Conservation Genet Resour (2014) 6:33–35 DOI 10.1007/s12686-013-0061-z
TECHNICAL NOTE
Development and characterization of thirteen polymorphic microsatellite markers in the fish Osteochilus salsburyi Li-Wei Su • Zhi-Zhi Liu • Wen-Qiao Tang • Bao-Long Bao • Dong Liu • Jin-Quan Yang
Received: 15 August 2013 / Accepted: 26 September 2013 / Published online: 1 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Osteochilus salsburyi (Cypriniformes, Cyprinidae) is a small-sized fish of significant economic value. In this study, thirteen polymorphic microsatellite markers were developed in O. salsburyi and were examined on twenty individuals collected from the Wanquan River in the Hainan Island. The number of alleles (NA), polymorphism information content, observed and expected heterozygosity per locus ranged from 10 to 21, 0.8020 to 0.9354, 0.7500 to 1.0000 and 0.8436 to 0.9628, respectively. No locus was found to significantly deviate from Hardy–Weinberg equilibrium. These markers would be suitable for studies on conservation genetics and population structure of O. salsburyi. Keywords Conservation genetics Microsatellite markers Osteochilus salsburyi
Osteochilus salsburyi (Cypriniformes, Cyprinidae) is a small-sized fish of significant economic value that prefers the slow running water region in either medium-sized rivers or streams with rocky bottom. This species has broad distribution area, including Southern China, Vietnam, and Greater Sunda Islands. In China, population sizes of O. salsburyi have dropped remarkably due to water pollution and constructing dams in recent years (Kang et al. 2009). Unfortunately, this species is poorly known and there is a Li-Wei Su and Zhi-Zhi Liu have contributed equally to this work. L.-W. Su Z.-Z. Liu W.-Q. Tang B.-L. Bao D. Liu J.-Q. Yang (&) Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, 999 Huchenghuan Road, Shanghai 201306, China e-mail:
[email protected]
great need of sound management strategy of genetic resources. In this study, we have developed and characterized thirty-one polymorphic microsatellite markers in O. salsburyi. These markers will be useful and applicable in studying genetic diversity, population structure, and conservation genetics of this species. The samples of O. salsburyi in the present study were collected from the Wanquan River in Hainan Island. Genomic DNA was extracted from fins according to traditional phenol–chloroform procedure. The microsatellite loci were isolated through the magnetic beads enrichment method (Su et al. 2013). Similarly, biotinylated probes (CA)15 were also used to capture the repeat motifs. A total of 142 clones containing insertion fragments ranged from 300 to 1,000 bp were then directly sequenced on ABI PRISM 3730 sequencer, of which 95 clones were finally determined showing microsatellite signals (number of repeats C8). Thirty-one pairs of primers were designed by using primer premier 5.0 and synthetized. All of them could lead to successfully PCR amplification. Eight individuals were first randomly picked for the preliminary polymorphism assessment among all microsatellite markers. PCR reactions were performed in a 10 lL final volume with 1 lL DNA, 5 lL 2 9 Taq PCR Master-Mix, and 0.4 lL primers. PCRs were as follows: 94 °C for 4 min followed by 35 cycles at 94 °C for 30, 30 s at primer-specific annealing temperature (Table 1), 72 °C for 1 min and a final extension step at 72 °C for 6 min. The PCR products were visualized on 8 % nondenaturing polyacrylamide gel with silver staining. Among the thirty-one primer pairs tested, thirteen pairs showed high levels of polymorphism and were further used to evaluate levels of polymorphism on total individuals. Then, we used twenty individuals to quantify the levels of polymorphism of the thirteen loci.
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Conservation Genet Resour (2014) 6:33–35
Table 1 Characterization of 13 microsatellite loci for Osteochilus salsburyi Loci
GenBank accession no.
Primer sequences (50 –30 )
Repeat motif
Ta (°C)
Size range (bp)
NA
PIC
HO
HE
H–W
Ost1
KF469267
F: CAGATAAGAGAAACGACC
(CA)28
57
152–210
14
0.8626
1.0000
0.8962
0.6673
Ost2
KF469268
(TG)17CA(TG)7
56
172–216
17
0.9146
1.0000
0.9436
0.6944
(TG)5TT(TG)17
49.5
135–185
16
0.8914
0.9500
0.9218
0.2290
F: ATGGAGTGAAACCAGAGC R:CAAAACACCCAAGCAACG
(AC)38
56
121–173
12
0.8034
0.9474
0.8464
0.0920
F: CTGTAGTAAATCTTGACG
(GT)12(GA)5
53.5
144–190
16
0.9117
0.9500
0.9410
0.4294
(CA)20
57.5
164–202
15
0.8960
0.9500
0.9269
0.6734
(TG)25
51
202–242
14
0.8902
1.0000
0.9218
0.7578
(GT)18
57.5
115–141
10
0.8020
0.7500
0.8436
0.2245
(CA)15
53.5
130–170
14
0.8550
0.9000
0.8897
0.3548
(AC)16
53.5
145–201
13
0.8600
0.7500
0.8949
0.0697
R: GTGACTCCCATCAGACG F: CGGACTAAACCCACATCT R: TCTGCCTAAGCATTGAAA Ost3
KF469269
F: ATGGTGTCAGATTCCT R: GCATTTATCATCCTTC
Ost4
KF469270
Ost5
KF469271
R: TAACTCCCAGGAATGTG Ost6
KF469272
Ost7
KF469273
F: CCAGCCCTCAGAACAATG R:GACAACTGCTAAAATCCTAT F: CACCTTCAACACTCTT R: ACACACATCATCCTTC
Ost8
KF469274
F: TGTGTGTTCGGCTGAGTC R: TGCTTGCTTCCAGTTCTT
Ost9
KF469275
Ost10
KF469276
F: AGGAAACAAGGAGACGA R: AGAGTAATGTGAAGAATG F:TCAAGAGTATCTAAGACAAT R: AGTAAAAGCGATGGGAAG
Ost11 Ost12
KF469277
F: GCTGTAGTAAATCTTGACG
(GT)12(GA)5
53.5
145–189
15
0.9185
0.9500
0.9474
0.6406
KF469278
R: TAACTCCCAGGAATGTGA F: CACAATCACACAAGAAC
(TG)28C(GT)7
51
191–243
21
0.9354
1.0000
0.9628
0.7409
(TG)39
53.5
171–253
21
0.9162
1.0000
0.9449
0.6531
R: AACTCCAAGGACACTAA Ost13
KF469279
F: TAAAACACTTCCATCTTCTG R: ACTATTCTGCCACCAACTA
Ta annealing temperature, NA the number of allele, PIC polymorphic information content, HO observed heterozygosity, HE expected heterozygosity, H–E Hardy–Weinberg equilibrium
The PCR products were analyzed automatically by the QIAxcel system using OM700 in combination with QIAxcel DNA High Resolution Kit (QIAGEN, German). MICRO-CHECKER v.2.2.3 (Van Oosterhout et al. 2004) was used to assess null alleles and possible scoring errors at each locus. Allele frequency, observed (HO) and expected (HE) heterozygosity, Hardy–Weinberg equilibrium (H–W) (fisher’s exact test) and pairwise tests for linkage disequilibrium (LD) (fisher’s exact test) were performed using GENEPOP 3.4 software (Raymond and Rousset 1995). Polymorphism information content (PIC) was calculated by CERVUS 3.0 (Kalinowski et al. 2007). The characterization of thirteen microsatellite loci for O. salsburyi was shown in Table 1. The number of alleles (NA) and PIC on each locus ranged from 10 to 21 (average 15.2308) and from 0.8020 to 0.9354, respectively. The observed (HO) and expected (HE) heterozygosity ranged
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from 0.7500 to 1.0000 (average 0.9344) and from 0.8436 to 0.9628 (average 0.9139), respectively. MICROCHECKER analysis showed no evidence for scoring error or technical or statistical artifacts (Van Oosterhout et al. 2004) as well as. No locus was found to deviate from Hardy–Weinberg equilibrium. After applying sequential Bonferroni corrections to compensate for multiple statistical tests, no evidence of genotypic linkage disequilibrium (LD) was found between all pairs of these thirteen loci (P [ 0.0038). These thirteen loci will be useful in genetic diversity, population structure and other relevant studies of this species. Acknowledgments This study is supported by the research grants to Jin-Quan Yang from the National Natural Science Foundation of China (No. 31172066) and Shanghai Universities First-class Disciplines Project of Fisheries and by the Funding Program for Outstanding Postgraduate Dissertations to Li-Wei Su from SHOU.
Conservation Genet Resour (2014) 6:33–35
References Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Mol Ecol 16: 1099–1106 Kang B, He DM, Perrett L et al (2009) Fish and fisheries in the upper mekong: current assessment of the fish community, threats and conservation. Rev Fish Biol Fish 19:465–480
35 Raymond M, Rousset F (1995) GENEPOP (version1.2): population genetics software for exact tests and ecumenicism. J Hered 86:248–249 Su LW, Liu ZZ, Wang CT et al (2013) Isolation and characterization of polymorphic microsatellite markers in the fish Garra orientalis (oriental sucking barb). Conserv Genet Resour 5:231–233 Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538
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