ГЕНЕТИКА, 2011, том 47, № 7, с. 996-999
КРАТКИЕ СООБЩЕНИЯ
УДК 575.1:599.824
ISOLATION AND CHARACTERIZATION OF TWENTY-ONE POLYMORPHIC MICROSATELLITE LOCI IN THE TIBETAN MACAQUE (Macaca thibetana)* © 2011 X. D. Jia1, 2, B. D. Yang1, B. S. Yue3, H. L. Yin4, H. X. Wang5, X. Y. Zhang1
1Sichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University,
Chengdu, Sichuan 610064, China e-mail: zhangxy317@126.com 2College of Resource and Environmental Engineering, Mianyang Normal University, Mianyang, Sichuan 621000, China 3Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), Sichuan University,
Chengdu, Sichuan 610064, China 4Laboratory Animal Center, Sichuan University, Chengdu, Sichuan 610041, China 5Institute of Laboratory Animal Science, Academy of Medical Sciences, Chengdu, Sichuan 610065, China
Received November 11, 2010
Twenty-one microsatellite loci were isolated from AC-enriched library of Tibetan macaque (Macaca thibetana). The number of alleles at the 21 microsatellite loci ranged from 8 to 15, with an average of12.2 per locus. Polymorphism information content (PIC) ranged from 0.805 to 0.910 with an average of 0.873. The observed and expected heterozygosities ranged from 0.208 to 0.792 and from 0.843 to 0.938, respectively. These microsatellite loci will be useful for future studies that relate to the genetic diversity and population structure of Tibetan macaque.
The Tibetan macaque (Macaca thibetana), also known as the Chinese stump-tailed macaque or Milne-edwards' macaque, which belongs to the family of Cercopithecid-ae, is mainly distributed in Sichuan, Guizhou, Anhui, Guangdong, Fujian, Guangxi, Jiangxi, Zhejiang, Hunan, and Yunnan provinces, China [1]. This species is usually considered an endemic monkey to China and mainly found in subtropical, deciduous and broadleaf evergreen forest, ranging from 800 to 2500 m in elevation [2]. However, it has also been reported in Arunachal Pradesh, India, in 2005 [3]. Due to many anthropogenic disturbances, such as serious deforestation, farmland development and illegal poaching, the wild populations ofM. thibetana have markedly declined in recent years. Now, this species is listed under CITES Appendix II and also classified as a "near threatened" species in the IUCN Red List of Threatened Species. Meanwhile, the Tibetan macaque can be used as an animal model for studies related to human health and disease [4]. So, some captive populations of Tibetan macaque were established to facilitate medical research, but there is limited genetic information available for these captive populations. In order to provide appropriate genetic data for artificial propagation and breeding, it is essential to study their genetic diversity and population genetic structure. In the recent years, microsatellites have become one of the most popular molecular markers used in research ofpopulation structure and genetic background. However, there is no works involve in isolation of microsatellites and their application in investigation ofge-netic structure of the Tibetan macaque. In this study, we
* Xiaodong Jia, Biao Yang contributed equally to this work.
isolated 21 polymorphic microsatellite loci and represented their characterizations.
A total of 24 Tibetan macaque individuals were included in this research. They belonged to the captive population in the Laboratory Animal Center in Jian-yang County of Sichuan Province. The blood samples for DNA extraction were obtained from the 24 individuals during medical examinations. The samples were stored at —80°C in laboratory.
Total genomic DNA was extracted from the blood sample following phenol-chloroform method according to Sambrook and Russell [5]. An enrichment DNA library for AC-repeats was constructed using the protocols described by Bloor et al. and Zou et al. [6, 7]. Genomic DNA was digested with the Sau3AI restriction enzyme (TaKaRa, China). The digested DNA fragments of 300 to 900 bp were purified from the gel using E.Z.N.A. Gel Extraction Kit (OMEGA, USA) and ligated to Sau3AI adaptors (annealed products of oligo A, 5'-GGCCAGAGACCCCAAGCTTCG-3' and oligo B, 5'-pGATCCGAAGCTTGGGGTCTCTGGC-3') using T4 DNA ligase (TaKaRa, China). The ligated fragments were amplified using oligo A as the primer in order to test successful ligation. The adaptor-ligated fragments were hybridized with biotinylated (AC)12 probe at 60°C for 30 min, and incubated with streptavi-din-coated magnetic beads (Dynal, Norway) at 70°C for 3 h. Enriched DNA fragments were amplified by PCR using oligo A as the primer. The products were purified and cloned into pMD18-T vector (TaKaRa, China), and then transformed into competent JM109 Escherichia coli cells. The positive clones with microsatellites were selected by PCR using oligo A and nonbiotin-
Primer sequences and characteristics of 21 microsatellite loci isolated in Macaca thibetana
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Locus/ GenBank accession no. Primer sequence (5— 3') Repeat motif ra(°C) N Size range Ho He PIC
Mat006/HQ238939 F-TTATGACACTCACATAA R-AGCTACTTCAACCATC (AC)16 62 24 12 320-356 0.583 0.892 0.861
Mat013/HQ238940 F-CCCAAGACTGGGCAATTT R-AAAGAGGTTTACTGG (AC)21 60.5 23 10 386-422 0.522 0.843 0.805
Mat018/HQ238941 F-AGTGGGGGACTGCATTC R-AAATCCAGGCCATT (AC)15 59.5 24 10 184-206 0.583 0.875 0.841
Mat023/HQ238942 F-CAAAACAAATGAATACAG R-TCATGCCAAGATGGCAAAGGATG (AC)19 60.5 22 8 176-190 0.318 0.869 0.831
Mat030/HQ238943 F-ATAAAAGAGTTATCTTACCTTGTCT R-AGAAAGCTAGCAATTTAGA (TC)3TT(TC)6CC(TC)5(AC)12AG(AC)6 59 24 12 424-462 0.208 0.907 0.878
Mat033/HQ238944 F-TTTCTGGGTTTCCCCCATTC R-TTGCTTTCCTTTCTACTGCT (AT)3(AC)8GC(AC)14AT(AC)2(AT)5 59 24 11 218-246 0.417 0.910 0.881
Mat034/HQ238945 F-ACCAGATAAAAACTGGTAGCTTC R-AATGTTAATTTCCATTTGAC (AC)19 61.3 24 14 302-378 0.708 0.925 0.898
Mat036/HQ238946 F-AACCACCTACCTACCTACCTAG R-CTGCAGTTCATTCACACTGT (AC)17 60 24 13 418-470 0.542 0.917 0.889
Mat037/HQ238947 F-ACAGAGTGAGACCTTGTCTCACGT R-AAAATAAAATATTGGTATTG (AC)22 59 24 12 324-358 0.417 0.876 0.845
Mat045/HQ238948 F-CAATAAGTTTTGGATGAGACATT R-CCCAGGTGACCATAAAA (AT)2GG(AG)4(AT)2T(AT)4GTGTAT(AC)12TC(AC)6(AI)5 59 24 12 268-292 0.500 0.905 0.876
Mat049/HQ238949 F-TATTCTTGTACCTCTAAGACAACAA R-GGTGTATATCTTTTCCTTT (AC)16 59.4 24 11 328-360 0.333 0.887 0.856
Mat057/HQ238950 F-GACCTTTGGTTCTAACCACAGACAG R-CCCATCCTTAGACACTTCCT (AC)7GC(AC)16 59 22 15 294-334 0.455 0.938 0.910
Mat059/HQ238951 F-GAAAGAGTGGGACAAGTGAAG R-GCAGCTTTGAGAAAGGGCG (AC)13(AG)n 58 23 13 286-320 0.609 0.902 0.872
Mat060/HQ238952 F-CTTCTAAGAAGAGGAAGACAGAT R-AAGGTTATGTGAGCACAGGCG (TC)5TGTG(TC)7C(AC)5ATGC(AC)3ATGC(AC)7 62 24 11 176-198 0.792 0.904* 0.874
Mat062/HQ238953 F-AGCTTCCTCTGTCTGCTTC R-CCTCTCATCCCATGACTACT (AC)22 58 23 14 320-382 0.522 0.920 0.891
Mat064/HQ238954 F-AAGCTGACTACTGTTACCA R-TTCCAGTGGCTGAACATAATAGTTC (ACACAG)3(AC)20 58.3 21 12 298-336 0.476 0.909 0.877
Mat065/HQ238955 F-TTTTACTCAGTTAAGACACAT R-CCCAAGGAGAGAAGTACATAATTAAT (AC)i3 63.9 23 12 194-218 0.304 0.923 0.894
Mat066/HQ238956 F-CTACCTTGCAGGATTAGAAGGT R-AGGTATTCATACACATATACAT (AC)21 58.3 24 14 374-440 0.542 0.917 0.889
Mat078/HQ238958 F-TATGTGCCAGGTGCACATGC R-ATGCATTAGAAGCCACTTAGAGAA (AC)1s 58.3 23 14 300-346 0.522 0.917 0.888
Mat080/HQ238959 F-AGTAGCAGGCCAAGAACTGCT R-ATAGAGTAATTAACTACAGAGGA (AC)16(AG)2(AC)4AG(AC)10 65.3 21 14 424-478 0.429 0.911 0.879
Mat089/HQ238960 F-AATCTAAATTATTCCCCCCCAA R-GATGCTGTCCCCTGAATAAAA (AC)1S 65.3 24 13 298-326 0.625 0.931 0.905
Note: 7"a, annealing temperature of primer pair; N, number of individuals genotyped; iVa, number of alleles; HQ, observed heterozygosity; He, expected heterozygosity; PIC, polymorphism information content; * indicated conformed to Hardy—X^inbeig expectations.
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labelled (AC)12 as primers. Insert fragments of positive clones were sequenced using ABI 3730 Genetic Analyser (Applied Biosystems).
A total of 96 positive clones were sequenced, 73 of which contained AC-repeat microsatellites, and 59 had sufficient flanking region to design primers. The design of primers was performed using the software primer 3.0 [8], and PCR conditions of each primers were optimized with a gradient PCR iCycler (Bio-Rad, USA).
PCR was carried out in a 25 |L reaction volume containing 50 ng genomic DNA, 1xPCR buffer (TaKaRa, China), 1.0-2.5 mM MgCl2, 0.5 |M of each primer, 50.0 |M of each dNTP and 1.0 U TaqDNA polymerase (TaKaRa, China). The final optimized PCR conditions were as follows: 95°C for 5 min, followed by 35 cycles consisting of 94°C for 30 s, a primer-specific annealing temperature (Table) for 50 s, 72°C for 50 s, and a final extension at 72°C for 10 min.
We used the 24 Tibetan macaque individuals to assess polymorphisms of the obtained loci. PCR products were separated on 8% urea-polyacrylamide gel along with the DNA ladder pUC19 DNA/MspI (HapII) (MBI Fermentas) and visualized with the silver staining protocol described by Zou et al. [7].
The size and number of alleles was determined with GeneTool software (BioTools Incorporated, Canada). Observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were calculated using CERVUS 2.0 [9]. Hardy-Wein-berg equilibrium (HWE) and linkage disequilibrium (LD) test were carried out using Genepop 3.3 [10].
The result indicated that 21 loci showed high polymorphisms. The number of alleles per locus ranged from 8 to 15, with an average of 12.2. The observed and expected heterozygosities ranged from 0.208 to 0.792 and 0.843 to 0.938, respectively (Table). Polymorphism information content (PIC) ranged from 0.805 to 0.910 with an average of0.873. Each pair of loci was tested for LD across populations and four pairs of loci showed linkage (Mat033-Mat036, Mat036-Mat037, Mat036-Mat062 and Mat059-Mat065) (P < 0.05), so these markers should be used selectively. HWE tests showed that all but one (Mat060) deviated significantly from Hardy-Weinberg expectations (P < 0.05) and all the
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