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

蔷薇科果实表现由S单倍型控制的配子体自交不亲和性，该单倍型含有雌蕊和花粉S基因。由雌蕊S基因编码的S核糖核酸酶降解自交花粉管的核糖核糖核酸，抑制花粉管的生长。花粉S基因产物与S核糖核酸酶之间的相互作用引起了花粉粒互作中的自我/非我识别。在李属植物(甜樱桃、杏、杏)中，花粉S基因编码F-box蛋白，位于S核糖核酸酶下游380~38kb之间，但在苹果、日本梨中尚未被鉴定。为了分离日本梨花粉中的S基因，本研究从从日本梨中分离的S_4纯合子(S2S_4)中构建了细菌人工染色体文库，并启动了从S4-核糖核酸酶中走染色体的过程。在S4-RNase上游395kb和下游615kb处组装了一个BAC重叠群。自交不亲和品种‘Osa-Nijisseiki’是‘Nijisseiki’(S2S4)的花蕾突变体，其花柱部分突变体S4^&lt；sm&gt；-单倍型缺失了S_4-核糖核酸酶基因，但保留了花粉S等位基因。从S_4和S_4纯合子基因组中扩增出S4-核糖核酸酶236kb的缺失片段。这表明花粉S基因位于S核糖核酸酶上游48kb和下游188kb的缺失断裂点外。在缺失区中发现了5个花粉特异表达的F-box基因，表明这5个基因是花粉S的候选基因。S单倍型特异标记是为早期筛选具有S单倍型的自交亲和品种而开发的。为了充分理解自交不亲和性，需要分析花粉管抑制与花柱的细胞相互作用。对穿透花柱组织的花粉管进行了超微结构研究，通过处理一系列的半薄切片来检测标本的合适位置。

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.