Map-based Cloning Of Gene Conferring Wide Compatibility In Rice (Oryza Sativa L.)

The wide-compatibility (WC) is one of the most important traits in rice, which can break the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize great yield potential of inter-subspecific hybrid. The S₅ⁿ gene located on chromosome 6 has been previously reported to responsible for the wide-compatibility in rice. Here we reported the precise location of the S₅ⁿ gene, the structure and expression of the candidate genes, construction of the BAC library, the cloning and transformation of the candidate genes. The S₅ⁿ was preciously mapped into a 50Kb region, in which two candidate genes were determined. The full-length cDNA sequences of the two candidate genes were built up using the RACE (Rapid Amplified cDNA ends) method. The structures of two candidate genes were revealed by comparing their cDNA sequences with genomic sequences. Meanwhile, the sequences of candidate genes from different WCVs were also investigated. A BAC library was constructed from the WCV '02428' and the positive clone covering the whole S₅ⁿ region was obtained by screening, the library. The express constructs have been constructed for complementary tests. The results were described in detail as followings.1. Mapping of the S₅ⁿ gene. The Ss" was previously reported to reside between the RFLP markers R1954 and R2349 on chromosome 6. Based on this location, we conducted the fine mapping of the S₅ⁿ locus using the narrow down strategy. Firstly, high-density molecular markers have been developed in the target region based on thesequence divergences between the indica rice cv. '93-11' and the japonica rice cv. 'Nipponbare'. Then primers were designed on regions flanking the divergence to amplify the parents of a given cross. A marker was successfully developed if the amplicons were polymorphic between the parents. The S₅ⁿ gene in WCVs '02428V'Dular' was introduced into the non-WCVs 'Nanjingll'/'Balilla' by backcrossing for over ten generations, followed by self pollinating for one or two' generations. This produced 549 isogenic lines for fine mapping of the S₅ⁿ gene. In the first-pass mapping, a total of 17 markers (including 12 STS markers and 5 SSR markers) covering the Ss" region were used to screen a sub-population consisting of 242 isogenic lines. A total of eight recombinants have been identified downstream the S₅ⁿ locus. The crossing-over nearest to the S₅ⁿ locus occurred between markers J17 and J18, and hence delimiting the right border to the marker J18. There were three recombinants upstream the S₅ⁿ locus, restricted its right border to the marker J2. In summary, the S₅ⁿ locus has been roughly delimited within an interval between J2 and J18 with the physical distance of 200 Kb. In the fine mapping, 14 more markers were developed within the 200 Kb harboring the S₅ⁿ locus to assay the remaining 307 isogenic lines. Five recombinants left to and two recombinants right to the S₅ⁿ locus were identified. For all recombinants, their phenotypes and genotypes were repeatedly assayed to be sure no mistaken happened. Eventually the Ss" locus was restricted within a 50 Kb region flanked by the markers Jl3 and J17.2. Identification of the candidate S₅ⁿ genes and their expression profiling. Two putative genes were predicted within the 50Kb region, and their hypothetical functions were related to development of flower organs. Considering the S₅ⁿ gene functions to restore the fertilities for both the male and female gamates, and it can be assumed that S₅ⁿ gene.is involved in the development of both megaspore and pollen. Based on both mapping and functional analysis, the two putative genes predicted were considered as the candidate S₅ⁿ genes. The RACE method was employed to investigate the expression' profilings and to build up full-length cDNA sequences for the two candidate genes. The RACE reaction was carried out by firstly extracting the mRNA from the young spikelets of the respective WCVs '02428' and 'Dular', followed by reverse transcription to get the first-strand cDNAs. The results in...