Identification And Map-based Cloning A New Yellow Green Leaf Gene YGL9 In Rice(Oryza Sativa L.)

Leaf color mutants are ideal materials in illuminating molecular mechanism of photosynthesis, photosynthetic pigment metabolic pathway and chloroplast development. The novel mutant ygl9 was isolated from the progeny of ethyl methane sulfonate(EMS) treated Jinhui10(Oryza sativa L.ssp. indica) and displayed yellow-green leaves. We studied the morphological identification of the ygl9 mutant, main agronomic traits, content of photosynthetic pigments, photosynthetic characteristics and stoma characteristics. At the same time, mesophyll cell and chloroplast ultrastructures were observed by using transmission electron microscopy.The F1 generation was hybrided between xinong 1A of normal phenotypes and mutant ygl9. According to leaf color phenotype of the F1 generation and leaf color separation condition of the F2 population, genetic analysis of the mutant ygl9, molecular mapping and map-based cloning were taken. Using qRT–PCR technology, the transcription expression levels of some related genes were analyzed. This study provided a foundation for function confirmation and functional analysis of YGL9 gene. The main results were as follows: 1 morphology identification and main agronomic traitsThe novel mutant ygl9 displayed yellow-green leaves from seedling stage to jointing stage while changed gradually to light green at the heading stage.From seedling stage to jointing stage, leaf blade of ygl9 was yellow-green, and up to heading stage, leaf color changed gradually to light green until maturity stage. Compared to the wild type, some traits of ygl9 were very significantly reduced, such as plant height, primary branch number, spike length, grain number per panicle, filled grain number per panicle, seed setting rate and 1000-grain weight, while no significant difference was identified of effective panicles. 2 the analysis of photosynthetic pigment contentAt seedling stage and tillering stage, the content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid content of ygl9 mutants decreased very significantly compared to that of the wild type. Because of greening of leaf color at heading stage, photosynthetic pigments content reduced only significantly compared with wild type. 3 determination of photosynthetic characteristics and SEM analysis of stomataThe characteristics of stomata length, stomatal conductance and transpiration rate increased significantly in the ygl9, while no obvious changing for net photosynthetic rate between the wild type and the mutant. Scanning electron microscope(SEM) showed that stomata of ygl9 was integrity in structure, distribution neatly and regularly, and no density difference was observed. But stomata length of ygl9 was 1.17 times longer than that of wild type, reaching a very significantly level.4 leaf cell ultrastructure observationMesophyll cells of ygl9 were fully developed. The chloroplasts were at a similar size to the wild type, and distributed regularly stick to the cell wall, packaged completely in the membrane in a same way as the wild type. In addition, more osmiophilic granules, fuzzy grana and fewer/looser stroma lamella were observed in ygl9. 5 Genetic analysisThe F1 progeny derived from a hybridization between Xinong1 A and the ygl9 mutant showed a normal, wild-type phenotype. The F1 plants were selfed to obtain F2 plants. In the F2 progeny, there were 2397 normal plants and 759 yellow-green mutant plants. Segregation of the normal and mutant phenotypes showed no significant difference from the separation ratio of 3:1(χ2 = 1.14<χ20.05 = 3.84). This finding showed that the ygl9 phenotype was controlled by a recessive nuclear gene. 6 gene mapping and candidate gene analysisAccording to the initial-mapping and fine-mapping, the YGL9 locus was finally mapped on the short arm of chromosome 3 between SSR marker S03-1 and InDel marker Ind03-19 with genetic distances of 0.13 cM and 0.07 cM respectively, and the physical distance was only 63 kb which contained 11 open reading frames(ORFs).By sequencing and comparing DNA and cDNA sequences, a base mutation from Gâ†'A transformation was found in a chloroplast signal recognition particle gene, encoding a 43 kDa protein. The base transformation makes the 300 th encoding amino acids from tryptophan(Trp) variate to the TAG end, leading to the early termination of gene protein translation. These results could preliminarily determine the YGL9 was a chloroplast signal recognition particle(cpSRP43)(Loc_ Os03g03990). 7 expression analysis of some related genes qRT-PCRQuantitative analysis results showed that the transcription levels of some photosynthesis system related genes(PsaA(PSI), PsbA(PSII)) and cytochrome b6 f complexes genes(PetA, PetB, PetD) were upregulated in ygl9, comparing with the wild type. At the same time, the transcription levels of carotenoid metabolic pathways related genes(PSY1, PSY2) and rubisco subunits encoding genes(RbcL, RbcS) were downregulated in ygl9. In addition, the transcription levels of chlorophyll metabolic pathways related genes(HEMA, CHLD, CHLM, CAO1) showed no obvious difference.The above results show that mutation of YGL9 may influence gene expression level of carotenoid metabolism and photosynthesis in rice.