| Co-Investigator(Kenkyū-buntansha) |
TSUCHIYA Toru Mile University, Faculty of Bioresources, Assistant Professor, 生物資源学部, 助手 (30293806)
KAKEDA Katsuyuki Mie University, Faculty of Bioresources, Associate Professor, 生物資源学部, 助教授 (50221867)
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| Research Abstract |
The present study aims to reveal a molecular genetic mechanism of self-incompatibility in a wild species of sweet potato, Ipomoea trifida. The self-incompatibility of this plant is a sporophytic type and controlled by multiallelic S-locus genes. Interaction between stigma cell and pollen after self-pollination results in a complete failure of pollen germination on the stigma surface. To construct a fine genetic linkage map around the S-locus, identification and mapping of DNA markers were performed by RFLP analysis of S-allele segregating population. From the experimental results, 6 DNA markers were found to link to the S-locus within 1cM, among which AAM-68 marker is most tightly linked to the S-locus in a distance of 0cM.For map-based cloning of S-locus genes, a BAC library consisting of 40,000 clones was constructed using large size of genomic DNA isolated from leaf-cell nuclei. From the BAC library, several clones were screened using 4 DNA markers around the S-locus as probes. Terminal sequencing of the BAC clones revealed the overlapping region in some clones, those were aligned with the linkage map. Using the terminal sequences of BAC clones as DNA markers, further fine map was constructed in the vicinity of the S-locus. From this fine map, physical limitation spanning the S-locus was defined within 0.1cM.One of the BAC clones screened with AAM-68 marker, No.683 (ca.50kbp), was selected as a most promising candidate that includes S-locus genes in the genomic DNA, and is now used for DNA sequencing with shotgun method. In the present study, self-compatible mutants of I.trifida were also genetically analyzed. Radiation-induced mutants were found to be due to a chromosomal deletion around the S-locus, and spontaneous mutant was shown to confer a dominant-negative effect for normal S-alleles. These mutants are useful for transgenic plant assay of S-locus genes.
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