Genetic mapping in Japanese coniferous tree species using microsatellite DNA markers

为建立高效的SSR(Simple Sequence Repeats)DNA区域选择体系，研究了取代分子杂交法的三重亲和捕获法(TAC)、改进的基于PCR的微卫星阵列分离(PIMA)等方法。结果如下：1.利用插入了[CT]-重复序列([CT]_&lt；10&gt；，[CT]_&lt；15&gt；，[CT]_&lt；20&gt；和[CT]_&lt；25&gt；)的模型DNA，对反应的pH和温度条件进行了TAC优化。在pH 6.4-7.0的条件下，对原有的PIMA方法进行了改进，使之能够可靠地筛选到插入SSR的重组菌落。3.将改进后的TAC和PIMA方法应用于柳杉、黑松、木通和马占相思基因组中SSR的实际筛选。本研究选择了300个左右的SSR片段，并根据其序列设计了SCAR标记。4.构建连锁图谱的家系材料分别来自日本锦鸡儿(Sawa-1的单倍体胚乳家系)、松柏(司马-的单倍体胚乳家系)和钝顶锦鸡儿(Aira-32与温州蜜柑的异花授粉家系)。

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).