Genetic Analysis And Molecular Mapping Of A White Stripe Leaf Gene St(k) In Rice

The block occurs in chlorophyll biological pathways or the chloroplast development process mostly can result in the formation of varies mutants of leaf color in rice. Both of the chlorophyll biosynthesis and the chloroplast development are the consequence of the interaction between the nucleus genes and cytoplasm genes. And most of leaf color mutants in rice are mainly controlled by nuclear genes, the effect and regulation of cytoplasm gene can be neglected. Most of the leaf color mutants of rice were induced by artificial mutation, and the EMS was used as a common chemical mutagen for mutation. Genetic resources serve as the basis of genetic breeding, the study on leaf color mutant of rice has a great significance for functional genomics research and rice breeding.A white stripe leaf mutant, with a stable phenotype, was isolated from a indica variety of rice induced by the common chemical mutagen EMS, is named st(k) temporarily. Subsequently, we have analyzed it on the aspects of the major agronomic traits、phenotype、the physiological and biochemical process, and have built two F2 genetic groups, st(k)/NIP and st(k)l gu guang zhan, for the fine mapping of the target gene, then pick out the candidate genes.The main research results are as follows:The phenotype characteristics and main agronomic traits of the mutant st(k):Compared with the wild type, in the whole growth period, the mutant st(k) is characterized by the obvious white stripe leaf, and there was considerable difference between wild type WT and mutant st(k). When it was in the first leaf stage, the color of stem and leaf were more shallow than the wild type, under the second leaf growth, the mutant leaves appeared white stripe, more shallow than the wild type. The growth period of the mutant st(k) was earlier than wild type. After the investigation of the main agronomic traits of the mutant st(k),the plant height、the sword leaf length、 the effective tillers、panicle length、spikelet numbers、grain weights, we found that the differences of the plant height、effective tillers and spikelet numbers were significant between the mutant st(k) and the wild type WT.The photosynthetic pigment contents of the mutant st(k):There were six different periods of materials for the photosynthetic pigment contents measure, seedling stage,10 days、20days、30days、40days、50 days after transplantation. Compared with wild type, there were 54.2%,43.5%,49.8%,46.1 54.0%,44.3% reductions of the chlorophyll of mutant st(k) in six different periods, separately. And all differences of each period reach the significant level.The content of chlorophyll a (chla),chlorophyll b (chlb) and carotenoid (caro) all reduced, the differences were significant, above all, the decline degree of chlorophyll b content was the largest one.The genetic analysis of the mutant st(k):The mutant st(k) as the female parent was crossed with NIP、gu guang zhan as the male parents to build F2 genetic groups, respectively.At the same time it was back-crossed with NIP and guguangzhan as the female parents, respectively. The leaves of the F1 plants were both presented normal green, which shows the trait was not affected by cytoplasmic inheritance. In two F2 genetic groups, after the investigation of the numbers of individuals with normal leaf color and individuals with white stripe color leaf, the separation ratio fit the expected ratio 3:1 in the chi-squareχ(c2) test. It demonstrated that the white stripe leaf trait of the mutant st(k) was controlled by a pair of recessive nuclear genes.The fine mapping of the mutant st(k) and the analysis of candidate gene:We chose two mapping groups, the one F2 group from the mutant st(k)/NlP, the other F2 group from the mutant st(k) / gu guang zhan, to map the locus. Then we Selected 380 pairs of SSR markers uniformly distributed on 12 chromosomes for two parents polymorphism amplification. Subsequently, we designed Indel markers according to the vacant sites of genome sequence and synthetized SSR markers needed from Gramene Marker information released online (http://www.gramen e.org/microsat/).Finally,the mutant st(k) gene was mapped between the marker RM19652 and RM8270 on the short arm of Chr6, respectively, the physical distance was about 100 Kb.We analyzed the the candidate genes at the limited genomic region according to the database,TIGR-MSU Rice Genome Annotation (Osal) Release 7.1, it contained eight candidate genes, five genes function are known, one is expressing protein, two are hypothetical proteins. Through the subcellular localization according to the database of online predicting Plant-Ploc, it predicted four proteins of candidate genes,LOC_Os06g12240,LOC_Os06g12260,LOC_Os06g12270,LOC_Os06g12300, were positioned on the chloroplast. We cloned the exon genome sequence of these four genes from the mutant st(k) and the wild type. It demonstrated that the differences existed in the coding regions of LOC_Os06g12300 between WT and the mutant st(k). There was one locus of single base mutation (the 137th:T�'C). Ultimately, the base mutation caused into the coding amino acid mutation(the 46th:threonine�'methionine). In conclusion, the st(k) gene,a candidate gene of white stripe leaf traits, is a new gene never been reported before.