Genetic Analysis And Gene Mapping Of A Rice Yellow-to-green Mutant K₂

Rice is an important grain crops in the world.Improving photosynthetic efficiency is one of the keys to increase rice yield.As the main place of photosynthesis,the chloroplast has been a hotspot in the formation,the development and the structural characteristics.By using the luminous energy that chlorophyll absorbs from the light,plant turns carbon dioxide into carbohydrates through a series of reactions.Thus,it is the great interest to study the formation,the development and the structural characteristics of chloroplast.In addition,the leaf color mutant can be used as selection marker for plant breeding application.We obtained a yellow mutant k2 from the tissue-cultured rice Kitaake.The yellow phenotype is stably inherited in the mutant k2 through many generations from self-crossing.In this study,we characterized the leaf color phenotype,and comparatively determined the chlorophyll content,the agronomic traits between this mutant and its wild type Kitaake.We then performed genetic analysis,molecular mapping,and candidates prediction of the gene,in which the mutation leads to the yellow phenotype in the k2 mutant.The main results are summarized below.1.Phenotypic characterization and agronomic traits determination in the mutant k2 The yellow mutant k2 exhibits white color in the first three leaves post seed germination.When the 3rd leaf comes out,the first two leaves start to turn green from the leaf tips to their whole leaves.The leaf color of entire plant turns green almost completely at last with the plant development.At the maturation stage,we investigated the main agronomic traits and found that the plant height,ear length,ripening rate,and tiller number of mutant were all obviously reduced compared to the wild type.2.Determination of photosynthetic pigment content in the k2mutant:We performed comparative determination on the pigment content between the mutant k2 and the WT at different stages,respectively.Results showed that the total chlorophyll of the k2 mutant is only 25.94%of the wild type in the two-leaf stage.It rises up to 57.14%of the wild type in the four-leaf stage,and up to 85.71%of the wild type in the six-leaf stage.These results suggest that the total of chlorophyll gradually increases to the level of the wild type along with the development of the K2 mutant plants.3.Disease resistance of the mutant k2:According tosome researches,the leaf color mutant not only changes the leaf color but also have an impact on disease resistance.We inoculated the k2 mutant and the wild type with rice blast fungus pathogens.Through quantitative real-time PCR(qRT-PCR)analysis,we found that the expression of the pathogenesis-related genes,PR1 and PR10,was increased post inoculation with blast pathogen.4.Genetic analysis:We performed reciprocal crosses between the k2 mutant and the rice variety Jodan or C418.All the F1 plants from the crosses between k2 and Jodan or C418 showed the wild type phenotype.However,in the F2 population,plants with the green-revertible yellow and the wild type phenotypes were segregated and the ratio between the number of the green-revertible yellow plants and the number of the normal green color phenotype matched the value of 1:3 under an X2-test.These results suggest that the green-revertible yellow phenotype in thek2 mutant is controlled by a single recessive nuclear gene.4.Gene mapping and candidates prediction:Through analysis on the F2 plants derived from the cross of k2/Jodan using molecular markers,SSR and InDel,we determined the k2 gene between the marker,RM333 and RM24898,on chromosome 10 with a physical distance of about 250kb.According to the gene annotations from Gramene(http://www.gramene.or)and Rice Genome Project(hptp://www.plantbiology.msu.edu/),we found that a total of 16 genes were predicted in this 250 kb region.Out of these genes,11 were functionally annotated,in which the potential candidates of k2 were selected for further study.