Map-based Cloning Of Two Yellow-green Leaf Genes In Rice

Two yellow-green leaf mutants, designated as w390 and g160, was found from the progeny of 60Co-γ-treated indica cultivar ZH8015. In this study, the morphological and physiological character of the each mutant was compared with its wild type, and the two mutant genes were isolated by map-based cloning. The main results were summarized as follow:The w390 exhibited a stable yellow-green leaf phenotype throughout the whole life cycle. The chlorophyll b was not detected in w390, and the chlorophyll a and carotenoids content of w390 were reduced by 50.6% and 44.8%, compared with the wild-type. The transmission electron microscope assay demonstrated that the number of thylakoids decreased and granas were poorly stacked in the mutant, resulting in underdevelopment of chloroplasts. The plant height, the number of panicle per plant, the number of spikelets per panicle, the number of grains per panicle of W390 were reduced by 12.0%, 22.3%,18.5%, 27.6%, respectively. A F2 population was constructed from the cross of w390 mutant and Nipponbare. Genetic analys is conducted using the population and the result showed that the yellow-green leaf phenotype was controlled by a s ingle recessive gene. Using mutant type individual of the F2 population, the gene was delimitated to a region of 71.8-kb on the long arm of chromosome 10, which contained OsCAO1 involved in leaf color. Sequence analysis revealed that the mutant carried two nucleotide substitutions in the eighth exon of OsCAO1, which led to the substitution of leucine and glycine for histidine and glutamic acid, respectively. This implied that w390 might be a novel allele of OsCAO1 gene. The expression of 15 leaf color related genes were exanimated by real-time PCR. The signif icant distinction between w390 and ZH8015 were hardly observed for these genes, with only one marginal difference detected for OsCRTIS, a key gene involved in carotenoid synthesis(P=0.0367).The g160 also showed a stable yellow-green leaf phenotype throughout the whole life cycle. The photosynthetic pigment, chlorophyll a and chlorophyll b content of g160 were reduced by 38.6%, 44.7% and 41.7%, compared with the ZH8015, while the significant difference was not detected for carotenoids content between g160 and ZH8015. The net photosynthetic rate,Fv/Fm, Y(II) and ETR of g160 were reduced by 56.3%、37.7%、60.0% and 55.5%. A F2 population and a F3 population were constructed from the cross of g160 mutant and 02428. Genetic analysis conducted using the F2 population and the result showed that the yellow-green leaf phenotype was controlled by a single recessive gene. Us ing mutant type individual of the F2 and F3 populations the gene was delimitated to a region of 121.5-kb on the long arm of chromosome 7. Twenty four putative ORFs were predicted in this region, and their c DNA in g160 and ZH8015 were compared. An 8-bp deletion was identif ied in the fifth exon of ORF23 in g160, which caused a frame shift. The genomic fragment of ORF23 from ZH8015 was introduced to g160 and transform T0 plants was obtained, and T0 plants of ORF23 overexpression and RNAi also constructed. They will use to functional verification of g160.