Development of EST-derived microsatellite markers in Ophiorrhiza pumila (Rubiaceae) and their application in congeners

In East and Southeast Asian islands, forest area is decreasing and population size of multiple Ophiorrhiza species (Rubiaceae) is diminishing. Here we report the first expressed sequence tag-derived microsatellite markers, which can be applied to wide range of species of the genus. We developed 38 tri- and tetra-nucleotide microsatellite markers based on nonredundant unigenes of O. pumila and tested polymorphism of nine markers using the phylogenetically distant congener O. japonica (n = 96). The number of alleles ranged from 4 to 9. Observed and expected heterozygosities ranged 0.044–0.631 and 0.096–0.674, respectively. The polymorphic information content ranged 0.094–0.626. No null allele, significant deviation from Hardy–Weinberg equilibrium, or significant linkage disequilibrium was observed. Additional tests of cross-species amplification with three other congeners were successful in the nine markers. These markers will be used to investigate genetic diversity and differentiation of island populations of multiple Ophiorrhiza species to build conservation plans.