Genetic variation among pelt sheep population using microsatellite markers

1 Department of Animal Science, Faculty of Agriculture, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2 Department of Animal Biotechnology, Animal Science Research Institute of Iran, Karaj, Iran. 3 Department of Molecular Biology, Iran University of Medical Sciences, Tehran, Iran. 4 Animal Science Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. 5 Department of Animal breeding, Animal Science Research Institute of Iran, Karaj, Iran.


INTRODUCTION
Awareness of the value of animal genetic resource is currently prompting deliberate efforts at optimum utilization and conservation of the species.The maintenance of genetic diversity is a key to the long-term survival of most species (Hall and Bardley, 1995).The genetic polymor-population of 500,000, 500,000 and 300,000, respectively (ASRI, 2004).In the past, the Gray Shiraz, Karakul and Zandi were selected for production of good quality pelts mainly for exports.Now, due to the increased demand of the meat in the country, these breeds are mainly oriented towards the meat and lamb production (Moradpour, 1993).The Gray Shiraz and Karakul sheep breeds are from the same origin.Gray Shiraz has a slightly smaller size than Karakul (Tavakolian, 1999).Gray Shiraz is found in South of Iran (Fars province).Karakul is very resistant to harsh condition and is raised mostly under the semi nomadic system of management.Karakul breed is mainly found in the plain of Sarakhs in the province of Khorasan, neighboring Turkmenistan.Zandi breed is similar to Gray Shiraz and Karakul (Moradpour, 1993).It has a slightly smaller size than both two mentioned breeds.This breed is mainly found in parts of Tehran, Semnan, Ghom provinces and some parts of the Central province.These breeds are medium size, meat and pelt type, with coarse wool breed (Tavakolian, 1999).Information about population genetics is one of the most important factors in animal breeding.In order to estimate genetic diversity in the populations, molecular markers like microsatellites are useful tools (Esmaeilkhanian and Banabazi, 2006).Microsatellites are stable, polymorphic and easy to analyze; this occurs regularly throughout an animal genome as well.
Microsatellites are co-dominant markers, so that all alleles can be scored.Several studies had investigated the genetic diversity in sheep using microsatellites (El Nahas, 2008;Dalvit et al., 2008;Mahmoudi and Babayev, 2009;Sharifi sidani et al., 2009;Kusza et al., 2010).The aim of this study is to use the molecular data to evaluate genetic variability, gene flow and inbreeding in sheep flocks, and also for designing a breeding strategy aimed at incrementing genetic diversity within and between pelt sheep breeds.

Blood sampling and DNA extraction
Blood samples of 360 sheep of both sexes, were randomly collected from three pelt breeds: Gray Shiraz, Zandi and Karakul from distant located experimental stations belonging to the Animal Science research Institute.Genomic DNA was extracted from fresh or frozen blood using modified salting-out (Miller et al., 1988) and DNA Extraction kit (Diatom DNA Prep 100).DNA concentration was determined using a UV spectrophotometer.

Microsatellite polymorphism detection
Fifteen microsatellite markers were selected in respect of polymorphisms, a non-linkage criterion for syntenic loci and criterion of location on different chromosomes (Table 1).Microsatellites were amplified with polymerase chain reaction (PCR) using genomic DNA extracted from individual animals.The PCR was performed for each locus in 15 µl reaction mixture consisting: 1X buffer, 200 µM dNTPs, 1.5 -4.5 mM MgCl2, 0.25 µM of each Primers , 1 units Taq polymerase and 100 -200 ng DNA.The reactions were done with Gradient Master Cycler Ependorf.The cycling protocol was as follows: 5 min denaturing at 95°C followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 52 -63°C (depending on primers) for 30 s, extension at 72°C for 45 s and the final extension at 72°C for 5 min.Amplification products were electrophoresed on 8% denaturing polyacrylamide gels and stained according to rapid silver staining procedure.The gel was photographed using Gel-Doc XR (BioRad).Patterns of the different genotypes for each microsatellite locus were analyzed using Gel-Pro analyzer, version 3.1 for windowsTM, which determines the allele's sizes in each animal.

Statistical analysis
Allele frequencies at each locus for each breed were calculated using a computer program GenAlEx 6 (Peakall and Smouse, 2006).GenAlEx 6 software program was also used for calculating the observed and expected heterozygosity overall loci in each of the three breeds (Levene, 1949;Nei, 1978).Test of departure from Hardy-Weinberg equilibrium at each locus overall breeds were performed using Chi-square and Likelihood ratio or G-square test (Hedrick, 2000) using GenAlEx 6 program and POPGENE software version1.31(Yeh et al., 1999), respectively.GenAlEx 6 program was also used to calculate F-statistics that include three indices: Fit, Fst (genetic differentiation) and Fis (within breed heterozygosity deficit).Polymorphism information content (PIC) was calculated using HET software version 1.8 (Ott, 2001).The genetic distance (DS) Nei (1972) was calculated using the GenAlEx 6 program.Nei genetic distances was used to construct phylogenetic tree using the neighbor-joining method (NJ) and the unweighted pair group method with arithmetic mean (UPGMA) using MEGA software version 3 (Kumar, 2004).

RESULTS
The number of alleles for each of the fifteen microsatellite loci in each of the three breeds is presented in Table 2.The total number of detected alleles varied from 6 (BM1815, BM1815, OARCP26, OARFCB20 and MAF64) to 12 (MCMA2 and BMS678).The mean numbers of alleles per locus are 8.1, 8 and 8.1 in Gray, Zandi and Karakul, respectively.The mean number of alleles shared between Gray and Zandi, Gray and Karakul and also Zandi and Karakul are 7.7, 7.9 and 7.8, respectively, whereas the mean number of the alleles shared by the three breeds is 7.6.It is worth mentioning that all alleles representing ten loci are present in the three breeds and the only difference found is at BMS460, OARCP26, OARAE129, MAF64 and BM6444 loci where some alleles at loci are not present in Gray, Zandi or Karakul.
Table 3 presents the alleles frequency distribution at the analyzed loci in the three breeds.At loci MCMA26, the same alleles are at the highest frequency in Gray, Zandi and Karakul breeds.Gray and Karakul breeds have similar alleles at the highest allele frequency at six loci.Whereas Gray and Zandi; Zandi and Karakul breeds show similar alleles at the highest allele frequency at three loci.Gray and Zandi breeds have the highest allele frequency at OARCP26 locus.
The average direct count of heterozygosity (observed      heterozygosity) overall loci in Gray, Zandi and Karakul breeds are 0.984, 0.986 and 0.988, respectively.Whereas the average expected heterozygosity overall loci in the three breeds are 0.837, 0.830 and 0.831, respectively (Table 4).These results show more heterozygosity than expected in each breed.Mean PIC overall loci in Gray, Zandi and Karakul breeds are 0.8155, 0.8077 and 0.8081, respectively (Table 4).As expected, fairly high level of genetic heterogeneity was further reflected within three breeds by a mean PIC value.These high estimates of PIC substantiated the suitability of used set of markers to applications such as parentage control, linkagemapping programs in addition to genetic polymorphism studies in Iranian sheep too.
The PIC values for all loci ranged from 0.680 (OARCP26) to 0.906 (MCMA2).The PIC values are very high, indicating that these loci are highly informative and suitable for genetic studies of sheep breeds (Table 5).
Tests of genotype frequencies for deviation from Hardy-Weinberg equilibrium (HWE), at each locus overall breeds, reveal significant departure from HWE (P > 0.001) (Table 5).G ST and F-statistics for each locus overall GST values of breed differentiation were similar and ranged from 0.003 (BMS332) to 0.042 (OARAE129).Per pair estimator of FST, which is the measure of differentiation among population, is 0.013 between Gray and Zandi, 0.013 between Gray and Karakul and 0.015 between Zandi and Karakul (Table 6).The calculated genetic distance matrix is shown in Table 7.The distance between Gray and Zandi, and Gray and Karakul was smaller than the distance between Zandi and Karakul.Nei' (1978) genetic distances among breeds are presented in Table 7, with the corresponding UPGMA dendrogram and neighbour-joining tree in Figures 1 and 2, respectively.The smallest genetic distance (0•139) was estimated between Gray and Karakul.There is little differentiation between the pelt sheep breeds with Nei distances ranging from 0•139 to 0•155.The cluster analysis shows that Gray and Karakul breeds cluster independently from Zandi breed.

DISCUSSION
Recently, the preservation of unique, genetically distinct breeds of domesticated animals, especially indigenous, has received much attention.Knowledge and information on genetic diversity, population structure and genetic relationships between populations are absolute prerequisites for defining and accomplishing effective preservation strategies (Kusza et al., 2010).This study aimed to characterize the genetic diversity and structure of pelt sheep populations by using fifteen microsatellites.Over the past decade, numerous studies on genetic diversity in domestic livestock (mainly in small ruminants), based on the analysis of microsatellite loci, have been carried out worldwide.Investigation of genetic variation in Nanekarani et al. 7443 Taleshi goat using microsatellite loci indicated substantial genetic variation based on their gene diversity and average number of alleles per locus (Mahmoudi and Babayev, 2009).Genetic characterization of Alpine sheep breeds was established on the basis of individual genotypes at microsatellite loci (Dalvit et al., 2008).Study of genetic relationships among bulgarian sheep breeds using microsatellite loci indicated a high level of variation in the tested breeds (Kusza et al., 2010).Arora et al. (2008) examines the genetic variability in Jalauni, an important sheep of northwestern arid and semi arid region of India, at 25 microsatellite loci.Also, genetic diversity and relationships within and among Magra, Marwari and Sonadi sheep breeds of India, were distinguished based on microsatellite markers (Arora et al., 2008).
In Iran, many studies were performed to evaluate the genetic diversity of Iranian sheep breeds.Esmail et al. (2007) used nineteen microsatellites to evaluate genetic variation within Baluchi sheep breed.Banabazi et al. (2007) studied the genetic variation within and between five Iranian sheep populations including Sanjabi, Kordi Kordistan, Kordi Khorasan, Mehraban and Moghani using six microsatellite Markers.Also, genetic variation among different ecotypes of the Iranian sanjabi sheep was investigated based on the analysis of microsatellite loci (Sharifi et al., 2009).In the present study, fifteen microsatellite loci were used to evaluate the genetic diversity within and between pelt sheep breeds reared in Iran.The fifteen microsatellites are all polymorphic in the three breeds.The total number of alleles was 122 at the 15 studied loci.Major differences between the three breeds were not observed.The average expected heterozygosity overall loci in Gray, Zandi and Karakul are 0.837, 0.830 and 0.831, respectively.High value of average expected heterozygosity within the breed could be attributed to the large allele numbers detected in the tested loci (Kalinwski, 2002).The average direct count of heterozygosity overall loci in each of the three sheep breeds is more than the expected heterozygosity.
All loci were derived from Hardy-Weinberg equilibrium (p < 0.001) due to excess of heterozygote individuals than homozygote individuals, migration, high mutation rate in microsatellite and artificial selection in all breeds (Aminafshar et al., 2008).Deviation from HWE at microsatellites loci have, also been reported in various studies (Barker et al., 2001;Laval et al., 2000;El Nahas, 2008;Aminafshar et al., 2008;Sharifi sidani et al., 2009).It is known that a population is considered to be within HWE only when it is able to maintain its relative allele frequencies.
The global inbreeding coefficients F IS (-0.19) and F IT (-0.168) observed in the present study indicate an excess of heterozygotes and so it does not probably encounter problems that results from inbreeding depression.This result may explain the observed high value of direct count of heterozygosity in each breed and the deviation from HWE which were detected in all loci overall breeds.According to Hartl (1980), per pair F ST value equals 0.05 is indicative for moderate differentiation between populations.The per pair FST values reported in the present investigation between all pairs of the tested breeds are less than 0.05 which may indicate a low differentiation between populations under investigation.The estimated F ST , which corresponds to the proportion of genetic variability accounted for by differences among breeds, was 0.018.These results indicate that genetic diversity quantified by microsatellite markers shows very little differentiation among pelt sheep breeds.Our results are similar to those reported for other sheep breeds, where F ST estimates range between 0.03 and 0.08 (Arranz et al., 1998;Alvarez et al., 2004;Rendo et al., 2004;Sodhi et al., 2006;Peter et al., 2007;El Nahas et al., 2008).The genetic similarity observed among three breeds is probably a result of migration among populations that may have a common origin and which have been selected mostly for morphological traits associated with the breed standard.Migration has a great effect on the reduction of genetic differentiation between popula-tions (Laval et al., 2000).Additionally, low genetic distance values (Ds = 0.139 -0.155) supported high genetic similarity between these three breeds and were in similar range with those cited by Arranz et al. (1998) and Sodhi et al. (2006) for closely related Spanish sheep breeds (Ds = 0.21 -0.36) and Nali and Chokla sheep (Ds = 0.229), respectively.In summary, the three breeds were found to be genetically and closely related to each other, although there was a significant geographical distance between the three populations.
In conclusion, the tested microsatellites, being all polymorphic in the three breeds, could be fruitfully used for the differentiation between breeds.The evaluation of

Figure 1 .
Figure 1.UPGMA phylogenetic radial tree based on Nei's standard genetic distances of the three sheep breeds.

Table 1 .
Characteristics of the microsatellites under investigation.

Table 2 .
Number of alleles at each microsatellite locus in the three breeds and the number of alleles shared between breeds.

Table 3 .
Allele frequencies at each microsatellite locus in the three sheep breeds.

Table 4 .
Mean heterozygosity and polymorphism information content (PIC) in the three sheep breeds.

Table 5 .
PIC, FIT, FST, GST and FIS values, and chi-square and G-square test for HWE for each locus over all breeds.

Table 6 .
Per pair Fst values between all pairs of the tested breeds.

Table 7 .
Nei's genetic distances between the studied breeds.

Table 5 .
The global F IS , F ST and F IT are -0.19,0.018 and -0.168, respectively.All markers had negative values of F IS , showing an excess of heterozygotes.F ST values of genetic differentiation and