ГЕНЕТИКА, 2015, том 51, № 10, с. 1212-1216
КРАТКИЕ СООБЩЕНИЯ =
ISOLATION AND CHARACTERIZATION OF 15 MICROSATELLITE LOCI FOR Ophicephalus argus Cantor © 2015 M. Xiao, H. Xia, and F. Bao
College of Life Sciences, Anhui Science and Technology University, Fengyang 233100, China
e-mail: email@example.com Received November 7, 2014
The isolation and development of 15 polymorphic dinucleotide microsatellite loci were described for Ophicephalus argus from the Huaihe River. All loci were polymorphic in the 30 individuals tested. The number of alleles per variable locus ranged from nine to seventeen, with a mean of 12.00. These novel microsatellite loci showed high level of polymorphism. Observed and expected heterozygosities ranged from 0.793 to 0.929 and from 0.841 to 0.952, respectively. Two loci were found deviated from HWE in the sampled population after Bonferroni correction. These microsatellite loci will be useful for revealing population structure, genetic diversity, and phylogeography of Ophicephalus argus.
The Channa argus Ophicephalus argus Cantor is an air-breathing teleost which is widely distributed in the lower Yellow and Yangtze river systems in eastern China, Amur drainage in southeastern Russia and eastern China, and various rivers of the Korean peninsula and usually inhabits stagnant swamps, pools and creeks [1—3]. The Ophicephalus argus is a high commercially valuable aquaculture fish in some regions of China. In recent years, due to the worsening environmental pollution, water conservancy projects and human activities on the uncontrolled exploitation of natural resources, has made the Ophicephalus argus dwindling natural resources, and even some large waters have become extinct on the fishery resources caused irreversible damage [4—6]. As an important aquaculture resource in China, the aquaculture production of Ophi-cephalus argus has been increasing in past decades. Knowledge of the population genetic structure is important for management and sustainable utilization of this economic but threatened species. However, although a number of studies have been conducted on biology, artificial breeding, behavior, and physiology [7—13], only little information on molecular population genetics is available at present. Attempts to characterize population structure of Ophicephalus argus using isozymes and mitochondrial DNA D-loop sequence data have been hampered by the low variability of these genetic markers [6, 14, 15].
Neutral genetic markers with greater polymorphism would be ideal for providing better resolution of fine-scale population structure and phylogeography. Microsatellite is a kind of marker which meets the criteria . However, no Ophicephalus argus microsatellite loci have yet been published. Here, we cloned a suite of fifteen microsatellite markers from Ophiceph-
alus argus. These microsatellite primers should be useful in a wide range of population and evolutionary studies of this species.
Thirty individuals were collected from the downstream of the Huaihe River. All sampled fishes were captured by angling, gillnetting, or electro-fishing. The voucher specimens were preserved in 95% ethanol and stored at —20° C freezer at the College of Life Sciences, Anhui Science and Technology University. Genomic DNA was extracted from muscle tissues using the DNeasy Tissue Kit (QIAGEN). Briefly, 5 |g DNA of an adult fish was pooled and digested with 5 U Sau3AI restriction enzyme (New England Biolabs) at 37°C for 2 h and 300- to 800-bp fragments were excised from agarose gels. Size-selected fragments (300— 800 bp) were ligated to Sau3AI adaptors: oligo A: 5'-GGCCAGAGACCCCAAGCTTCG-3' and oligo B: 5'-pGATCCGAAGCTTGGGGTCTCTGGCC-3' . Then 10 |L of the ligated fragments were hybridized with 1.0 |L (10 |mol/L) 5' biotinylated probe (CA)15 at room temperature for 30 min and then captured by 100 |L of streptavidin-coated magnetic beads (Streptavidin magnesphere Paramagnetic Particles, Promega) . Nonspecific binding and unbound DNA was removed by several nonstringent and stringent washes. It follows that 100 |L of fresh magnetic beads were re-suspended and washed three times with 300 |L 6x SSC and 0.1% SDS. The 30 |L of beads-probe-DNA complex was separated by Magnetic Separation Stand (Promega). The supernatant was removed by applying a magnetic field to precipitate the beads, which were washed once at 68°C and twice at room temperature with 400 |L of 6x SSC, 0.1% SDS for 10 min for each wash, respectively. They were further washed twice with 3x SSC and twice with 1x SSC
ISOLATION AND CHARACTERIZATION
at room temperature for 10 min per wash. After removing the final wash, the targeted DNA fragments were eluted with 40 |L of pure water by denaturing the beads at 95°C for 5 min. These microsatellite-en-riched DNA fragments were amplified again by polymerase chain reaction (PCR), the PCR cycling scheme included an initial denaturation of 3 min at 94°C followed by 30 cycles of 94°C for 45 s, 57°C for 45 s, 72°C for 45 s, and a final extension at 72°C for 10 min, and then ligated into pGEM-T Easy vectors (Promega) and transformed into JM109 chemically competent cells . Transformed cells grew at 37°C for 16 h on LB agar plate containing ampicillin, X-gal and IPTG for blue/white selection. Forty positive clones were screened and sequenced using BigDye termination (Applied Biosystems) with the products being resolved on an ABI3730 sequencer.
Twenty five primer sets were designed through Primer 5.0 software  and synthesized. Instead of a 5' dye-labelled primer, a M13F (—29) sequence  was added to the 5' end of the forward primer or the reverse primer. PCR was carried out in a total volume of 20 |L containing 2.5 mM MgCl2, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM/L each dNTP, 0.4 |M each primer (M13F (-29)), 0.2 unit Ex-Taq DNA polymerase (Takara, Japan), and 100 ng DNA template. As a result, amplified DNA fragments all have the M13F (-29) sequence. To produce labeled DNA fragments, labelled M13F was added to the reaction. PCR amplifications were conducted in 25 -|L volumes containing 100 ng template DNA, 12.5 |L Ex Taq premix buffer (Ex Taq premix buffer consists of 20 mM Tris HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% Nonidet P-40 and 50% Glycerol) (TaKaRa), 5 pm of each primer, and 0.5 pmol of fluorescently labelled M13 primer (either IRD700 or IRD800 (LI-COR)). The conditions for amplification were 5 min at 95°C followed by 30 cycles of 30 s at 95°C, 30 s at the annealing temperature (table) and 30 s at 72°C with a final extension time of 10 min at 72°C. PCR products were separated on denaturing 6.5% polyacrylamide gels using a LI-COR 4300 automated DNA sequencer. IRDye® 800 Sizing Standard — 50-350 bp (LI-COR Biosciences) was used and the fragment size analysis was carried out with LI-COR SAGAGT software.
Isolation of microsatellite with enrichment by magnetic beads could yield approximately 48% ofpos-itive clones in Ophicephalus argus. Forty positive clones were screened and sequenced using BigDye termination with the products being resolved on an ABI3730 sequencer, from which 30 (75%) sequences showed at least a short microsatellite motif. Twenty five primer sets were designed through Primer 5.0 software  and synthesized. Upon PCR amplification, three loci showed monomorphic patterns, seven loci exhibited unspecific or uneven amplifications, and fif-
teen loci were successfully standardized for popula-tional analysis. The fifteen isolated loci were given names that started with the SS prefix (from Simple Sequence Repeats) followed by the clone number: SS7, SS11, SS23, SS33, SS34, SS36, SS44, SS58, SS89, SS90, SS102, SS182, SS219, SS225 and SS239 (table). Amplified fragments ranged from 147—348 bp.
The primers were tested for polymorphism on 30 individuals collected from the upstream to downstream of the Huaihe River. Genomic DNA from those 30 individuals was extracted using standard overnight proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation . The number of alleles at each polymorphic locus, their size range, and observed and expected heterozygosities was calculated using CERVUS 2.0 software  and was shown in table. The number of alleles per locus ranged from nine to seventeen, with a mean of 12.00. These novel microsatellite loci showed high level of polymorphism. Observed and expected heterozygosities ranged from 0.793 to 0.929 and from 0.841 to 0.952, respectively. Deviation from Hardy—Weinberg equilibrium and linkage disequilibrium at each locus was calculated using Genepop 3.4 . Two loci deviated from HWE in the sampled population after Bonferroni correction (adjusted P value = 0.0033, table), and the remaining 13 loci were in HWE (table).
The Ophicephalus argus genomic library developed in this study showed a higher percentage of positive clones (48%) than the average (3.1%) reported by Zane et al.  for fish, when using traditional methods similar to those employed here. Rivera et al.  observed that both the number of alleles and the observed heterozygosity should only be considered preliminary estimates if the sample size was small. Assessment of more individuals may reveal additional alleles for both the variable and monomorphic loci reported. So in the present study, we can not rule out the possibility that the presence of these loci specific alleles may be due to the limited samples size. Two loci deviated from HWE in the sampled population after Bonferroni correction. These deviations from expectations may be due to insufficient sample size, the occurrence of null alleles, or sampling of individuals from multiple distinct populations since they were collected throughout a very large river system. Further, null alleles were not found in 15 loci detected with MicroChecker utility
 (P < 0.05), and stuttering
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