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

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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.