Development of microsatellite DNA markers for breeding of main domestic tree species

本项目的目的是开发微卫星标记用于主要营林树种赤松、短叶松和柳杉的育种，并建立一种省时省力的微卫星培育新方法。首先，我们通过一种改进的方法，包括含有DNA片段的微卫星的富集化过程，开发了几个密花青霉和钝顶青霉的微卫星标记。另一方面，我们探索了一种新的技术思路，建立了一种新的简便的微卫星标记开发方法。该方法由两个阶段组成：第一阶段，确定一个与微卫星相邻的序列，并根据该序列设计一条引物。第二阶段，利用抑制性聚合酶链式反应技术确定一个包含微卫星附近两个序列的序列，并设计另一个用于扩增微卫星区域的引物。在第一阶段，可以获得大量包含不同微卫星区域的亚克隆，这表明该方法对于开发多个标记是有效的。该方法除适用于树种外，还适用于真菌、昆虫、线虫等植物。这种方法将使主要营林树种的微卫星育种标记的大规模生产变得更容易。

The aim of this project was to develop microsatellite markers for breeding of the main silvicultural tree species, Pinus densiflora, Chamaecyparis obtusa and Cryptomeria japonica, and to establish a novel method for microsatellite development, which needs less time and labor. First of all we developed several microsatellite markers of P. densiflora and C. obtusa by an improved method including an enrichment process of microsatellite containing DNA fragments. On the other hand, we investigated a new technical idea, and established a novel simple method for development of microsatellite markers. This method was composed of two stages; in the first stage, a sequence neighboring a microsatellite was determined and -one primer was designed from the sequence. The second stage, a sequence including both sequences neighboring the microsatellite were determined by use of a suppression PCR technique, and designed the other primer for PCR amplification of the microsatellite region. In the first stage, a lot of sub-clones including different microsatellite regions could be obtained, indicating that this method is effective for developing a number of markers. This methods were also applicable to species of fungus insect, nematode in addition to the tree species. This method will enable mass production of microsatellite breeding markers for the main silvicultural tree species more easily.