ГЕНЕТИКА, 2015, том 51, № 9, с. 1066-1074
GENETIC DIVERSITY, PARENTAGE VERIFICATION AND GENETIC BOTTLENECKS EVALUATION IN IRANIAN TURKMEN HORSE BREED © 2015 G. Rahimi-Mianji1, A. Nejati-Javaremi2, and A. Farhadi1
laboratory for Molecular Genetics and Animal Biotechnology, Department of Animal Science, Sari Agricultural Sciences
and Natural Resources University, Sari, Iran e-mail: email@example.com 2Department of Animal Sciences, University of Tehran, Karaj, Iran Received October 01, 2014
The present study was undertaken to genetically evaluate Turkmen horses for genetic diversity and to evaluate whether they have experienced any recent genetic bottlenecks. A total of 565 individuals from Turkmen horses were characterized for within breed diversity using 12 microsatellite markers. The estimated mean allelic diversity was (9.42 ± 1.78) per locus, with a total of 131 alleles in genotyped samples. A high level of genetic variability within this breed was observed in terms of high values of effective number of alleles (4.70 ± 1.36), observed heterozygosity (0.757 ± 0.19), expected Nei's heterozygosity (0.765 ± 0.13), and polymorphism information content (0.776 ± 0.17). The estimated cumulative probability of exclusion of wrongly named parents (PE) was high, with an average value of 99.96% that indicates the effectiveness of applied markers in resolving of parentage typing in Turkmen horse population. The paternity testing results did not show any mis-identification and all selected animals were qualified based on genotypic information using a likelihood-based method. Low values of Wright's fixation index, FIS (0.012) indicated low levels of inbreeding. A significant heterozygote excess on the basis of different models, as revealed from Sign and Wilcoxon sign rank test suggested that Turkmen horse population is not in mutation-drift equilibrium. But, the Mode-shift indicator test showed a normal 'L' shaped distribution for allelic class and proportion of alleles, thus indicating the absence of bottleneck events in the recent past history of this breed. Further research work should be carrying out to clarify the cause of discrepancy observed for bottleneck results in this breed. In conclusion, despite unplanned breeding in Turkmen horse population, this breed still has sufficient genetic variability and could provide a valuable source of genetic material that may use for meeting the demands of future breeding programs.
Archeological documents hold Iran as one of the oldest domestication and breeding center of the horse in the world. On the basis of geographical localization, at least seven separate populations of native horses, Caspian, Kurdish, Gharabagh, Dareshouri, Baluchi, Asil and Turkmen (Fig. 1) are distributed in Iran. The Turkmen horse breed is considered as one of the most ancient horse breed in the world. It is breed in the north, east of Caspian Sea, Golestan and Northern Khorasan provinces, mainly in Raz and Jargalan regions at 100 km to Boojnord city, the center of Northern Khorasan province (Fig. 2). Steadily and unfortunately our attentions to these valuable animals decline, because it has not been used so widely as a means of transport in war and peace, communications as well as agricultural progress. This situation has been reduced horse keepers and breeders in the world and then in Iran as the other countries the horse populations are taken the risk of endangered species. The horse breeds that have suffered a substantial decline in population size may have elevated levels of inbreeding which can lead to an overall decline in fitness (inbreeding depression) and increased risk of extinction.
These effects are believed to be the result of increased homozygosity leading to the increased expression of deleterious recessive alleles. In recent years, the issue of maintaining biodiversity as a major element of environment preservation has been discussed globally. To this end, preserving biodiversity of indigenous species, especially of those of economic interest must represent a relevant aspect in the scientific research activity. For four decades now, FAO (Food and Agriculture Organization) has included in its agenda the problem of preserving, evaluating and using animal genetic resources . One of the difficulties in implementing a selective breeding program in horse stocks is maintaining pedigree information. A correct pedigree is important for any domestic horse breed whether rare or not. For breeds that are common, an incorrect pedigree can frustrate breeding plans for selective improvement of the breed. For rare breeds, correct pedigrees are important for developing breeding strategies. Another difficulty in managing a selective breeding program is loss of genetic variability and increases in inbreeding as a result of the inadvertent mating of related individuals. The effects of inbreeding in horses will result in a
genetic diversity; parentage verification
Fig. 1. A typical Turkmen horse [copyright Mobarak
decrease in genetic diversity, which will limit the potential for genetic gain from artificial selection. Once reliable pedigree information is available, mating can be arranged in order to minimize inbreeding . In livestock, the use of paternity testing primarily aims to confirm the relationship between individuals and to help in criminal investigations [3—8]. Such assays have wide acceptance among the entities responsible for keeping pedigree registries. Genetic characterization is the first step in the conservation of breeds. This information could serve as a guideline for future breeding strategies [4, 9]. Genetic analysis using molecular markers can provide valuable information about current levels of genetic variation. This information can then be used to make predictions about how particular management strategies will influence genetic variation in the herd.
As an introduction of foreign horse breed in the country and also lack of sound breeding programs for conservation of native horse breed, presently only a few hundred true Turkmen horses are in existence. To avoid further loss of potential unique genes and to preserve the genetic diversity within breed, characterization of genetic structure based on molecular genetic markers is of priority. Application of microsatellite markers in assessment of the biodiversity levels in Iranian Turkmen horses breed has not been done yet and this is the first research work for characterization of genetic structure and parentage verifications based on tests with ISAG's (international society for animal genetics) standards microsatellite paternity markers in this breed.
Fig. 2. Main geographical location of the Turkmen horse breed sampled at the present study.
MATERIALS AND METHODS
Genomic DNA isolation
The animals were randomly chosen by their breeders who were able to document their pedigrees (parents, offspring). Blood samples were collected from 565 individuals of Turkmen horses in EDTA treated tubes as an anticoagulant. The samples were kept in a cooling chain, transferred to the laboratory and stored at —20°C until further analysis. Genomic DNA was extracted using NucleoSpin Blood Quick Pure Kit (Macherey & Nagel, Düren, Germany) according to the manufacture's recommendations.
Microsatellite markers and multiplex PCR
Turkmen horses were characterized for within breed diversity using 12 microsatellite markers. Multiplex PCR reactions were carried out in a total volume of 30 |L, containing 5 |L 10x PCR buffer, 0.17 mM dNTP-mix, 2.5 mM MgCl2, 100 ng DNA and 2 units Taq DNA polymerase (ABGene). In the 8-plex PCR the following microsatellite markers, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, AHT4 and VHL20 [10—14] were used (Table 1). The markers AHT5, ASB2, HMS2 and HTG10 [10, 12, 13, 15] were used in the 4-plex PCR with a total volume of15 |L (Table 2). For each PCR reaction one primer was 5'-end labeled with commercially fluorescent labels TAMRA, FAM and HEX. The thermo-cycling conditions included an initial denaturation at 95°C for 15 min, followed by 30 cycles of 1 min at 94°C, 1 min at 60 °C for annealing temperature and 1 min at 72°C. A final elongation step was carried out at 72° C for 10 min. The cycling conditions were the same in both multiplex PCR. DNA sequencing was performed with the Dye Primer Cycle Sequencing Ready Reaction-21-M13 kit (Applied
Table 1. Primer sequences were used in an 8-plex PCR for amplification of the microsatellite loci in Turkmen horse population
Locus Chromosome number Fluorescent label Primer sequences [5'—3'] Reference
HMS3 9 TAMRA F: CCA ACT CTT TGT CAC ATA ACA AGA R: CCA TCC TCA CTT TTT CAC TTT GTT 
HMS6 4 HEX F: GAA GCT GCC AGT ATT CAA CCA TTG R: CTC CAT CTT GTG AAG TGT AAC TCA 
HMS7 1 FAM F: AAC CGC CTG AGC AAG GAA GT R: GCT CCC AGA GAG TTT ACC CT 
HTG4 9 FAM F: CTA TCT CAG TCT TGA TTG CAG GAC R: CTC CCT CCC TCC CTC TGT TCT C 
HTG6 15 HEX F: CCT GCT TGG AGG CTG TGA TAA GAT R: GTT CAC TGA ATG TCA AAT TCT GCT 
HTG7 4 TAMRA F: CCT GAA GCA GAA CAT CCC TCC TTG R: ATA AAG TGT CTG GGC AGA GCT GCT 
AHT4 24 FAM F: AAC CGC CTG AGC AAG GAA GT R: GCT CCC AGA GAG TTT ACC CT 
VHL20 30 FAM F: CAA GTC CTC TTA CTT GAA GAC TA R: AAC TCA GGG AGA ATC TTC CTC AG 
Table 2. Primer sequences were used in a 4-plex PCR for amplification of the microsatellite loci in Turkmen horse population
Locus Chromosome number Fluorescent label Primer sequences [5'—3'] Reference
AHT5 8 HEX F: ACG GAC ACA TCC CTG CCT GC R: GCA GGC TAA GGG GGC TCA GC 
ASB2 15 HEX F: CCA CTA AGT GTC GTT TCA GAA GG R: CAC AAC TGA GTT CTC TGA TAG G 
HTG10 21 TAMRA F: CAA TTC CCG CCC CAC CCC CGG CA R: TTT TTA TTC TGA TCT GTC ACA TTT 
HMS2 10 TAMRA F: CTT GCA GTC GAA TGT GTA TTA AAT G R: ACG GTG GCA ACT GCC AAG GAA G 
Biosystems) following the supplied protocol. The sequencing products we
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