Study on the molecular genetic mechanism of self-incompatibility in a wild species of sweet potato.

本研究旨在揭示一种野生甘薯（Ipomoea trifida）自交不亲和的分子遗传机制。该植物的自交不亲和性是由多等位s位点基因控制的孢子生型植物。自花授粉后柱头细胞与花粉的相互作用导致花粉在柱头表面完全不能萌发。为了构建s位点周围的精细遗传连锁图谱，对s等位基因分离群体进行了RFLP分析，对DNA标记进行了鉴定和定位。从实验结果来看，有6个DNA标记在1cM范围内与S-locus连锁，其中AAM-68标记在0cM范围内与S-locus连锁最为紧密。为了进行s位点基因的图谱克隆，利用从叶细胞核中分离的大片段基因组DNA构建了包含40000个克隆的BAC文库。从BAC文库中，以s座周围的4个DNA标记为探针，筛选了几个克隆。对BAC克隆进行末端测序，发现部分克隆存在重叠区，与连锁图谱一致。以BAC克隆末端序列为DNA标记，在s位点附近进一步构建精细图谱。从这张精细的地图上，跨越s位点的物理限制在0.1cM以内。其中一个用AAM-68标记筛选的BAC克隆No.683 （ca.50kbp）被选为最有希望的候选克隆，该克隆在基因组DNA中包含s位点基因，现在使用霰弹枪法进行DNA测序。本研究还对三叶草的自亲和突变体进行了遗传分析。辐射诱发的突变是由于s位点周围的染色体缺失引起的，自发突变被证明对正常的s等位基因具有显性负作用。这些突变体可用于s位点基因的转基因植物检测。

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.