Genetic mapping in Japanese coniferous tree species using microsatellite DNA markers

为了建立一种高效的SSR（简单重复序列）DNA区域选择系统，研究了取代分子杂交方法的新方法TAC （triplex affinity capture）、改进的PIMA（基于pcr的微卫星阵列分离）等。实验结果如下：1.实验结果表明：以[CT]-重复序列（[CT]_<10>,[CT]_<15>,[CT]_<20>, [CT]_<25>）为模型dna，对反应的pH和温度条件进行TAC优化。在pH 6.4 -7.0中，高重复的DNA片段被有效地选择。对原PIMA方法进行了改进，以可靠地筛选插入SSR的重组菌落。将改进的TAC和PIMA应用于从柳杉、松、黄杉和相思的基因组中实际筛选SSRs。在粳稻中，选取了约300个SSR片段，并根据其序列设计了SCAR （sequence characteristic扩增区）标记。构建连锁图谱的科系材料包括：c.a japonica （Sawara-1的单倍体-胚乳科）、p.t unbergii（Sima-64的单倍体-胚乳科）和c.b obtuse （Aira-32和Satsuma-8之间的异花授粉科）。

To establish an efficient selection system of SSR (simple sequence repeats) DNA regions, TAC (triplex affinity capture) that is a new method instead of a molecular hybridization method, a modified PIMA (PCR-based isolation of microsatellite arrays) and so on was investigated. The results were as follows:1.Using model DNAs in which [CT]-repeats ([CT]_<10>,[CT]_<15>,[CT]_<20>, and [CT]_<25>) were inserted, an optimization of TAC was executed on pH and temperature conditions of the reaction. In pH 6.4 -7.0, highly repeated DNA fragments were selected efficiently.2.The original PIMA method was modified for the reliable screening of the recombinant colony in which SSR was inserted.3.The modified TAC and PIMA were applied into actual screening of SSRs from the genomes of Cryptomeria japonica, Pinus thunbergii, Chamaecyparis obtuse, and Acacia mangium. In C.japonica, About 300 SSR fragments were selected, and SCAR (sequence characterized amplified region) markers were designed based on their sequences.4.The materials of families for constructing linkage-maps were made in C.japonica (a haploid-endospermic family of Sawara-1), P.thunbergii(a haploid-endospermic family of Sima-64), and C.obtuse (a cross-pollinated family between Aira-32 and Satsuma-8).