Genetic Analysis And Gene Cloning Of Panicle Degradation Mutant Spd11 And Green-revertible Yellow Mutant D78 In Rice

1. Genetic analysis and fine mapping of panicle degradation mutant spdll in riceThe grain yield potential of rice can be dissected into four major components:effective panicle per unit area, grain number per panicle, grain weight, and ratio of filled grains. Exploring, cloning and functional studying on genes related to panicle traits are helpful to understand the regulation mechanism of the development of young panicle. And this will be of great importance in guiding the high yield breeding in rice. In our study, a panicle degradation mutant, spdll, was isolated from the mutant library of japonica rice dense and erect mutant dep2 of Zhonghua 11 by ethyl methanesulfonate mutagenesis. Studies on agronomic traits, inheritance pattern, gene localization and candidate gene were conducted afterwards. The results were as follows:(1) Under field conditions, spd11 exhibited long blade length of flag leaf compared to its counterpart parent. The length of primary branches were shortened along with the branch numbers decreased by 58%. The florets of spdll almost degenerated to white floc, though incompletely degenerated florets which were only consisted by a completely closed husk could be found occasionally. Other agronomic traits such as tiller numbers and blade width of flag leaf were little difference.(2) The spd11 mutant phenotype was controlled by a single recessive nuclear gene. The mutant gene was mapped to a region of 43.2 kb between two In/Del markers ch1-2295 and ch1-2299 on the long arm of chromosome 1, and the genetic distance were 0.23 cM and 0.46 cM, respectively.(3) Sequencing analysis revealed that a single base was changed (G116A) in the coding region of the OsLOG gene which encodes a cytokinin-activating enzyme, causing a missense mutation (C39Y). Comprehensive analysis of the mutation sites and the phenotypes of reported log allelic mutants hinted that this site (C39Y) may be a more key amino acid of OsLOG. SPD11 may be allelic to OsLOG gene.2. Genetic analysis and gene cloning of green-revertible yellow mutant d78 in riceAs important pigment participating in photosynthesis, the chlorophyll of higher plants has great effects on the development and yield formation of crops. Leaf color mutant is the ideal material for studying on photosynthesis. And it plays important role in fundamental researches such as the biosynthesis of photosynthetic pigment, the differentiation and development of chloroplast. In our study, a green-revertible yellow mutant, d78, was isolated from the mutant library of japonica rice variety Nipponbare by EMS mutagenesis. Studies on agronomic traits, content of photosynthetic pigment, structure of chloroplast, inheritance pattern, gene localization and cloning and complementation test were conducted afterwards. The results were as follows:(1) d78 displayed a yellowish phenotype in seedlings. Then the yellowish leaves gradually turned green following the development progress. But the leaf color was still trend to be yellow by maturation stage. In addition, d78 growed slowly at the seedling stage, and its heading stage was delayed. Compared to its wild type, the effective panicles per plant, panicle length and thousand grain weight of d78 decreased by 39.9%,7.1% and 10.7%, respectively. Other agronomic traits such as plant height, grains per panicle and setting rate were little difference.(2) Compared to its wild type, the amount of total chlorophyll, Chi a, Chi b and carotenoids in d78 decreased by 26.4%-41.9%,25.3%-42.9%,30.7%-37.9% and 14.8%-20.3% from seedling stage to heading stage, respectively. The shape and size of chloroplast between d78 and the wild type had no difference. But the grana stacks in the mutant were reduced and less dense. The mutant chloroplasts contained a distorted thylakoid membrane system and swollen lumens. And there also showed an increase number of osmiophilic plastoglobuli in mutant.(3) The d78 mutant phenotype was controlled by a single recessive nuclear gene. The mutant gene was mapped to a region of 66.0 kb between two In/Del markers Z2 and Z3 on the long arm of chromosome 1, and the genetic distance were 0.19 cM and 0.10 cM, respectively.(4) Within the Z2 and Z3 region,15 predicted genes had been annotated in Rice Genome Annotation Project. And we found one of them encoding a putative chloroplast precursor. Sequencing analysis revealed that a single base was changed (G741A) in the coding region of this gene, causing the codon of trp-250 converted to a stop codon in its encoded protein. D78 encodes a 393-amino acid protein, which contains an apparent chloroplast-targeting sequence of 23 amino acid residues at its N terminus and a transmembrane domain of 23 amino acid residues.(5) A full-length cDNA fragment encoding D78 were amplified by reverse transcription (RT)-PCR from the wild type and ligated into the binary expression vector pCAMBIA2300-actin. At last, we transformed D78 of wild type in d78 mutant by Agrobacterium-mediated transformation method, and successfully obtained positive transgenic lines which all displayed green leaf phenotype, suggesting that the d78 phenotype was rescued by transformation with the wild type D78 gene.