Abstract: In order to investigate genetic variation of 3 ecotypes of Sanjabi sheep (Zardi, Kajal, Kolul), an experiment was planned, using 10 microsatellite markers (INRA023, SPS113, HSC, D5S2, McM527, MAF65, INRA005, INRA063, oarFCB304 and oarFCB11). Genomic DNA was extracted from 150 blood samples, using modified Salting out method. All loci were amplified by PCR except INRA023. Loci in all of the ecotypes were highly polymorphic. Significant departures from HWE were detected for all loci in all population, except for oarFCB11 in Kolul ecotype (p<0.05). INRA063 was the most polymorphic marker according to its effective number of alleles (equal to 6.58 in Zardi), expected heterozygosity (equal to 0.86 in Zardi), Shannon information index (equal to 2.01 in Zardi) and Polymorphism information content (equal to 0.83 in Zardi). However, MAF65 showed the lowest effective number of alleles (equal to 2.91 in Kajal), expected heterozygosity (equal to 0.66 in Kajal), Shannon information index (equal to 1.18 in Kajal) and Polymorphism information content (equal to 0.59 in Kajal). According to the observations, the Zardi ecotype was more polymorphic and showed highest genetic variation than the others as well as Kolul was lowest. The phylogenetic clusters presented that Zardi and Kolul ecotypes have minimum Genetic Distance to each other. Results showed that, high level of genetic diversity was observed in all ecotypes of Sanjabi breed and this breed was not at risk for conservation concept.

INTRODUCTION

The history of breeding of native sheep's in Iran back to thousands of years ago. However some factors like, the mutational process, random drift, gene flow and selection may be affected genetic variation during the time but different breeds with high level of polymorphism observe in Iran, due to their extensive production system during the history (Saadatnoori and Siahmansur, 1993). The population size of the Sanjabi breed is more than other breeds of sheep (except Baluchi breed) in Iran and 3 ecotypes were detected for this breed (Tavakkolian, 2000).

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). There were several researches about population genetic of sheep, used the molecular markers, in many countries (Gizaw et al., 2007; Esmaeilkhanian and Banabazi,, 2006). Therefore, the objective of this study is about genetic variation and phylogenetic relationship between all ecotypes of Sanjabi sheep, by microsatellite markers.

MATERIALS AND METHODS

One hundred and fifty blood sample of Sanjabi sheeps (47, 57 and 46 sample from Zardi, Kajal and Kolul ecotypes, respectively) were collected randomly. DNA was extracted from whole blood using modified Salting-out method (Miller et al., 1988). Genomic DNA was amplified in following condition of PCR; the PCR buffer in final concentration of 1X, 5 μM MgCl₂, 200 μM dNTPs, 1 unit per reaction Taq DNA polymerase, 150 ng genomic DNA and 0.25 μM of each primers in a total volume of 15 μL. The reactions were done with Gradient Master Cycler Eppendorf. The cycling protocol was as a follows; 2.5 min denaturing at 95°C followed by 32 cycles of denaturation at 95°C for 30 sec, Annealing at 50-63°C (depending on primers) for 30 sec, extension at 72°C for 30 sec and the final extension step at 72°C for 2.5 min.

Table 1	Characterizations of microsatellites used in the analysis

PCR products were visualized by silver staining after electrophoresis on 8% Acryl amide gels and genotypes of animals were determined. Genotype and allele frequency were calculated by direct counting. Test of departure from Hardy-Weinberg equilibrium was performed using Chi-square (χ²_t) and Likelihood Ratio or G-square (G²_t) test (Hedrick, 2000). The observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho) and expected heterozygosity (He), were estimated (Hedrick, 2000; Aminafshar et al., 2008), using POPGENE software, version 1.31 (Yeh et al., 1999).

Since the maximum amount of heterozygosity is equal to 1, so for polymorphic markers like microsatellites, these values are not sufficiently sensitive to show increasing the variation. To prevalence on this deficiency, we used the Shannon information Index (I), as the following equation:

Polymorphic Information Index (PIC) was estimated using HET software, version 1.8 (Ott, 2001) as the following equation:

Genetic Distance between ecotypes was estimated using Nei´s genetic Distance method as a follow equation:

Barker (1994) suggests some standards to select microsatellites in diversity studies, such as selection loci with at least 4 different alleles, selection loci that was used in mapping studies previously and preferably to be unlinked, selection different forms of microsatellite that have the Mendelian inheritance and selection loci that was used in some relative species such as bovine and ovine. Microsatellite markers were chosen according to all above criteria and their characteristic was shown in Table 1 (Hoffmann et al., 2004).

RESULTS AND DISCUSSION

All microsatellite loci were amplified except INRA023. The range of allele size was between 81 bp (oarFCB11) to 319 bp (HSC). The size of amplified alleles (Table 2) was higher than reported allele size (Maddox et al., 2001). Difference in genetic structure and existence of the new allele may be the main reasons.

All loci were deviate from Hardy-Weinberg equilibrium (p<0.05) due to excess of heterozygote individuals than homozygote individuals, migration, high mutation rate in microsatellites and artificial selection in all ecotypes (Aminafshar et al., 2008).

Table 2:	Annealing temperatures, reported and observed size of microsatellite Alleles

Table 3:	Population genetic parameter for each microsatellite marker in all ecotypes

Table 4:	Average of population genetic parameter in all ecotypes

Table 5:	Genetic distance (upper elements) and similarity (lower elements)

The population genetic parameters were showed in Table 3. According to the Table 3, maximum effective number of alleles (Ne), expected Heterozgosity (He), Polymorphism Information Content (PIC) and Shannon information index were equal to 6.58, 0.86, 0.83 and 2.01 at locus INRA063 in Zardi ecotype. However, minimum effective number of alleles (Ne), expected Heterozgosity (He), Polymorphism Information Content (PIC) and Shannon information index was equal to 2.91, 0.66, 0.59 and 1.18 at locus MAF65 in Kajal ecotype, respectively.

The average of genetic parameters was a little higher in Zardi ecotype than Kajal and Kolul, according to the Table 4. It seems, the differences are likely related to the sampling error not real differences among population.

According to the Table 5, the genetic distance between all ecotypes was very low and varies from 2-3%. By the other hand, all of the ecotypes are the same in this study. However, the minimum genetic distance was observed between Zardi and Kolul and it was equal to 0.02.

CONCLUSION

Finally, it was concluded, the Sanjabi breed possessed a considerable amount of genetic diversity due to the extensive production system, low pressure of artificial selection and possibility of random mating. The genetic diversity of that breed was higher in compared with genetic diversity in Indian sheep (Mukesh et al., 2006; Arora et al., 2008) and this breed was not at risk for conservation concept.

ACKNOWLEDGEMENTS

The current study was supported by Animal Science Research Institute, Karaj, Iran. We thank Mr. Kamanger and Mr. Nazokkar Maher, due to their help in laboratory. However, we are grateful of all staff of Animal Science Research Institute who helps us in this study.