| The study on rice floral development's genetic and molecular mechanism has become one of important subjects for rice developmental biology. On the one hand, the study has very important theoretical significance in reveal both the molecular mechanism of rice reproductive development. On the other hand, because rice yield and quality depend on the floral development directly, it has a potential production meaning. In this study, we obtained a multi-pistil mutant from Nipponbare(O.sativa subsp. japonica cv.) T-DNA inserted mutant library. In order to facilitate the description, the mutant gene was designated as dcr. Compared with the wild type, mutant plants have no significant difference in plant type and heading date, while a single mutant floret has produced more than 2 seeds. Therefore, we named the mutant as dcr. After hygromycin tests, the mutant could not survive, which confirmed that the mutant was not caused by T-DNA insertion mutations, probably in tissue culture, or natural Daejeon induced mutations.We studied the floral anatomical structures, histological sections, multi-pistil trait's genetic analysis and molecular mapping of dcr gene. The main results as followed:One, phenotype characteristic of dcr mutant:Compared with the wild type floral organs, these dcr mutants showed multi-pistil phenotypes. Stamens were almost normal, only 10% of the spikelet number was not six stamens. The lemma, palea and lodicules were normal. The pistils in a single mutant floret were fertile, could produced two or more seeds but each seed was smaller than wild-type one.Two, genetic analysis and preliminary mapping of dcr mutant:The mutant was reciprocal crossed to Nipponbare (the wild type) and Dular (a wide compatibility variety). The F1 generations from those crosses all showed wild-type phenotypes, and the F2 populations of all crosses showed the normal Mendel's segregation ratio, indicating that the dcr mutant was controlled by a single recessive gene. Randomly selected 62 mutant-phenotype individuals from dcr / Dular F2 generation segregating population, we mapped the dcr gene to chromosome 11 between SSR markers RM26931 and RM27289 at the genetic distance 9.1 and 28cM respectively.Three, fine mapping of dcr mutant and the candidate gene:Based on the preliminary mapping, we design 18 pairs of InDel marks for fine mapping. All the mutant-phenotype individuals in 1083 were used for analysis. The dcr gene was finally mapped between the InDel tags JUN11 and JUN12 with distances of 0.2cM and 0.1cM respectively,291kb in physical distance on chromosome 11. Annotation of this region, we found a candidate gene FON4. FON4 gene encodes a hypothetical signaling protein, belonging to CLE gene family, which are very important to plant morphogenesis and cell signal transduction.Four, dcr gene's candidate gene sequencing analysis:FON4 gene contains three exons and two introns, and the CLE domain located in the third exon. Based on already published Nipponbare sequence, we designed InDel marked J3, amplified dcr mutant dcr candidate gene sequences. Results showed that, dcr candidate genes in the second intron of total first 621 base pairs changed from A (adenine) to G (thymine).Five, target gene dcr's vicinity sequence is a reorganization of cold:It was found that on both sides of the dcr gene were closed linked between InDel tags JUN11 and JUN12. The dcr was limited to the context of genetic distance 0.3cM, while the physical distance between InDel marks JUN11 and JUN12 were about 291kb. By analysis through the Gramene web site (http://www.nebi.gramene.com) reported marked information, the ratio of its physical distance and genetic distance is as about 4 times as the rice genome-wide estimate in mind the value of 244kb/cM (Chen,2002). |
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