Development of Seven Microsatellite Markers Using Next Generation Sequencing for the Conservation on the Korean Population of Dorcus hopei (E. Saunders, 1854) (Coleoptera, Lucanidae).

Tae Kang,Sun Park,Sang Han

doi:10.3390/ijms160921330

Abstract

We developed microsatellite markers for genetic structural analyses of Dorcus hopei, a stag beetle species, using next generation sequencing and polymerase chain reaction (PCR)-based genotyping for regional populations. A total of 407,070,351 base pairs of genomic DNA containing >4000 microsatellite loci except AT repeats were sequenced. From 76 loci selected for primer design, 27 were polymorphic. Of these 27 markers, 10 were tested on three regional populations: two Chinese (Shichuan and Guangxi) and one Korean (Wanju). Three markers were excluded due to inconsistent amplification, genotyping errors, and Hardy-Weinberg equilibrium (HWE). By multi-locus genotyping, the allele number, observed heterozygosity and polymorphism information content of seven microsatellite loci were ranged 2‒10, 0.1333‒1.0000, and 0.1228‒0.8509, respectively. In an analysis on the genetic differentiation among regional populations including one Japanese population and one cross-breeding population, the individual colored bar-plots showed that both Chinese populations were closer to each other than to the Far East Asian populations. In Far East Asian populations, Wanju and Nirasaki populations could not be distinguished from each other because the frequency of genetic contents was very similar in some individuals of two populations. Moreover, the cross-breeding population contained all patterns of genetic contents shown in Chinese, Korean, and Japanese populations, compared with the genetic content frequency of each regional population. As a result, we examined whether the cross-breeding population might be a hybrid population, and might contain a possibility of interbreeding with Chinese populations in parental generations. Therefore, these markers will be useful for analyses of genetic diversity in populations, genetic relationships between regional populations, genetic structure analyses, and origin tests.

Highlights

Dorcus hopei [1] (Lucanidae, Coleoptera), belonging to the group of stag beetles, is widely distributed throughout China and Japan
By polymerase chain reaction (PCR) analysis, 48 loci showed polymorphic amplification; among these, 12 loci showed ambiguous amplification and nine primer sets did not produce any visible amplicon from some regional populations
The remaining 27 primer sets were deemed as candidate loci for use in genetic diversity and relatedness analyses for each of the regional populations (Table S1)

Summary

Introduction

Dorcus hopei [1] (Lucanidae, Coleoptera), belonging to the group of stag beetles, is widely distributed throughout China and Japan. The species is divided into two subspecies, D. hopei hopei, which is distributed throughout central China, and D. hopei binodulosus [2], which is found in northeastern China, Korea, and Japan [3,4]. D. hopei can be distinguished from other closely-related species by its mandible shape (thick mandible with one inner tooth) in males, and the elyral puncture line in females. D. hopei is one of the largest Coleoptera species in East Asia: males can grow up to 76 mm in the wild [3,4,5]. It was recently reported to be a rare species in Korea and Japan [3,6].

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Conclusion