بررسي تنوع ژنتيكي در جمعيت اسب‌هاي كرد ايران

Evaluation of the genetic diversity of Iranian Kurdish horses

زمينه مطالعاتي: نژادهاي مختلفي از اسب‌ها وجود دارد و اين نژاد‌ها را مي‌توان با استفاده از شاخص‌هاي مورفولوژيكي از يكديگر تمييز داد. با اين وجود اين روش دقيق نيست و با خطا همراه است. براي جبران اين كاستي امروزه پژوهشگران از ريزماهواره‌ها به عنوان شاخص تعيين نژاد و مطالعه جمعيت اسب‌ها استفاده مي‌كنند زيرا اين روش بسيار دقيق مي‌باشد. هدف: در اين مطالعه تنوع ژنتيكي جمعيت اسب‌هاي كرد ايران با استفاده از ريزماهواره‌ها مورد بررسي قرار گرفته شد. روش كار: در اين مطالعه تنوع ژنتيكي 52 اسب نژاد كرد مورد بررسي قرار گرفته است. براي اين منظور از ماركر‌هاي ريزماهواره پيشنهادي ISAG استفاده شد. اين ماركر‌ها شامل ريزماهواره هاي VHL20، HTG4، AHT4 وHMS7 مي‌باشد. اين جايگاه‌ها توسط روش مولتي پلكس PCR با چهار جفت پرايمر نشاندار به رنگ فلورسانس تكثير شدند و اندازه محصولات حاصل از PCR توسط الكتروفورز مويينه جداسازي و مورد بررسي قرار گرفتند. نتايج: داده‌ها نشان داد كه تنوع ژنتيكي بالايي در جمعيت اسب‌هاي كرد وجود دارد. تعداد آلل‌هاي مشاهده شده براي هر جايگاه از 8 تا 13 متغير بوده است و ماركر AHT4 با 13 آلل داراي بيشترين تعداد آلل و بيشترين هتروزيگوسيتي مي‌باشد. جايگاه‌هاي HTG4 و HMS7 داراي 8 آلل مي باشند كه كمترين تعداد آلل در ميان جايگاه‌هاي بررسي شده را دارا مي‌باشند و جايگاه HTG4 داراي پايين‌ترين مقدار هتروزيگوسيتي مي‌باشد. نتيجه ­گيري نهايي: نتايج حاصل از اين مطالعه نشان دهنده فراواني بالاي تنوع ژنتيكي جمعيت اسب هاي كرد در مقايسه با ساير نژادهاي اسب مي باشد.

Introduction:The horse is a large land mammal notable for its speed, strength, and endurance. Horses are members of the Equidae family, the horse ’ s influence on human history and civilization make it one of the most important domestic animals . The h orse has various breeds and these breeds can be distinguished from each other based on morphological characteristics ( Mahrous et al. 1994 ) . However, this method is not accurate enough. Among molecular markers, microsatellites are suitable for biodiversity evaluation owing to their codominant inheritance, high heterozygosity and distribution across the gen ome, ease and reliability of scoring . Microsatellites or Short Tandem Repeats (STRs) are used as useful markers found in the DNAs of most species. They are defined as tandem repeats of 2 - 10 base pair units and are often present as perfect or imperfect repe ats. The number of repeats found in any given microsatellite region is sometimes highly variable, with as many as hundreds of copies of the repeat unit in each microsatellite. As a result of their highly polymorphic nature, microsatellites are informative molecular markers that can be applied to research in the fields of pop ulation genetics, medical genetics, forensic science, evolutionary biology, and plant breeding. Once potentially useful (i.e. polymorphic) microsatellites are found, PCR primers are constructed from the DNA sequences flanking the microsatellite regions Thu s researchers are using microsatellite markers for parentage testing as well as for population genetics studies. In the last decade microsatellite markers have been widely used to assess genetic variability within and between different horse breed s ( Nichol as et al. 2009 ) . In the current work genetic diversity of the Kurdish horse breed was studied using 52 individual horses. Material and method s : S ampling from Kurdish horse was done and their DNAs extracted based on salting out method ( Miller et al. 1988). Extracted DNAs was run in an agarose gel and concentration and quality of DNAs was measured by N ano - drop. We used four microsatellite markers that all have been recommended by ISAG for parentage testing. These markers include VHL20, HTG4, AHT 4, and HMS7. These loci were amplified by multiplex PCR with fluorescent dye - labeled primers. Polymerase chain reaction was performed using 25 micro liters for each sample and PCR products were separated and analyzed with capillary electrophoresis and the outputs were analyzed using G enmarker software. R esult s and d iscussion : The results showed there are high genetic diversity within the Kurdish horse population. At VHL20 locus 9 allele s was seen and t he most frequent allele at VHL20 locus was 170 bp and the lowest frequent allele was 90 bp also the biggest allele in this locus was 107 bp and the smallest allele was 88 bp , observed heterozygosity in VHL20 locus was 0/75 however the expected heterozygosity was 0/84. At HTG4 locus 8 allele was seen and the most frequent allele at HTG4 locus was 132 bp and the allele with 126 bp had a lowest allelic frequency , the allele with 138bp was the biggest allele at this locus and the smalle st allele was 124bp . At this locus observed heterozygosity was 0/73 and expected heterozygosity was 0/72. At AHT4 locus 13 allele was seen a lso the allele with 156 bp had a high est allelic frequency and the allele s with 147 and 150 bp had a lowest allelic frequency , a t this locus the biggest allele was 156bp and the allele with 140 bp was the smallest allele in this locus . Observed and expected heterozygosity a t this locus were 0/80 and 0/85 At HMS7 locus 8 allele s was seen and the allele with 183 bp was th e most frequent and the allele with 183 bp had a low allelic frequency . At HMS7 locus the biggest allele was 183 bp and smallest allele was 169 bp. Observed and expected heterozygosity was calculated 0 /75 and 0/73. In average 9/5 allele per each locus seen in this population. The number of alleles w as between 8 and 13. The AHT4 marker had 13 alleles with the highest allelic frequency and the highest heterozygosity. Either of HTG4 and HMS7 markers had 8 alleles and had the lowest allelic frequency and heterozygosity. C onclusion : R esults of this study show ed high frequency genetic diversity in Iranian Kurdish population in compare with the other horse breeds . The non - standard mating and mixing of Kurdish horses with other b reeds can be the reason of the high diversity.