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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