Genetic mapping in Japanese coniferous tree species using microsatellite DNA markers

为了建立一个高效的SSR（simple sequence repeats）DNA区域选择体系，研究了一种新的代替分子杂交的方法--三重亲和捕获法（TAC）和一种改进的微卫星阵列分离法（PIMA）等。结果如下：1.利用插入了[CT]-重复序列（[CT]_<10>，[CT]_，[CT]<15>_，[<20>CT]_<25>）的模型DNA，对TAC的pH和温度条件进行了优化。在pH6.4 ~ 7.0范围内，有效筛选出高重复片段; 2.对PIMA法进行了改进，使其能可靠地筛选到插入SSR的重组菌落; 3.将改进的TAC法和PIMA法应用于柳杉、黑松、钝头Chamaaparis obuse和马占相思基因组SSR的实际筛选。在粳稻中，筛选出300多个SSR片段，并根据其序列设计了SCAR标记。4.以粳稻（Sawara-1的单倍体胚乳家系）、雷松（Sima-64的单倍体胚乳家系）和钝头粳稻（Aira-32和Satsuma-8的异花授粉家系）为家系材料构建连锁图谱。

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).