosynthetic system,but down-regulated or stable for genes related to heme synthesis in the leaves of ygl-15 mutant.Histochemical staining experiments showed that reactive oxygen species（ROS）including O^2-and H₂O₂,were found accumulated in the spotted leaves of ygl-15 plant at the tillering stage.To explore the ROS metabolic process in ygl-15 plants,enzymatic activities of the plant anti-oxidative enzymes superoxide dismutase（SOD）and catalase（CAT）were examined in the WT and ygl-15 mutant leaves.Results showed that SOD activity in ygl-15 spotted leaves was significantly up-regulated to be higher than that in the WT leaves.However,CAT activity in ygl-15 leaves was significantly lower than that in the WT leaves.The increase of SOD activity suggests that the ygl-15 mutant may actively respond to the O^2-accumulation and produce more H₂O₂,while the decrease of CAT activity may be not enough to scavenge the additional H₂O₂,leading to its accumulation in ygl-15.Accumulated ROS may play several important roles in the leaf spot formation process of ygl-15.