Identification And Gene Mapping-based Clonging Of Two Chalkniess Mutants In Rice

Rice is the most important staple food crop and a model monocot with the smallest genome maintaining world food security.Chalkiness is a kind of white and opaque phenomenon in rice endosperm,which not only affects the appearance quality of rice,but also has great influence on processing quality and taste quality.Therefore,it is very helpful to improve the rice yield and quality of rice via mining endosperm mutants,understanding the gene regulation route and molecular of major cereals in the world.The production and quality of rice play an important role in mechanism of endosperm development.Here,we identified two chalkiness mutants,eb6 and eb7 which derived from EMS-treated japonica rice Zhonghua11.And their physical and chemical properties were analyzed.Fine mapping,functional verification of candidate genes and gene polymorphism analysis were performed.The results of the study revealed the mechanism of chalky phenotypic.Results from the study are summarized as follows:?1?Mutants showed white and opaque in central part of endosperm.Compared with wild type,there were no significant differences for plant height,tiller number,heading date,seed length,grain width,grain thickness and 1000-grain weight in mutant eb6 and eb7.Scanning electron micrograph?SEM?of cross-section of endosperm of eb6 and eb7 showed that The central parts of them were white and opaque,with loosely,smaller and irregularly arranged compound starch granules?SGs?.Whereas,the marginal part of endosperms of WT and mutants and the central parts of WT were filled with densely packed,similar sized polyhedral SGs.?2?The filling rate of mutant eb7 was consistent and the grain weight without water was not significantly different with wild type during the whole process of grain development.Simultaneously,the content of glucose and fructose was the same as that of wild type.In the middle to later stage of grain filling,the sucrose content of eb7 was higher than that of wild type.The results of physical and chemical analysis showed that amylose content,viscosity and gel consistency of mutants were dramatically lower than that of the wild-type.However,protein content were significantly increased.total starch content,amylopectin content and fat content did not change significantly,but the proportions of chains with degree of polymerization?DP?of amylopectin has changed.?3?Genetic analysis indicates that a single recessive gene controls chalkiness phenotype of mutants.The target genes of the two chalky mutants were located at the long arm of chromosome1 based on the F₂ population derived from the cross between eb6/NanJing11 and eb7/9311.the gene was finally narrowed down to a 86.6 kb physical region using 970 individual selected from F₂ seeds of eb6/NanJing11.Os01g0633100?OsAGPL2?was predicted to be the target gene.it was found that a single base in the third exon of the gene was replaced in the mutant eb6.In the eb7,a single base was replaced in the same gene.The two mutants were crossed reciprocally.Seeds from F₁ and F₂ were similar to the chalky phenotype of the parent.Thus,we identified OsAGPL2 as the target gene for controlling the chalky phenotype in both mutants.?4?OsAGPL2 was mainly transcribed in the middle and late stages of grain filling.And the same results were obtained by in situ hybridization.The relative expression of genes related to starch synthesis was analyzed in different grain filling stages.It was found that The expression of several genes were up-regulated and down-regulated.And some of them were the same in wild type.?5?To know nucleic acid sequence polymorphism of OsAGPL2,we have acquired sequence for103 rice core germplasm.There are 25 SNPs and 2 Indel in the genomic region of about 4.6 kb,and there is only one SNP in the exon region of the OsAGPL2.But the amino acid did not change.These results indicate that nucleic acid polymorphism of the gene has notably decreased by the natural and artificial selection,the gene function in wild rice and cultivated rice is exactly the same.