Isolation and Characterization of Microsatellite Markers for the Sable, Martes zibellina (Mammalia: Mustelidae)

Twenty two M. zibellina individuals, including specimens, bodies of natural death and road killing, were collected from two populations in Northeast China. Among them, 12 individuals of M. zibellina princeps were collected in Daxinganling (50°10′- 53°33′ N, 121°12′- 127°00′ E), and other 10 individuals (M. zibellina linkouensis) were collected in Xiaoxinganling (48°52′-49°11′ N, 126°45′-127°25′ E). All the muscle tissues were stored at -80°C. Total genomic DNA was extracted using DNeasy® Blood & Tissue Kit (QIAGEN GmbH) following the manufacturer’s guidelines. All DNAs showed qualified after the detection of 1% agarose gel electrophoresis.

In accordance with the FIASCO protocol (Zane et al., 2002), we constructed a partial genomic library and used 5’-biotinylated (AAAC)8 oligonucleptide probe for enrichment selection from it. Isolated DNA was digested by MseI and ligated to double strand AFLP linkers (MseI F: 5’-TACTCAGGACTCAT-3’ and MseI R: 5’-GACGATGAGTCCTGAG-3’). The hybrids strands were captured with Dynabeads® M-280 Streptavidin (Invitrogen) and were amplified to recover the dsDNA. The enriched fragments were ligated into pMD18-T simple cloning vector (TaKaRa) and transferred into Trans1-T1 chemically competent cells (TransGen Biotech). Recombinants were screened by PCR with three primers: vector primers (M13F and M13R) and (AAAC)8 oligonucleotide. Then, the PCR products of clones were examined by 1% agarose gel electrophoresis, and lanes showing multiple bands were considered as positive clones. The positive clones were sequenced by Sangon Biotech (Shanghai) with ABI3730XL Genetic Analyser (Applied Biosystems, Carlsbad, CA, USA).

A total of 196 positive clones were identified from 432 plasmids. Then 100 selected positive clones were sequenced and 79% of them had tandem repeats.

Thirty specific-primers were designed based on conserved regions using the software Primer Premier 5.0 (Lalitha, 2000) and eight primers were successfully yielded clear bands. PCRs were performed in a 25-µl reaction volume containing 7.5 µl 2×Easy Taq PCR Supermix (TransGen Biotech), 100–200 ng genomic DNA and 2.0 pM of each primer pair (forward primer fluorescently labeled with FAM, HEX). PCR-amplifications were carried out in an Applied Biosystems (ABI) 9700 thermal cycler under the following conditions: a first denaturation at 95°C for 5 min, 35 cycles at 95°C for 30 s, 30 s at the annealing temperature (Table I), and 72°C for 30 s, followed by a last cycle at 72°C for 10 min, and a hold step at 4°C. PCR products were analysed on an ABI 3500 genetic analyser (Applied Biosystems) with a LIZ600 size standard, and the allele sizes were estimated using the software GeneMapper (version 5.0, ABI).

The detection of potential scoring errors (e.g. stutter bands, large allele dropout and null alleles) for the data was analysed using Micro-Checker 2.2.1 (van Oosterhout et al., 2004). The number of alleles (NA), observed heterozygosities (HO), expected heterozygosities (HE) and Principal Coordinate analysis (PCoA) were estimated using GenAlEx 6.5 (Peakall and Smouse, 2012). Linkage disequilibrium (LD) between pairs of microsatellite loci and deviations from the Hardy-Weinberg equilibrium (HWE) were calculated by GENEPOP v4.2 (Rousset, 2008). Cervus 3.0 was used to assess the polymorphism information content (PIC) and the frequency of null alleles (Kalinowski et al., 2007). Inbreeding coefficients (Fis) were determined by FSTAT v2.9.3.2 (Goudet, 1995).

Ta, annealing temperature; NA, number of alleles; HE, expected heterozygosity; HO, observed heterozygosity; PIC, polymorphic information content value; F, null allele frequency.

N, number of individuals tested; NA, number of alleles; NE, number of effective alleles; HE, expected heterozygosity; HO, observed heterozygosity; Fis, inbreeding coefficient.

Although a high proportion of sequences contained repeats, the majority of them was unusable for primer design for several reasons such as the higher or lower annealing temperature (Zhi et al., 2014). Thirty primer pairs were designed, of which eight successfully yielded clear bands. Futhermore, only six markers yielded polymorphic amplification products in the test for polymorphisms across 22 individuals from two natural populations of M. zibellina. DNA sequences of the six microsatellites were deposited into Genbank (accessions KX712105- KX712110).

NA per locus varied from two to five, with an average of 3.67 (Table I), which showed a lower level than previous study (Kashtanov et al., 2011). At the species level, HO and HE ranged from 0.227 to 0.545 and from 0.201 to 0.630, with an average of 0.447 and 0.453, respectively (Table I).

None of these loci were found to deviate from the HWE (Table I), and significant LD was detected between the locus Mzf49 and Mzf56 after Bonferroni correction (p<0.05), which indicated significant allelic association between the two markers. The PIC value ranged from 0.181 to 0.573. Locus Mzf58 showed low polymorphism (PIC<0.25) and Mzf61 showed high polymorphism (PIC>0.5), while the rest were moderately polymorphic (0.25<PIC<0.5). The frequency of null alleles varied from -0.0548 to 0.0563 and none of these loci showed evidence of null alleles. None of the loci showed evidence for large allele dropout or stutter bands. Genetic diversity parameters in each population are presented in Table II. Numbers of effective alleles (NE) were all less than 3, and ranged from 1.180 to 2.824 in the two populations. The locus Mzf61 showed the highest inbreeding coefficient (Fis=0.267) in Daxinganling population. Furthermore, using the 6 microsatellites, PCoA permitted the discrimination of the populations of Daxinganling and Xiaoxinganling, although any overlap was observed. It suggested that the sets of loci proved to correctly discriminate populations (Fig. 1).

In conclusion, microsatellite DNA is an effective molecular marker for studying the genetic diversity of sables. The FIASCO protocol is fast and simple, and its utility is better than traditional enrichment methods. The six microsatellite markers show polymorphism in the two populations, which is helpful for the research and conservation of M. zibellina in the future.

This work was supported by the Special Fund for Forest Scientific Research in the Public Welfare (201404420), the National Natural Science Fund of China (31672313, 31372220), Science and technology research plan of Shandong Province (2013GSF11707).

Authors have declared no conflict of interest.