270 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) ISSN (Print) 2313-4410, ISSN (Online) 2313-4402 © Global Society of Scientific Research and Researchers http://asrjetsjournal.org/ Genetic Diversity of Endangered Sheep Breed of Valley Swat, Pakistan, Based on Microsatellite Analysis Naveed Ullaha, Muhammad Tariq Zebb*, Mirza Ali Khanc, Muhammad Tahir Khand, Sibghat Ullahe, Muhammad Nauman-ul-Islamf, Azizullah Noorg, Ihsanullah Khanh, Muhammad Idreesi, Sohail Ahmadj, Ihsan uddink a,jDepartment of Animal Breeding and Genetics, The University of Agriculture, Peshawar, Pakistan. b,kCenter of Microbiology and Biotechnology, Veterinary Research and Disease Investigation Center, Swat, Pakistan. cCenter of Microbiology and Biotechnology, Veterinary Research Institute, Peshawar, Pakistan. dDepartment of Bioinformatics, Mohammad Ali Jinnah University, Islamabad, Pakistan. eFoot and Mouth Disease Research Centre, Veterinary Research Institute, Peshawar, Pakistan. fLivestock Research and developmental Station surezai, Peshawar, Pakistan. gHazara University, Mansehra, Pakistan. hLivestock and Dairy Development Department, Khyber Pakhtunkhwa, Peshawar, Pakistan. iCenter for Biotechnology and Microbiology, The University of Swat, Pakistan. bEmail: drtariqzeb@yahoo.com Abstract Morphologically, Kutta breed is black coat color sheep, with short tail and having thick wool fiber diameter. Weight differences in Kutta sheep exists as animals found in plain areas are slightly heavier and bigger than those found in hilly and remote areas of upper Kalam and Malamjaba of valley Swat with an average body weight of 23.2 ±0.34 kg. Aim of the study is to provide information on the genetic structure of the Kutta breed. A set of 31 FAO recommended microsatellite (SSR) markers was used to access the genetic variability in 21 DNA samples extracted. ------------------------------------------------------------------------ * Corresponding author. http://asrjetsjournal.org/ American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 271 The breed showed high level of genetic diversity. Out of the total 31 FAO recommended markers, 23 were fully amplified during Polymerase Chain reactions (PCR), having a total of 151 different alleles obtained, with an effective allele of (6.57) per locus. Similarly high level of average heterozygosity (0.77) and gene diversity (0.80) was observed. The within breed estimate (FIS) in breed was found extremely low (0.002). Shannon information Index (I) was found (1.65). The mean Chi-square value for the Hardy Weinberg Equilibrium (0.10) indicates that the population has lost its equilibrium on the overall basis. Majority of the markers were found neutral when test on Evens-Watterson, which suggest that decreases in heterozygosity in the population was not due to selection. Test for mutation drift equilibrium in the population showed that the population had preserved its effective size and has not experienced genetic bottleneck at least in the recent past. Keywords: Kutta sheep; Endangered; Microsatellite; Inbreeding estimate; Heterozygosity. 1. Introduction Sheep survive very well under harsh climatic condition. It is well adapted to various climatic conditions i.e. mountainous habitat to desert. Sheep was domesticated between 9000 and 11000 years ago. It is assumed that domesticated sheep descended from the wild sheep of Asia and Europe. Domestic sheep evolved from two wild stocks viz. European Moufflon (Ovismusimon) and Eastern moufflon (Ovisorientalis).Among the local European breed; remarkable population of moufflon is still found in certain parts of Europe. Both of the Moufflon stocks are considered as ancestor of domestic sheep [14]. Pakistani sheep breeds are assumed to be descended from Urail of Afghanistan, Argali of India and Marco polo of China however, recent cytological evidences decline it. Moreover, evidence of domestication exist in Bolan valley of Mehargar [2]. A total 28 indigenous breeds of sheep are found in Pakistan. Punjab and Khyber Pakhtunkhwa have seven each; Balochistan and Azad Jammu and Kashmir (AJK) have four each while Sindh and Northern Areas have three breeds each. In Pakistan sheep are kept for wool and mutton. However, majority Pakistani sheep’s have coarse wool texture [17]. Commercially important sheep breeds for commercial purposes in Pakistan are Rambouillet, Awassi, Kaghani, Damani, Kachhi and Lohi [17]. Kutta sheep is characteristically black coat; thin short tail sheep with an average weight of 23.2±0.34 kg. Utilization of the breed is limited to wool production that is used for manufacturing hand woven cloth; locally called “Lamsay & sharri”. The breed show remarkable resistance to enterotoxamia and Foot Mouth diseases. Delicious taste of its meat quality has another bright aspect which need to be highlighted [1]. However, as a result of indiscriminate crossing with other exotic breeds of sheep, the population is at extremely endangered. According to recently assessment report (September-October, 2012), less than 300 specimens remain in the entire region of Swat, of them 274 were ewes and only 23 were breeding rams. As nearly 20% of the documented breeds are considered to be under the threat of extinction. So far, of 7600 breeds documented in the databank animal Genetic Resources (AnGr) 11% got extinct and only 38% are safe from extinction [18]. Kutta is also among those breed which is going to extinct [1]. Keeping in view the endangered status of Kutta sheep; the present study was designed to genotypically characterize Kutta sheep using FAO recommended SSR markers. This will facilitate the conservation process for the breed. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 272 2. Material and methods 2.1 Blood sampling for DNA isolation Animal belonging to pure breed of Kutta sheep were selected from their breeding tract for blood collection. Three milliliter of blood sample was collected randomly from each individual. For this purpose jugular vein was punctured using disposable syringe and the blood was transferred to the labeled vacutainer. Before DNA isolation, the samples were carefully placed in a refrigerator at -20 ºC. 2.2 Genomic DNA extraction from blood and PCR DNA was extracted from blood following phenol chloroform extraction procedure. The details of procedure given elsewhere [12]. PCR reaction volume was 15 μL with 100ng total genomic DNA, 0.25 μM of each primer (forward and reverse), 200 µM of dNTP, 1 X PCR buffer (Fermentas), 1.5mM MgCl2and 1.0 unit of Taq DNA polymerase. Amplification condition were an initial denaturation at 94 ºC, followed by 30 cycles each consisting of a denaturation step of 1 minute at 94 ºC, annealing step of 1 minute, and an extension step of 2 minutes at 72 ºC. The last cycle was followed by 10 minutes extension at 72 ºC. All amplification reactions were carried out using Gene Amp PCR System 2700 (Applied Biosystem). For each and every DNA sample 31 ovine specific SSR markers (given in supplementary Table 1) were applied. The PCR product was analyzed on 10% polyacrylamide gel along with 1 Kbp DNA ladder. The gel was stained in ethidium bromide solution for twenty minutes and then was visualized under UV light. Each allele bands were manually scored from the gel. 2.3 Statistical Analysis Variations at different SSR markers among pure Kutta were analyzed using different computer software program. Each individual genotype banding pattern was scored manually from PAGE banding pattern. Observed and expected heterozygosity, effective number of alleles, observed number of alleles, allele’s frequencies and linkage disequilibrium between various combinations of loci was calculated using POPGENE software version 1.32 [21]. Allelic richness and estimates of inbreeding at different loci were taken to calculate gene diversity using [8]. Frequencies of null alleles were calculated using GENEPOP version 4.0 [15]. Polymorphism information (PIC) was calculated for each locus with the following mathematical equation [6] using allele frequency data. 3. Results Twenty one unrelated animals true to Kutta breed were selected randomly from their breeding tract. Blood samples from selected individuals were analyzed for 31 SSR markers at Institute of Biotechnology and Genetic Engineering, The Agricultural University Peshawar to characterize the population at molecular level. The level of genetic diversity found in Kutta population of Swat valley is presented. Total of 151 different alleles were examined at 23 polymorphic loci in which 109.3 with an average of 4.766 in Kutta population (Table 1). Observed number of alleles (Na) ranged from 4 in (in BM1824 HUJ616 and (OARVH 72 primers) to 9 (in American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 273 OARJMP58 primer). Effective number of allele (Ne) was very less than (Na) ranging from 2.348 (in OARFCB226) to 7.606 (in MAF70) (Table 1). Shannon information index (I) value ranged from 1.103 (in primer BM1824) to 2.108 (in primer MAF 70) with an average of 1.645 (Table 1). The Observed heterozygosity at different loci was different ranging from 0.222 (in OARFCB226) to 1 (in ILSTS5, SRCRSP1, MAF70, and MCM527) as indicated in Table 1. However, expected heterozygosity ranged from 0.590 in primer OARFCB226 to 0.900 in primer SRCRSP9. The overall average heterozygosity was 0.767 ranging from 0.574 in (OARFCB226) to 0.868 (MAF70). However, the observed heterozygosity (0.797) was almost similar to the expected heterozygosity (0.798) (Table 1). At about eight loci the expected heterozygosity exceeded the observed heterozygosity that included SRCRSP9, INRA063, SRCRSP5, OARFCB304, BM1824, ILSTS11 and ILSTS28 (Table 1). Table 1: Sample size, Number of observed and effective alleles, Shannon information index, Observed, Expected and Average Heterozygosity at 23 Microsatellite loci in Kutta sheep. Locus Sample size Na Ne I Ho He Hav SRCRSP9 8 8 6.400 1.960 0.625 0.900 0.844 MAF214 19 7 5.014 1.734 0.842 0.822 0.800 OARCP38 11 8 6.368 1.933 0.818 0.883 0.843 INRA063 8 6 4.129 1.560 0.750 0.808 0.758 SRCRSP5 12 8 6.698 1.969 0.583 0.888 0.850 ILSTS5 12 7 3.600 1.536 1.000 0.754 0.722 OARFCB304 17 8 6.084 1.903 0.706 0.861 0.836 OARFCB128 19 7 4.126 1.633 0.947 0.778 0.758 DYMS1 18 7 5.734 1.826 0.944 0.849 0.826 OARCP34 20 7 5.063 1.766 0.900 0.823 0.802 BM8125 19 5 4.057 1.496 0.895 0.774 0.753 BM1824 8 4 2.509 1.103 0.500 0.641 0.602 OARJMP58 19 9 6.623 2.010 0.947 0.876 0.849 SRCRSPI 17 7 4.313 1.616 1.000 0.791 0.768 MAF70 16 9 7.606 2.108 1.000 0.895 0.868 MCM527 19 7 5.429 1.779 1.000 0.838 0.816 ILSTS11 18 8 5.268 1.795 0.611 0.833 0.810 ILSTS28 15 6 4.500 1.603 0.800 0.805 0.778 BM1329 20 5 4.324 1.539 0.950 0.788 0.769 HUJ616 5 4 2.778 1.168 0.800 0.711 0.640 MAF209 16 5 3.160 1.370 0.687 0.706 0.684 OARFCB226 18 5 2.348 1.133 0.222 0.590 0.574 OARVH72 10 4 3.226 1.275 0.800 0.726 0.690 MEAN 15 6.565 4.766 1.645 0.797 0.798 0.767 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 274 With inbreeding the expected homozygosity increases at the expense of heterozygosity. The overall inbreeding estimate is indicated as (Fis) which was 0.002 for the Kutta population understudy (Table 2).The mean PIC value of the population was 0.678 per locus; ranging from 0.218 in primer (OARFCB226) to 0.850 in (MAF70) (Table 2 and Table 4). This value indicates that the set of marker used for this population has high degree of relevance for providing information regarding the genetic diversity of this population. The mean chi-square value for the Hardy Weinberg Equilibrium (0.097) indicates that the population has lost its equilibrium in general (Table 2). However, out of 23 loci 19 were in the state of equilibrium. The four loci viz. ILSTS28, BM1824, INRA063 andHUJ616 had exhibited a high degree of disequilibrium 0.361, 0.451, 0.458 and0.796 respectively (Table 2 and Table 4). Table 2: Genetic diversity, Allelic richness, Inbreeding estimate, Polymorphism Information Content and HWE P value at 23 Microsatellite loci in Kutta sheep. Locus Gdiv AR Fis PIC HWE P SRCRSP9 0.920 6.456 0.320* 0.801 0.049 MAF214 0.822 5.004 -0.025 0.747 0.018 OARCP38 0886 5.950 0.077 0.814 0.002 INRA063 0.813 4.837 0.077 0.646 0.458 SRCRSP5 0.902 6.058 0.353* 0.815 0.012 ILSTS5 0.742 4.580 -0.347 0.572 0.036 OARFCB304 0.866 5.611 0.185 0.748 0.006 OARFCB128 0.773 4.703 -0.225 0.644 0.000 DYMS1 0.846 5.358 -0.116 0.768 0.000 OARCP34 0.821 5.149 -0.096 0.736 0.000 BM8125 0.770 4.306 -0.101 0.677 0.005 BM1824 0.652 3.492 0.233 0.301 0.451 OARJMP58 0.870 5.922 -0.089 0.808 0.000 SRCRSPI 0.785 4.613 -0.274 0.678 0.001 MAF70 0.898 6.483 -0.114 0.850 0.001 MCM527 0.833 5.165 -0.200 0.775 0.000 ILSTS11 0.840 5.158 0.272* 0.742 0.049 ILSTS28 0.805 4.632 0.006 0.689 0.361 BM1329 0.784 4.437 -0.211 0.727 0.000 HUJ616 0.700 4.000 -0.143 0.580 0.796 MAF209 0.706 4.117 0.027 0.643 0.002 OARFCB226 0.601 3.471 0.630* 0.218 0.000 OARVH72 0.722 3.773 -0.108 0.628 0.001 MEAN 0.798 4.925 0.002 0.678 0.097 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 275 Test for non-random association between alleles belonging to different loci showed that 22 of the allele combinations were at frequency deviating significantly from expected (p < 0.05) (Table 3). Table 3: Linkage disequilibrium among different combinations of alleles at significance level (p<0.05) Locus Alleles Locus Alleles Co-relation Chi- square P-value SRCRSP9 C INRA063 E 2.0000 8.00 0.0047 SRCRSP9 G BM1824 C -1.3333 5.33 0.0209 MAF214 A OARFCB128 D 0.5000 4.25 0.0393 MAE214 A OARFCB128 G 0.5000 4.25 0.0393 MAF214 A MAF70 I 0.5000 4.00 0.0455 OARCP38 A BM1824 C -2.0000 8.00 0.0047 OARCP38 D BM1824 B -2.7080 14.67 0.0001 OARCP38 D BM1824 C 4.6904 44.00 0.0000 OARCP38 E BM1824 C -2.0000 8.00 0.0047 OARFCB128 D ILSTS11 H 0.5000 4.00 0.0455 OARFCB128 G ILSTS11 H 0.5000 4.00 0.0455 OARFCB128 A OARFCB226 E 0.5000 4.00 0.0455 OARCP34 A MAF70 A 0.5000 4.00 0.0455 OARCP34 A MAF70 C 0.5000 4.00 0.0455 BM1824 C HUJ616 C -2.0000 8.00 0.0047 OARJMP58 F SRCRSP1 A 0.5000 4.00 0.0455 OARJMP58 F SRCRSP1 C 0.5000 4.00 0.0455 OARJMP58 A ILSTS11 A 0.5000 4.25 0.0393 OARJMP58 C ILSTS11 A 0.5000 4.25 0.0393 SRCRSP1 A OARFCB226 E 0.5000 4.00 0.0455 SRCRSP1 B OARFCB226 A 0.5155 4.25 0.0392 SRCRSP1 C OARFCB226 E 0.5000 4.00 0.0455 Allele frequencies at 23 polymorphic loci in Kutta population are given in Table 4. Null alleles also called dis- functional allele, results due incomplete amplification during PCR were absent at four loci (ILSTS5, DYMS1, SRCRSP1 and MAF 70). Null alleles were present at very low rate in just six loci (INRA063, OARCP34, OARJMP58, ILSTS28, BM1329 and MAF209); however, majority is of higher value (twelve loci) with highest frequency of 0.264 at marker OARFCB226 (Table 4). American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 276 Table 4: Alleles frequency at different loci and Size of alleles in base pair observed at different loci in Kutta population Locus Null Allele size range A B C D E F G H I SRCRSP9 0.214 331-457 0.062 0.062 0.125 0.062 0.125 0.187 0.250 0.125 MAF214 0.200 177-240 0.078 0.052 0.289 0.157 0.157 0.026 0.236 OARCP38 0.169 351-457 0.181 0.045 0.136 0.181 0.045 0.045 0.181 0.181 INRA063 0.076 184-263 0.062 0.062 0.312 0.187 0.312 0.062 SRCRSP5 0.186 158-211 0.166 0.208 0.041 0.166 0.125 0.125 0.125 0.041 ILSTS5 0.000 200-240 0.041 0.041 0.291 0.083 0.416 0.083 0.041 OARFCB304 0.100 151-200 0.029 0.250 0.147 0.147 0.235 0.117 0.088 0.029 OARFCB128 0.015 116-174 0.052 0.342 0.078 0.052 0.105 0.315 0.052 DYMS1 0.000 134-209 0.055 0.222 0.111 0.138 0.222 0.194 0.055 OARCP34 0.090 105-153 0.125 0.200 0.125 0.050 0.050 0.325 0.125 BM8125 0.189 316-381 0.105 0.342 0.131 0.131 0.289 BM1824 0.125 174-210 0.125 0.562 0.250 0.062 OARJMP58 0.057 120-176 0.026 0.157 0.026 0.236 0.105 0.078 0.184 0.078 0.105 SRCRSP1 0.000 135-182 0.029 0.029 0.029 0.205 0.352 0.176 0.176 MAF70 0.000 166-219 0.156 0.062 0.156 0.093 0.062 0.093 0.093 0.187 0.093 MCM527 0.142 145-199 0.052 0.026 0.131 0.210 0.157 0.157 0.263 ILSTS11 0.188 257-334 0.027 0.222 0.250 0.027 0.222 0.138 0.027 0.083 ILSTS28 0.090 316-398 0.066 0.233 0.233 0.300 0.033 0.133 BM1329 0.084 209-263 0.350 0.175 0.125 0.150 0.200 HUJ616 0.198 135-152 0.500 0.100 0.100 0.300 MAF209 0.083 166-204 0.500 0.156 0.156 0.093 0.093 OARFCB226 0.264 122-162 0.083 0.611 0.194 0.083 0.027 OARVH72 0.106 250-351 0.450 0.250 0.150 0.150 Whether the population had gone bottleneck in recent past or not was tested by quantitative geographical method that elaborated the allele frequency spectra [11]. The microsatellite alleles were divided into 10 frequency classes in plot. Although alleles with low frequencies (0.01-0.1) were found abundantly, however, alleles distribution exhibited the normal L- shaped (Figure 1). The graphical illustration clearly showed that the population had not undergone bottleneck at least in the recent past. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 277 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.0 - 0.1 0.1 - 0.2 02 - 0.3 0.3 - 0.4 0.4 - 0.5 0.5 - 0.6 0.6 - 0.7 Proportion of alleles Allele frequency class Figure 1: Normal (L shape) distribution of alleles in allele frequency classes. 4. Discussion Color of Pakistani sheep breeds is mostly white, however brown, red or black color sheep are also common [17]. Morphology describing, Kutta sheep is black coated small sized thin tail breed. Males are horned and female are polled. In 2007 this breed has been classified as endangered species [1]. Because of this, it is necessary to gather information of the population’s genetic diversity distribution of the population to effectively design the conservation policy for this endangered species. Various alleles in diversity parameters were quite a high in the present study for Kutta sheep breed (Table 1); that is accordance with other domestic sheep breeds [3, 7, 16]. Kutta sheep breed yielded a total of 151 alleles in a sample population for 23 marker loci, averaging 6.565 observed and 4.766 expected alleles compared to Vembur sheep having a total of 147 alleles in 25 marker loci with a mean of 5.88 alleles per locus in the population [3]. Effective number of alleles (Ne) ranged from 2.348 (OARFCB226) to (MAF70) compared to effective allele mean per locus 0.405 in Shandi and Wadi breeds of China [22]. The observed number of allele in Garole sheep was (6.2) which is similar to the present study (ranged from four in (BM1824, HUJ616 and OARVH72) to nine (OARJMP58)) [13]. Kutta sheep have substantial genetic variation on the basis of gene diversity and average number of allele per locus. The gene diversity was (0.798), similar to the values reported for sheep breeds of Iran (Sanjabi 0.77, Zardi 0.77, Kajol 0.76 and Kolu 0.76) [19], Garole sheep 0.603 [20], Nali sheep 0.651, Chokla sheep 0.657 [13]. The average heterozygosity over all loci in Kutta sheep was (0.767). The high value of average heterozygosity within the breed could be attributed to high number of alleles (151) in present study [9]. In Kutta population the observed heterozygosity (0.797) was almost similar to the expected heterozygosity (0.798). At about seven loci, American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 278 the expected heterozygosity exceeded the observed heterozygosity i.e (SRCRSP9, INRA063, SRCRSP5, OARFCB304, BM1824, ILSTS11, and ILSTS28). In Kutta sheep breed the expected heterozygosity (0.798) was high from average heterozygosity (0.767) evidences for overall loss in heterozygosity [4].The observed number of allele was quite high than effective alleles at all of the loci, which justified the predominance of some alleles at almost each locus in a population [10]. However, in assessing diversity estimates from different studies, one should be careful not compare different values with each other, because different microsatellite markers have been used by different researchers. Therefore, these give just a suggestive indication of diversity in the population The overall inbreeding estimates (FIS) in Kutta sheep was (0.002) indicating a very low rate of inbreeding in the population. Higher heterozygosity values highlighted inbreeding of low level, slightly or without selection pressure and large number of alleles. The similar low values of FIS were also found in Muzzafarnagri sheep breed (0.058) [5]. In Ganjam sheep inbreeding estimate value was also found very low (0.087). Mean chi square value in Kutta sheep for HWE was (0.097) which indicates that the population has lost its equilibrium. Four loci i.e ILSTS28, BM1824, INRA063 and HUJ616 had exhibited a high degree of receptive disequilibrium (0.361, 0.451, 0.458, and 0.796). These results suggest ban of indiscriminate crossing with other exotic and local breeds to preserve this precious germplasm. This breed is under constant threat of invasion from Rambouillet and Bulkhi sheep breeds. 5. Conclusion and recommendation In conclusion the specimen size is drastically reduced in a recent past and efforts should be mobilized to conserve it before complete extinction of the breed might occur. It is one of the local sheep breed which possessed high genetic diversity. Its population has shrunken to an endangered level and drastic efforts are required for its in-situ and in-vivo conservation for future use. Policy may be designed and be strictly implemented not to permit cross breeding in the pure flocks at the habitat. 6. Funding/Support The study was conducted at the Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture, Peshawar, Pakistan. 7. Conflict of interest The authors confirm that there is no conflict of interest. Acknowledgments The authors of paper are thankful to Prof. Dr. Zahoor Ahmad Swati, (Ex-Director, IBGE), Vice Chancellor, The University Agriculture, Peshawar, Pakistan and Dr. Ijaz Ali, Principal Research Officer, Veterinary Research Institute, Peshawar, Pakistan for their moral and technical support. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2016) Volume 25, No 1, pp 270-280 279 References [1] Ahmad S, Jalil A, Pervaiz U, Khan H (2009) Morphological characterization of Kutta-the native sheep breed from Northern Pakistan. Sarhad Journal of Agriculture 25:279-284 [2] Ahmed M (2014) Ancient Pakistan-An Archaeological History: Volume II: A Prelude to Civilization. Amazon [3] Álvarez I, Royo L, Fernández I, Gutiérrez J, Gómez E, Goyache F (2004) Genetic relationships and admixture among sheep breeds from Northern Spain assessed using microsatellites. 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Ying yong sheng tai xue bao= The journal of applied ecology/Zhongguo sheng tai xue xue hui, Zhongguo ke xue yuan Shenyang ying yong sheng tai yan jiu suo zhu ban 17:1459-1464