Isolation and characterization of thirteen microsatellite loci for the ...

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Sep 28, 2013 - Isolation and characterization of thirteen microsatellite loci for the western black crested gibbon (Nomascus concolor) by high-throughput ...
Conservation Genet Resour (2014) 6:179–181 DOI 10.1007/s12686-013-0041-3

MICROSATELLITE LETTERS

Isolation and characterization of thirteen microsatellite loci for the western black crested gibbon (Nomascus concolor) by high-throughput sequencing Naiqing Hu • Orkin Joseph • Bei Huang Kai He • Xuelong Jiang



Received: 14 August 2013 / Accepted: 23 September 2013 / Published online: 28 September 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract The critically endangered western black crested gibbon (Nomascus concolor) lives only in isolated mountainous areas of China’s Yunnan province, northern Vietnam, and northwestern Laos. A lack of genetic samples and polymorphic molecular loci has limited our ability to investigate the genetic diversity of this species. We collected fecal samples from 12 free-ranging individuals in the Wuliang Mountains of Yunnan and developed 13 polymorphic microsatellite loci using 1/8 plate of a Roche 454 GSFLX sequencing run. The number of alleles ranged from

Hu Naiqing and Joseph Orkin have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s12686-013-0041-3) contains supplementary material, which is available to authorized users. N. Hu  B. Huang  K. He  X. Jiang (&) State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan 650223, People’s Republic of China e-mail: [email protected] N. Hu University of Chinese Academy of Sciences, Beijing, People’s Republic of China O. Joseph State Key Laboratory of Genetic Resources and Evolution, Ecology, Conservation, and Environment Center (ECEC), Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, People’s Republic of China O. Joseph Department of Anthropology, Washington University in St. Louis, St. Louis, MO, USA

three to eight per locus, and the observed and expected heterozygosities were 0.333–0.917 and 0.424–0.822, respectively. These loci will be useful tools not only for future studies of the social structure and population genetics of N. concolor, but also will facilitate an informed conservation management plan. Keywords Western black crested gibbon  Nomascus concolor  Microsatellite  High-throughput sequencing

Approximately 1,500 critically endangered western black crested gibbons (Nomascus concolor) remain in the world. To address ecological questions and facilitate their conservation, it will be crucial to quantify the genetic diversity within and among populations and to understand their dispersal. In this study, we shotgun sequenced whole genomic DNA from one individual and developed polymorphic microsatellite loci, which will allow for a meaningful contribution to their conservation. We extracted genomic DNA from a muscle sample of Nomascus concolor furvogaster. Genomic DNA was extracted by a standard phenol-chloroform method and underwent 454 sequencing with a GS-FLX (Roche, Germany) sequencer using 1/8 of a reaction plate. 91,426 reads with an average length of 342 bp obtained from the 454 run were screened for microsatellites, and 40 primer pairs were designed. 12 fresh fecal samples of N. concolor collected in the Wuliang Mountains were used for polymorphism screening. We desiccated fecal samples with silica after [24 h of storage in 99.9 % ethanol (Nsubuga et al. 2004) and extracted DNA using the 2CTAB/PCI method (Vallet et al. 2008). 2-step multiplex PCR was performed in 10 lL reactions following Arandjelovic et al. (2009), with an initial

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R: GCCTGTCACTGGCCAGGT

R(out): TTACTGATAATGTGCCTGTCACT

F: CCCCATCATGTGAGCTCAAAG

R: ACCGTCGCCTAGCCTCCT

R(out): ATGAGCCACCGTCGCCTAG

F: ACCCTGGGGCCTGTCAAT

R: GCAGGCAATTTATTCCAACAACAG

F:CTACTCTGAAGAACAAATCAGGTC

R: TAAATTGCACAGTCTTGGGTATG

F: GGTAACATCCACAGCTTGCTAAC

R: GTTAAGCCTCCCTCCAAACCC

F: CCTGACCACAGTAGGCACTC R(out): AAGTCAGTTAAGCCTCCCTCC

R: AGATTCCGCCATTGCACTCC

F: GAGTTCAGCCAGGTTTGGAC

R: GCGCCAATTGAAAGACTACCCTAT

R(out):GGGGGACACGAAAGCCAATT

F: CGACACATGCCCAGATTCC

R: AGAGGCAGATCACGCCACTG

R(out): CTTGAACCTGGGAGGCAGATC

F: ATCCAACCAGATCCAACATGAAG

R: GGTCATGCTCCATAATCATGTGAG

R(out): GACTTGAGTTTAGCTATGCCACT

F: TGCCCTCAGTCCTCCAATCTC

R: GGGGCAGTGTTAGCCGAGTAG

F: GGCACAACCAGTTACAGATTTCG

R(out): AGAACTGGAAGAACAGGACAAGC R: ACTGGAAGAACAGGACAAGCAGG

F: GCGAGTTACCAGCGAGATTG

R: AGCCAGACTCCACCTCAAAG

* significantly departed from HWE, * p \ 0.05; ** p \ 0.01

NC38

NC37

NC36

NC35

NC34

NC33

NC32

NC28

NC23

NC22

NC20

R(out):GCAGTGAGCCAAGATCATACC

F: TTCTCTTCTGGCTCTGCAGG

R: CCCTCGCTCCATTTGAAGAG

F: CCAAACAAAGGCATGACACTG

NC15

NC17

Primer sequence (5’-3’)

Locus

56–61

56–61

56–61

56–61

56–61

56–61

56–61

52–57

56–61

56–61

56–61

56–61

56–61

Ta (°C)

Table 1 Characteristics of 13 microsatellite loci for (N. concolor)

(AAT)n

(AAT)n

(AAAT)n

(AAAT)n

(AACC)n

(AGAT)n

(AGAT)n

(ACAT)n

(AGAT)n

(AAT)n

(AC)n

(AGAT)n

(ACTC)n

Array

243–255

210–246

206–237

218–230

202–210

119–143

153–165

226–250

182–202

227–240

160–182

5

6

8

4

3

5

4

4

6

5

6

2

4

150–162 176–180

Alleles

Size range (bp)

12

12

12

12

12

11

11

12

12

12

12

12

11

Individuals

0.750

0.750

0.667

0.750

0.500

0.545

0.727

0.500

0.917

0.750

0.667

0.333

0.545

HO

0.743

0.710

0.815

0.659

0.424

0.810

0.727

0.543

0.822

0.746

0.822

0.507

0.758

HE

0.665

0.643

0.755

0.561

0.371

0.736

0.640

0.451

0.756

0.661

0.759

0.368

0.673

PIC

0.500

0.783

0.014*

0.849

1.000

0.005**

0.637

0.453

0.775

0.549

0.008**

0.258

0.040*

PHW

-0.028

-0.040

0.070

-0.088

-0.131

0.155

-0.017

0.031

-0.080

-0.025

0.074

0.186

0.134

Null allele frequency

180 Conservation Genet Resour (2014) 6:179–181

Conservation Genet Resour (2014) 6:179–181

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Table 2 p values for test of linkage disequilibrium among microsatellite loci Microsatellite loci

NC15 NC17 NC20 NC22 NC23 NC28

NC15

NC17

NC20

NC22

NC23

NC28

NC32

NC33

NC34

NC35

NC36

NC37

NC38

-

0.835

1.000

0.289

1.000

0.646

1.000

1.000

0.874

0.519

0.149

0.277

1.000

-

0.900

0.923

1.000

0.002**

0.834

0.850

0.026*

0.407

0.477

0.195

1.000

-

1.000

1.000

0.860

1.000

1.000

0.936

0.780

1.000

0.437

0.556

-

0.260

0.392

1.000

0.436

0.191

0.245

1.000

0.470

0.465

-

1.000

0.208

0.157

1.000

1.000

1.000

1.000

0.296

-

0.818

1.000

0.604

0.098

0.434

0.104

1.000

-

1.000 -

0.354 0.872

0.583 1.000

1.000 1.000

1.000 1.000

1.000 1.000

-

0.493

0.553

0.616

0.128

-

1.000

0.746

0.788

-

0.168

1.000

-

1.000

NC32 NC33 NC34 NC35 NC36 NC37 NC38

-

* Significantly departed from linkage equilibrium, * p \ 0.05; ** p \ 0.01

denaturing of 15 min at 95 °C, followed by 30 cycles of 85 °C for 30 s, 57 °C for 3 min, and 72 °C for 60 s; a final extension was performed at 60 °C for 30 min. The second multiplex PCR was performed with the same conditions as above, using (6-FAM, HEX) labeled reverse primers and 1:100 dilution of the first PCR product. A negative control was processed with each set of PCRs. A locus was accepted as heterozygous if each allele was observed independently at least twice, and as homozygous if it occurred independently at least 5 times. PCR products were genotyped on an ABI 3730xl genetic analyzer (Applied Biosystems) and GeneMarker. Cervus 3.0 (Marshall et al. 1998) was used to calculate the number of alleles and observed and expected heterozygosities. Null gene frequency, deviations from Hardy-Weinberg equilibrium, and linkage disequilibrium tests were performed with GENPOP 4.1 (Rousset 2008). The characteristics of the 13 microsatellite loci are listed in Table 1. We observed no evidence of scoring errors. The number of observed alleles per locus ranged from three to eight. Observed and expected heterozygosities ranged from 0.333 to 0.917 and 0.424–0.822, respectively. Four loci deviated from Hardy-Weinberg equilibrium due to heterozygote deficit, likely the result of null alleles. Two pairs of loci showed significant linkage disequilibrium (Table 2), but when an additional 11 samples from five other sites in Yunnan were added to the dataset (not published), linkage equilibrium was maintained. Thus, the linkage disequilibrium might be resulting of population structure among gibbon subpopulations. We provide the first Nomascus-specific microsatellites, further expanding the number of potential microsatellites for hylobatids. The contribution of these loci provides a solid foundation for ongoing molecular ecological research into these apes.

Acknowledgments This study was supported by the National Natural Science Foundation of China (31070349, 31170498), Yunnan Provincial (2011FB105), Chinese Academy of Science (KSCX2-EWZ-4-1, GREKF11-04), the National Science Foundation (NSF OISE1015770, NSF BCS-1155904), The Leakey Foundation, The American Philosophical Society Lewis and Clark Fund, Lambda Alpha, and Washington University in St. Louis. Thanks are given to The Key Lab of Animal Ecology and Conservation Biology, Institute of Zoology, The Chinese Academy of Sciences, for technical guidance. Thanks to Douglas Yu, Ji Yinqiu, and Yang Yahan for valuable advice and assistance, and Alan Templeton for commenting on a draft of this paper. Special thanks to Fan Pengfei, Sun Guozheng, Guan Zhenhua, Ni Qingyong, Wang Xiaowei and Ning Wenhe for collecting samples. We thank the staff of the Jingdong Nature Reserve Management Bureaus for their much-needed support.

References Arandjelovic M, Guschanski K, Schubert G, Harris TR, Thalmann O, Siedel H, Vigilant L (2009) Two-step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples. Mol Ecol Resour 9(1):28–36 Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statistical confidence for likelihood-based paternity inference in natural populations. Mol Ecol 7(5):639–655 Nsubuga AM, Robbins MM, Roeder AD, Morin A, Boesch C, Vigilant L (2004) Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method. Mol Ecol 13:2089–2094 Rousset F (2008) GENEPOP’007: a complete re-implementation of the GENEPOP software for Windows and Linux. Mol Ecol Resour 8(1):103–106 Vallet D, Petit EJ, Gatti S, Levrero F, Menard N (2008) A new 2CTAB/PCI method improves DNA amplification success from faeces of Mediterranean (Barbary macaques) and tropical (lowland gorillas) primates. Conserv Genet 9:677–680

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