Development and characterization of EST-SSR markers from Thujopsis dolabrata var. hondae RNA-sequencing data

Thujopsis dolabrata Siebold et Zucc. var. hondae Makino (hiba), an endemic conifer of Japan, is naturally distributed in northern Honshu and southern Hokkaido. The species is one of the important woody materials for buildings, such as cultural assets, temples, and shrines, because of its long-term durability and has been planted since ancient times in Aomori, Niigata, and Ishikawa Prefectures. A linkage map is an important basis for marker-assisted selection and map-based cloning of desirable genes. In this study, to increase the number of available markers for linkage mapping, we used RNA-sequencing data for two parent individuals of the mapping family in T. dolabrata var. hondae to develop expressed sequenced tag simple sequence repeat (EST-SSR) microsatellite markers. Among 126 designed primer pairs, 62 showed amplifications. Sixteen of the 62 showed clear and stable allele peaks in 32 plus-trees and were used to assess the degree of polymorphism. The number of alleles per locus varied from 2 to 16, and the observed and expected heterozygosity values varied from 0.219 to 0.906 and from 0.242 to 0.897, respectively. All 16 markers developed in this study can be used in future linkage analysis. These EST-SSR markers will be useful for population genetic studies as well as linkage analysis in T. dolabrata var. hondae.