Many wild animal species lack informative genetic markers for analysing genetic variation and structure, which is essential for effective long term conservation and management. We present heterologous microsatellite markers in six Tanzanian antelope species including: grant’s gazelle, hartebeest, eland, roan, impala and topi. Thirty eight primer pairs from cattle, sheep, goat and wildebeest were tested. Thirty three revealed polymorphisms in one or more of the six antelope species. Six were polymorphic across all tested species, providing evidence for high genetic variability across species. These sets of microsatellites are of particular usage in population genetic analyses of antelope species.Key words: Antelopes, genetic study, microsatellites, Tanzania

Eblate, EM

Knut, RH

Lughano, KJ

Peter, ML

Sebastian, CD

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 10(56), pp. 11817-11820, 26 September, 2011     Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.793 ISSN 1684–5315 © 2011 Academic Journals      Full Length Research Paper  Polymorphic microsatellite markers for genetic studies of African antelope species  Ernest M. Eblate1,2*, Kusiluka J. Lughano3, Chenyambuga D. Sebastian3, Msoffe L. Peter3 and Røed H. Knut2  1Tanzania Wildlife Research Institute, P.O. Box 661, Arusha, Tanzania. 2Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, N-0033, Oslo, Norway. 3Department of Veterinary Medicine and Public Health, Sokoine University of Agriculture, P.O Box 3021,  Morogoro, Tanzania.  Accepted 7 July, 2011  Many wild animal species lack informative genetic markers for analysing genetic variation and structure, which is essential for effective long term conservation and management. We present heterologous microsatellite markers in six Tanzanian antelope species including: grant’s gazelle, hartebeest, eland, roan, impala and topi. Thirty eight primer pairs from cattle, sheep, goat and wildebeest were tested. Thirty three revealed polymorphisms in one or more of the six antelope species. Six were polymorphic across all tested species, providing evidence for high genetic variability across species. These sets of microsatellites are of particular usage in population genetic analyses of antelope species.  Key words: Antelopes, genetic study, microsatellites, Tanzania.    INTRODUCTION  In Tanzania, an increasing habitat loss and fragmentation is becoming a major conservation concern for many wildlife species (Bolger et al., 2008; Voeten et al., 2009). Studies have shown that wildlife is declining both inside and outside the National Parks (Caro et al., 1998; Caro and Scholte 2007; Stoner et al., 2007). The causes of these declines are principally anthropogenic, resulting from growth of human population, coupled with intensified agriculture and active elimination of wildlife on adjacent lands (Serneels and Lambin, 2001; Caro and Scholte, 2007; Bolger et al., 2008; Voeten et al., 2009). Consequently, many protected areas in Tanzania are rapidly becoming isolated, which may disrupt or prevent dispersal and gene flow (Kuehn et al., 2007; Heller et al., 2010). Small isolated populations are associated with increased genetic drift, inbreeding and loss of adaptive genetic variation (Soulé and Mills, 1992; Amos and Harwood, 1998; Keller and Waller, 2002), which  calls  for     *Corresponding author. E-mail: ernest2002eblate@yahoo.com. Fax: +25527 2548240. increased effort through genetic conservation programs. Establishment of conservation programs requires an identification of any grouping of a species into distinct populations and an understanding of their genetic differences. However, the analysis of the genetic diversity and structure of natural populations requires availability of genetic maker systems with a relatively high degree of variability, to be able to resolve differences between individuals and populations (Røed and Midthjell, 1998). Microsatellites have been described as ideal makers for this purpose. These markers are highly polymorphic, codominantly inherited, abundant throughout the genome, and are thus widely used in population genetics and conservation studies (Tautz, 1989; Vial et al., 2003; DeYoung and Honeycutt, 2005). Although having species-specific primers for genetic studies may be the most accurate technique (Cosse et al., 2007), the characterization of species-specific primers is relatively laborious and expensive, involving cloning and sequencing (Ostrander et al., 1992; Galan et al., 2003). Microsatellite flanking sequences offer, however, conserved regions across closely related species and even across families, an advantage that has been utilized  11818        Afr. J. Biotechnol.    by several cross-species amplification studies (Røed, 1998; Lorenzini, 2005; Lorenzen et al., 2006). Antelope species are the most abundant among wild ungulate species in Tanzania and the objective of this study was to develop a panel of highly polymorphic microsatellites by the use of cross-species primers that can be used to efficiently study the genetic structure of the Tanzanian antelopes: Grant’s gazelle (Nanger granti), hartebeest (Alcelaphus buselaphus), eland (Taurotragus oryx), roan antelope (Hippotragus equines), impala (Aepyceros melampus) and topi (Damaliscus lunatus).   MATERIALS AND METHODS  Study area and samples collection  Serengeti Ecosystem (SE) is a geographical region located in northern Tanzania between 34° 45' to 35° 50' E and 2° to 3° 20' S, often defined by high diversity of wildlife and seasonal ungulates migrations. The ecosystem mainly includes the Serengeti National Park (SNP), the Ngorongoro Conservation Area (NCA), the Maswa Game Reserves (MGR) and surrounding game controlled areas which act as important buffer zones between human and wildlife. Peripheral blood samples were obtained from 15 individuals of each of Grant’s gazelle, hartebeest, eland, impala and topi from Serengeti National Park (SNP) after immobilization using etorphine hydrochloride in combination with xylazine hydrochloride. Immobilization was done by a qualified veterinarian during park routine procedures, taking into consideration the animals’ welfare. Eight roan muscle samples were obtained from hunted animals in the MGR. Prior to laboratory procedures, blood samples were frozen in EDTA tubes, while muscle samples were stored in absolute ethanol at room temperature.   Laboratory procedures and data analysis    Genomic DNA was isolated using Qiagen®, DNeasy Blood and Tissue kit (Qiagen GmbH, Germany) following the manufacture’s instruction. Samples were genotyped using 38 microsatellites primer pairs, 16 from bovine, six from ovine/caprine and 16 from blue wildebeest. The bovine/ovine/caprine primers were selected from previous successful cross-species amplification studies, while the wildebeest primers were novel. PCR assays were performed in a final volume of 10 µl at approximately 30 to 40 ng genomic DNA template, 2.3 µl of 10 x buffer, 1 µl dNTP, 2 pm of each primer, 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl and 0.5 U of Taq polymerase (Amplicon). Amplification was done in Gene Amp® PCR 9700 system (Applied Biosystems), with annealing temperatures varying between 56 and 58°C to suit individual primers. Control samples (species specific for the primers used) were included in each PCR run. Forward primers were fluorescently labeled for electrophoresis on an ABI 3100 DNA (Applied Biosystems) sequencer. Commercially prepared size standard (ROX GENESCAN® 400HD) was run with every sample. Alleles were scored using GeneMapper v3.7 (ABI 3100, Applied Biosystems), and new PCR’s were performed for samples where genotypes were unclear.  Furthermore, 10% of all the samples were selected at random and PCR and electrophoresis on ABI 3100 processes was repeated to verify the consistence of genotype scoring.  Genetic diversity was estimated from the mean number of alleles per locus, allelic richness, observed and expected heterozygosity (Nei, 1978) using FSTAT 2.9.3 (Goudet, 2001). Deviations from Hardy-Weinberg equilibrium   (HWE)    were    assessed    using    GENEPOP    v4.0     (Rousset, 2008). The demenorization number was 10,000 and the number of batches was 5,000. The significance level was sequentially Bonferroni adjusted for repeated tests (Rice, 1989). We used Micro-Checker 2.2.3 (Van Oosterhout et al., 2004) to test for null alleles, scoring errors and allele drop-outs. Only readable and polymorphic loci were considered during analyses.   RESULTS AND DISCUSSION  Of the 38 microsatellite primers tested, 33 were amplified alleles of the expected size range and were polymorphic on at least one of the six antelope species tested. Six microsatellites revealed polymorphism across all tested antelope species (Table 1). Four wildebeest primer pairs (CT08, CT14, CT18 and CT30) and one bovine primer pair (BM4208) were not polymorphic in any of the antelope species tested, though amplified products were present in the control samples which were DNA from wildebeest and cattle. The percentage of polymorphic loci were relatively high in topi and hartebeest (71.1%), medium in eland (52.6%), roan (52.6%) and impala (47.4%) and low in grant gazelle (26.3%). Mean number of alleles per locus, observed and expected heterozygosity are given in Table 1. After Bonferroni adjustment, none of the loci showed deviation from HWE in topi, roan and grant gazelle, however two loci in eland and impala, three in hartebeest retained significant values (Table 1), and were generally due to an excess of homozygotes. The use of highly polymorphic microsatellites loci has greatly increased the potential for understanding population structure of species across the landscape. Despite the relatively low sample size, our results showed that the amount of genetic diversity was substantially high for all the six species analysed. Six loci (BM804, ETH10, BM1009, MCM38, BM2113 and TGLA122) were polymorphic across all tested species and five additional loci were polymorphic in five of the six species analyzed (BM1824, BMC3224, MAF209, MCM58 and SPS115). These sets of primers have proven to have sufficient levels of polymorphism and heterozygosity and they provide enough power to analyse population structure of antelope species in Tanzania. None of the wildebeest primers showed cross-species amplification success across all species despite the closer taxonomic relationship of the analyzed antelopes towards the wildebeest as compared with the bovine, ovine and caprine. This could be due to the fact that the wildebeest primers used in this study are novel (Røed et al., 2011) and have not so far been tested in any cross-species amplifications, while most bovine/ovine/caprine primers used here were not randomly chosen, but have proven to be useful also in other cross-species amplification studies (Vaiman et al., 1996; Lorenzen and Siegismund, 2004; Lorenzini, 2005; Lorenzen et al., 2006; Cosse et al., 2007). Since species specific microsatellites for many wild animals are lacking, and the cost of cloning and sequencing is relatively high, Eblate et al.        11819    Table 1. Polymorphisms of microsatellite loci in the six antelope species including marker name, number of alleles per locus (Na), observed (Ho) and expected (He) heterozygosity and their mean values for the polymorphic loci. The number of individuals is given in brackets.  Marker Topi (15)  Eland (15)  Hartebeest (15)  Grant Gazelle (15)  Impala (15)  Roan (8) Na Ho He  Na Ho He  Na Ho He  Na Ho He  Na Ho He  Na Ho He BM804†† 8 0.867 0.802  11 0.786 0.849  6 0.786 0.728  2 0.533 0.497  3 0.133 0.301  5 0.500 0.533 BM203†† 10 0.933 0.880  11 0.667 0.922*  10 0.867 0.768  - - -  3 0.267 0.246  3 0.571 0.615 BM1824†† 8 0.667 0.749  4 0.133 0.193  8 0.733 0.814*  - - -  3 0.133 0.297  3 0.125 0.242 BM757†† 2 0.133 0.129  - - -  6 0.857 0.735  - - -  8 0.800 0.834  - - - BM1009†† 2 0.133 0.239  8 0.800 0.830  2 0.667 0.522  4 0.733 0.706  5 0.733 0.729  6 0.875 0.850 BM2113†† 8 0.733 0.772  3 0.133 0.131  5 0.600 0.618  4 0.667 0.710  4 0.400 0.352  8 0.875 0.858 BMC3224§ 8 0.867 0.832  11 0.933 0.890  11 0.933 0.906  5 0.533 0.637  4 0.667 0.616  - - - BM1818†† - - -  10 0.750 0.909  4 0.900 0.600  - - -  - - -  - - - BM4107†† - - -  9 0.667 0.830  - - -  - - -  4 0.933 0.756  6 0.875 0.833 ETH10‡‡ 3 0.600 0.577  8 0.867 0.832  7 0.600 0.717  5 0.692 0.766  7 0.600 0.747  4 0.665 0.783 INRA23¥ 8 1.000 0.837  9 0.600 0.779  6 0.786 0.836  3 0.133 0.131  - - -  - - - TGLA53† 13 0.867 0.936  - - -  6 0.867 0.759  7 0.867 0.782  3 0.600 0.618  2 0.375 0.325 TGLA122† 9 0.867 0.890  13 1.000 0.940  2 0.200 0.186  5 0.857 0.791  6 0.667 0.692  7 0.750 0.892 CSSM003‡ 8 1.000 0.858  - - -  8 0.800 0.768  - - -  - - -  5 0.714 0.791 CSSM066** 2 0.071 0.071  - - -  4 0.500 0.492  6 0.933 0.763  - - -  7 0.500 0.775 SPS115§§ 7 0.600 0.818  6 0.929 0.852  8 0.867 0.860  - - -  3 0.643 0.685  3 0.500 0.508 MCM58¥¥ 7 0.667 0.823  7 0.933 0.747  6 0.867 0.798  - - -  8 0.667 0.834  6 0.750 0.792 MCM38¥¥ 10 0.867 0.917  9 1.000 0.902  8 0.933 0.825  4 0.733 0.692  5 0.571 0.696  5 0.625 0.792 MCM104# - - -  - - -  5 0.750 0.761  - - -  - - -  - - - MAF209 4 0.867 0.674  5 0.600 0.766  7 0.800 0.830  - - -  3 0.357 0.370  3 0.375 0.425 OarFCB48 7 0.692 0.855  - - -  - - -  - - -  - - -  - - - CT02 8 0.467 0.660  - - -  - - -  - - -  - - -  - - - CT03 - - -  10 0.667 0.903*  - - -  - - -  - - -  - - - CT07 10 0.800 0.871  - - -  6 0.733 0.644  - - -  - - -  9 0.750 0.917 CT10 8 0.733 0.669  5 0.533 0.497  - - -  - - -  4 0.692 0.711  5 0.625 0.775 CT12 2 0.133 0.129  - - -  7 0.467 0.738*  - - -  - - -  7 0.750 0.867 CT13 8 0.867 0.864  - - -  5 0.467 0.713  - - -  - - -  - - - CT17 7 0.615 0.849  9 0.733 0.846  7 0.692 0.818  - - -  - - -  3 0.375 0.625 CT19 - - -  - - -  3 0.143 0.624  - - -  - - -  - - - CT21 - - -  9 0.643 0.847  7 0.429 0.751*  - - -  - - -  - - - CT23 8 0.933 0.874  - - -  3 0.357 0.537  - - -  4 0.467 0.609  - - - CT25 8 0.417 0.855*  - - -  8 0.385 0.474  - - -  5 0.643 0.701*  5 0.625 0.767 CT27 9 0.733 0.846  5 0.429 0.709  - - -  - - -  - - -  - - - Mean 7.11 0.671 0.718  8.10 0.690 0.759  6.11 0.666 0.698  4.50 0.668 0.648  4.56 0.554 0.599  5.10 0.610 0.698 11820        Afr. J. Biotechnol.    Table 1 Contd.  SD 2.8 0.28 0.25  2.7 0.25 0.23  2.2 0.23 0.15  1.4 0.23 0.20  1.7 0.22 0.19  1.9 0.19 0.19  *Deviation from HWE. ††, Bishop et al. (1994); §, Kappes et al. (1997); ‡‡, Solinas-Toldo et al. (1993); ¥, Vaiman et al. (1994); †, Georges and Massey (1992); ‡ Moore et al. (1994); **, Barendse et al. (1994); §§, Moore and Byrne (1993); ¥¥, Hulme et al. (1994); #, Smith et al. (1995); , Buchanan et al. (1992); all CT primers: Røed et al. (2011).    the use of heterologous primer pairs may be a cost effective alternative in conservation manage-ment programs specifically in many developing regions.   ACKNOWLEDGEMENTS  We thank the Norwegian Programme for Development, Research and Education (NUFU) for funding the study. We also thank Liv Midthjell and all Antelope Conservation Project members for their assistance during this work. Conservation authorities in Tanzanian are acknowledged for the permission to sample wildlife.   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Polymorphic microsatellite markers for genetic studies of African antelope species

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Polymorphic microsatellite markers for genetic studies of African antelope species

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