Molecular and genetic analysis of pollen S gene controlled self-compatibility in Japanese pear

蔷薇科植物的果实具有配子体自交不亲和性（GSI），该自交不亲和性受雌蕊和花粉S基因的S单倍型控制。雌蕊S基因编码的S-RNase降解自花花粉管的rRNA，抑制花粉管生长。GSI中的自/非自识别是S-RNase与花粉S基因产物之间的相互作用。李属植物花粉S基因（甜樱桃、日本杏、杏仁）编码的F-box蛋白（SFB/SLF）位于S-RNase下游380 - 38 kb处，但在苹果亚科（Maloideae）物种中，（苹果、日本梨）的花粉S基因尚未被鉴定，为了分离日本梨花粉S基因，本研究本研究从日本精木分离的S_4纯合子（S2S_4）中构建了细菌人工染色体（BAC）文库，并以S4-RNase为启动子进行了染色体步移。组装跨越S4-RNase上游395 kb和下游615 kb的BAC重叠群。自交亲和品种Osa-Nijisseiki是'Nijisseiki'（S2S_4）的芽变体，其花柱部分突变体S4^<sm>-单倍型缺失S_4-RNase基因，但花粉S等位基因保留。S_4-和S_4^ -纯合子DNA的基因组PCR<sm>和BAC重叠群的DNA序列允许鉴定S4-RNase周围236 kb的缺失。在S_4-RNase上游48 kb和下游188 kb处的缺失断裂点外，推测有一个花粉S基因。在缺失区域外，发现了5个F-box基因，它们表现出花粉特异性表达，表明它们是花粉S基因的候选者。利用S^<sm>-单倍型特异性标记，可早期筛选具有S^<sm>-单倍型的自交亲和品种。为了全面了解自交不亲和性，需要分析花粉管抑制与花柱细胞相互作用的关系。采用半薄切片法对花粉管穿过花柱组织的超微结构进行了研究。

Rosaceae fruit exhibit a gametophytic self-incompatibility (GSI) controlled by the S-haplotypes, which contains pistil and pollen S genes. The S-RNase encoded by the pistil S gene degraded rRNA of self-pollen tube, arresting pollen tube growth. Self/non-self recognition in GSI is caused between S-RNase and a product of pollen S gene. The pollen S gene in Prunus species (sweet cherry, Japanese apricot, almond) encoded F-box protein (SFB/SLF) being located from 380 by to 38 kb downstream of the S-RNase, but in Maloideae species (apple, Japanese pear) have not been identified yet.To isolate a pollen S gene of Japanese pear, in this study, we constructed a bacterial artificial chromosome (BAC) library from an S_4-homozygote segregated from 'Nijisseiki' (S2S_4), and initiated chromosome walking from S4-RNase. A BAC contig was assembled spanning 395 kb upstream and 615 kb downstream of S4-RNase. A self-compatible cultivar `Osa-Nijisseiki', a bud mutant of 'Nijisseiki' (S2S_4), has a stylar-part mutant S4^<sm>-haplotype, which deletes S_4-RNase gene but remains pollen S allele. Genomic PCR of DNA from S_4-and S_4^<sm>-homozygotes and DNA sequence of BAC contigs allowed the identification of a deletion of 236 kb around of S4-RNase. This suggested that a pollen S gene is delineated out of the deletion breakpoint; 48 kb upstream and 188 kb downstream of S_4-RNase. Out of the deletion region, five F-box genes were found and exhibit pollen specific expression, proposing that these are pollen S gene candidates. S^<sm>-haplotype specific marker was developed for early selection of self-compatible cultivars haboring S^<sm>-haplotype. Toward the fully understanding of self-incompatibility, analysis of pollen tube inhibition is required in relation to the cellular interaction with style. Ultra structural studies of the pollen tube penetrated the stylar tissue were carried by handling the serial of semi-thin sections where to detect the appropriate position of specimens.