Zhiheng DU, Chunzhu XU and Fangyong NING

Northeast Agricultural University, Harbin 150030, Heilongjiang, China

Isolation and Characterization of 15 Microsatellite DNA Loci for the Alpine Stream Frog Scutiger boulengeri (Anura: Megophryidae)

Zhiheng DU, Chunzhu XU and Fangyong NING*

Northeast Agricultural University, Harbin 150030, Heilongjiang, China

Fifteen microsatellite DNA markers were developed from a transcriptome assembly of the alpine stream frog (Scutiger boulengeri).To characterize these loci, we genotyped 23 individuals collected from two sites. Eleven loci were variable, with the number of alleles ranging from one to six within each population. The expected and observed heterozygosities ranged from 0 to 0.78 and from 0 to 0.64, respectively. None of the loci was in linkage disequilibrium and one locus for one population was deviated from the Hardy-Weinberg equilibrium. We hope that these markers will offer useful tools in detecting population structure of S. boulengeri and in monitoring the fragile ecosystem of the Tibetan Plateau, where the species resides.

Alpine stream frogs, Microsatellite DNA, Tibetan Plateau, Scutiger boulengeri

Global climatic changes impose a major threat to species and ecosystem (The National Academies, 2009), and such changes are often amplifed at high altitudes (Rangwala et al., 2013). The Tibetan Plateau is the highest land on Earth and it maintains a unique biota. With the current global warming trend, its ecosystem is particularly vulnerable and establishing appropriate monitoring system is imperative. Amphibians are excellent environmental indicator species, and have been widely used in many ecosystem monitoring programs (e.g. Ryan et al., 2014).

Megophryid frogs of the genus Scutiger are the dominant amphibians of the Tibetan Plateau, and most of them have a restricted distribution and are endemic to this area (Fei, 1999; Frost, 2015). The alpine stream frog, Scutiger boulengeri, is the only widespread species in the genus. Its range extends from north to south along the eastern escarpment of the Plateau, and in the south, it further extends westward on top of the Plateau and reaches northwestern Nepal (Frost, 2015). The species occurs mostly in streams or ponds between elevations of 3300–5100 m, and populations may descend to 2200 m at its northern range (Fei et al., 2009; Li et al., 2009). It is relatively common although its population sizes tend to be small.

Microsatellite DNA markers are highly variable and have been widely used for population genetic and ecological studies (Selkoe and Toonen, 2006). Here, we report 15 microsatellite DNA markers that are specifcally designed for S. boulengeri. We hope that the availability of these genetic markers will facilitate the monitoring and conservation effects in the Tibetan region.

Microsatellite DNA loci were identified from an assembly of transcriptome sequences of S. boulengeri (Table 1). Sequences with repeat units greater than five were identifed and primers were automatically designed by QDD2 (Meglecz et al., 2010). A total of 40 pairs of primers with high primer scores and large numbers of repeats were selected. These primers were first tested and optimized for PCR parameters with five samples of S. boulengeri, particularly for annealing temperature and Mg2+concentration. Primer pairs that consistently produced the target products were characterized with 23 samples of S. boulengeri. These samples were collected from two sites, Daping (Gansu Province, China; n = 11; N34° 39.528', E104° 08.326', elevation 2830 m) and Linxia (Gansu Province, China; n = 12; N35° 28.215',E102° 54.373', elevation 2400 m).

We used a nested PCR method with tail-labeled primers (Schuelke, 2000). A “tail” (GAGTTTTCC CAGTCACGAC) was added to the 5' ends of all forward primers, and a NED fuorescent labeled tail was added to the PCR mix. All PCR were carried out in 10 μl reaction volume, including 30–50 ng template DNA, 0.8 pmol of forward primers, 2.9 pmol of reverse primers, 1.4 pmol of the labeled tail, 0.2 unit of R Taq polymerase (Takara), 0.15mM dNTPs, and standard PCR buffer (Takara). PCR reactions were performed with the following steps: 94 °C for 5 min, followed by 30 cycles of 30 s at 94 °C, 35 s at optimized annealing temperature (Ta), and 35 s at 72 °C, then 8 cycles of 30 s at 94 °C, 45 s at 53 °C (Tafor tail primer), and 45 s at 72 °C, and a fnal extension at 72 °C for 10 min. Products with fluorescent labels were electrophoresed on 6% denaturing polyacrylamide gels and visualized on an FMBIO laser scanner (Hitachi).

A total of 15 pairs of primers were characterized, of which eleven were polymorphic. The transcript sequences, which were used to design these primers, are deposited at NCBI (Accession numbers: KX024453, KX024454, KX024455, KX024456, KX024457, KX024458, KX024459, KX024460, KX024461, KX024462, KX024463, KX024464, KX024465, KX024466, KX024467). The primer sequences, repeat motifs, optimized annealing temperature (Ta), number of alleles, and heterozygosity for each locus are shown in Table 1. The number of alleles per locus (Na) ranged from 1 to 6 within each population. The expected and observed heterozygosities (HEand HO) ranged from 0 to 0.78 and from 0 to 0.64, respectively.

We used Genepop on the Web version 4.2 (Raymond and Rousset, 1995) to test departures from linkage equilibrium and the Hardy-Weinberg equilibrium (HWE) for each locus. No signifcant linkage disequilibrium was detected after Bonferroni correction for multiple tests. For HWE, we tested each population separately and combined. The combined tests increased the sample sizes and the detecting power, although it might introduce the Wahlund effect. Only one locus, SB3, was significantly deviated from HWE for the Daping population and combined. The same locus for the Linxia population was not signifcant (Table 1). Overall, these loci had relatively low numbers of alleles and four loci were monomorphic. The testing samples were from two peripheral populations at the species’ northernmost distribution range, which may have lower genetic diversity compared to central populations (Lesica and Allendorf, 1995). These markers will provide useful tools for detecting population structure of S. boulengeri and monitoring the environmental changes of the Tibetan Plateau.AcknowledgementsWe would like to thank Dr. B. LU for providing us access to his unpublished transcriptome data. This project is supported by a scholarship from the China Scholarship Council to Z. DU.

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*Corresponding authors: Dr. Fangyong NING, from Northeast Agricultural University, Harbin, Heilongjiang, China, with his research focusing on economic animal production.

E-mail: ningfangyong@sohu.com

Received: 15 December 2015 Accepted: 7 January 2016