مطالعه تنوع ژنتيكي اسب‌ها‌ي عرب و كاسپين با استفاده از ماركرها‌ي ريز ماهواره

The study of genetic variability of Arabian and Caspian horses using microsatellite

زمينه مطالعاتي: امروزه استفاده از ماركس‌هاي ژنتيكي جهت حفظ و مديريت تنوع در نژاد‌ها‌ي مختلف اسب در سراسر جهان بسيار مرسوم است. هدف: در اين تحقيق تنوع توالي تكراري كوتاه در نژاد‌‌‌‌ها‌ي اسب­ها‌ي عرب و كاسپين با استفاده از 4 جايگاه VHL20, HTG4, AHT4, HMS7موردتوافق انجمن ژنتيك حيوانات (ISAG) موردبررسي قرار گرفته و فراواني آللي تعيين شده است. روش كار: براي اين منظورDNA ژنومي از نمونه خوني اسب­ها توسط روش مايلر استخراج گرديد. DNA به‌وسيله واكنش زنجيره‌ا‌ي پليمراز چند‌گانه توسط پرايمر نشان­دار تكثير شد. محصولا‌ت حاصل توسط دستگاه الكتروفورز كاپيلاري مورد آناليز ژنتيكي قرار گرفت. نتايج: نتايج حاصل از ژنوتايپ، حاكي از وجود متوسط 7/5 آلل در اسب­ها‌ي عرب و7 آلل در اسب­ها‌ي كاسپين بود. متوسط هتروزيگوسيته مشاهده‌شده Ho)) 761/. در اسب عرب و 0/799 در اسب كاسپين و همچنين متوسط هتروزيگوسيته مورد انتظار (He) 0/779 در اسب عرب و 0/84 در اسب كاسپين بود. نتيجه‌گيري نهايي: نتايج نشان­ د‌هنده چند­شكلي و كارآمد‌ي بالاي چهار جايگاه مورد­استفاده در تعيين نژاد، تنوع و تست­ها‌ي ابوت مي‌باشد.

Introduction: Nowadays the genetic markers are very usual for the diversity and management in different horse breeds all around the world. One of the most important goals for horse breeders is conserving the typical phenotype and characters of horses. This conservation is performed based on selection and inbreeding ( Winton et al. 2015). In this regard, the main disadvantage is the homozygosity of undesirable alleles in the populations, which results in the reduced level of genetic va riation, that in turn leads to lots of defects and susceptibility to recessive diseases (Shahsavarani et al. 2010) . DNA genotyping by microsatellites is usually used for determining genetic diversity and parentage testing. However, there are inherited regions of DNA that can vary in different creatures. Variations in DNA sequence are named "polymorphisms". As findings show, sequences with the highest degree of polymorphism are very useful for DNA analysis in paternity verifications. This term is based on analyzing the inheritance of a class of DNA polymorphisms known as Short Tandem Repeats (STR). STRs are short sequences of DNA, normally of length 2 - 5 base pairs, that are repeated numerous times in a head - tail manner. The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population of i ndividuals. On the basis of different repeat units, STRs can be classified into different types. On the one hand, according to the length of the major repeat unit, STRs are classified into mono - , di - , tri - , tetra - , penta - , and hexanucleotide repeats. The t otal number of each type decreases as the size of the repeat unit increases. Short tandem repeats (STRs) like microsatellites represent lots of advantages including codominant inheritance and extreme polymorphism (Miller et al. 1988) . This study determined short tandem repeat (STR) and allele frequency of Arab and Caspian horse breeds in different regions of Iran using four loci (VHL20, AHT4, HTG4, HMS7) recommended by the (ISAG). Material and methods: Blood samples were collected from 37 Iranian horse breeds (East Azerbaijan and Gilan provinces). EDTA as an anticoagulant agent was used in blood tubes. Genomic DNA was extracted and purified by salting out method from whole blood (Miller et al. 1988) . Then, DNA concentration was evaluated using Nanodro‎p at 260 nm. Next, four microsatellite markers were used as labelled with fluorescent dye (6 - FAM) (Table1). Afterwards, multiplex PCR was performed in a total volume of 25 μl using the following cycling conditions: the first denaturation at 95 ̊C for 5 min followed by 25 cycles at 95 ̊C for 30 sec, 59 ̊C for 1 min, 72 ̊C for 30 sec, and a final extension at 72 ̊C for 5 min. PCR products were checked through electrophoresis on 1/8 % agarose gel. Then, they were genotyped by capillary electrophoresis on Genetic Analyzer ABI PRISM 3100 (Applied Biosystems, USA). The size of alleles was measured by fluorescent fragment analyzer Gene Marker genotype software and the alleles per locus we re calculated by counting. Results and discussion: Genotypes showed the mean number of alleles 7.5 in Arab horses and 7 in Caspian horses. As regards that mean observed heterozygosity (Ho) 0.761 in Arab and 0.799 in Caspian horse also expected heterozygos ity (He) calculated 0.779 in Arab and 0.84 in Caspian horse. The equine microsatellite was introduced by (Ellegren et al. 1992) . In the present study, the highest number of polymorphism was 10 for AHT4 locus in Arabian horses compared with 9 in Egyptian Native ho rses (Mahrous, Hassanane et al. 2011) . Two Iranian h orse populations (Caspian and Arabian) had high heterozygosity. Iranian Caspian pony heterozygosity (0.84) was higher than that of UK Pony (Winton, Plante et al. 2015) . This result was even higher than what was obtained in an other study 0.605 ( Amirinia et al. 2007) and lower than 0.8 (Shahsavarani et al. 2010) . Conclusion: Our findings were in agreement with other studies in that microsatellite DNA genotyping is useful for individual identification, and paternity and maternity verification on horse population. These kinds of studies help in assessing genetic diversity for conservation, management and breeding program in horse breeds. The number of alleles and heterozygosity level in four loci in our studied population showed higher genetic variability and polymorphism .