Isolation and characterization of 20 polynucleotide microsatellite markers in a vulnerable Korean snail, Ellobium chinense, using 454 pyrosequencing

Abstract

A small and air-breathing snail, Ellobium chinense (Ellobiidae), is a vulnerable species by International Union for Conservation of Nature (IUCN). To protect and manage habitat and population of E. chinense, microsatellite markers were developed using 454 pyrosequencing and 20 polymorphic microsatellite markers were identified. A total of 146,704 sequences containing a minimum of four repeat motifs (mean, 631 base pairs) were identified from 499,505 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5 %) produced strong PCR products, of which 20 were polymorphic among 48 samples of E. chinense. All loci exhibited high genetic variability, with an average of 18.9 alleles per locus, and the mean observed and expected heterozygosities were 0.65 and 0.90, respectively. In addition, cross-amplification was tested for all 20 loci in the same family species, Melampus sincaporensis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 20 newly developed microsatellites will be useful in future conservation genetic studies of this species.

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