Isolation and Characterization of Microsatellite Markers from Endangered Species (Camelus bactrianus)
محورهای موضوعی : Camelع. دانشور آملی 1 , م. امین افشار 2 , س.ا. شاهزاده فاضلی 3 , ن. امام جمعه کاشان 4 , ک. جمعه خالدی 5
1 - Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 - Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran
3 - Human and Animal Cell Bank, Iranian Biological Resource Center (IBRC), Academic Center for Education, Culture and Research (ACECR), Tehran, Iran|Department of Molecular and Cellular Biology, Faculty of Basic Science and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran
4 - Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran
5 - Department of Agriculture, Yadegar-e-Imam Khomeini (rah), Shahr-e-Rey Branch, Islamic Azad University, Tehran, Iran
کلید واژه: Conservation, Microsatellite, PIMA, <i>Camelus bactrianus</i>,
چکیده مقاله :
Iranian bactrian camel (Camelus bactrianus) is an endangered livestock breed with distribution in northwest of Iran. Microsatellites are a powerful marker for animal genetic and cell line identification and population genetic study. In this study, after producing more than 40 Camelus bactrianus fibroblast cell lines, microsatellites loci from the genome of Iranian Camelus bactrianus cell lines were identified using polymerase chain reaction (PCR)-based isolation of microsatellite arrays (PIMA) methods for genetic resources studies. PIMA is a relatively simple method which avoids not only library construction, but also radioactivity manipulation. Four polymorphic microsatellite loci (IBRC01 to IBRC04) were identified. Polymorphism of these new loci was assessed in 40 samples of Iranian bactrian camel. Number of alleles ranged from 3 to 5, observed and expected heterozygosity varied from 0.625 to 0.71 and from 0.642 to 0.807 and Shannon index from 0.89 to 1.603, respectively. The phylogeny analysis of all sequences indicated that breeds and species were grouped according to their geographic locations except a few sequences. These newly isolated polymorphic microsatellite markers would be useful tools for conservation genetic resources and population genetic studies and assessing genetic variations to establish a conservation strategy of this endangered species.
شتر دوکوهانه یکی از گونه‌های دامی در معرض انقراض ایران بوده که اکثراً در شمال غرب کشور پرورش داده می‌شود. ریز‌ماهواره‌ها یکی از نشانگرهای مناسب برای مطالعات ژنتیک جمعیت و تعیین هویت جانوران و رده‌های سلولی هستند. در این تحقیق نشانگرهای ریزماهواره با استفاده از 40 رده سلولی فیبروبلاستی شتر دوکوهانه و به روش PIMA (PCR Isolation of Microsatellite Arrays) جداسازی، شناسایی و بررسی گردیدند. PIMA روش مناسبی است که نیازی به تشکیل کتابخانه ژنومی و استفاده از مواد رادیواکتیو ندارد. پس از بررسی توالی‌ها، تعداد 4 جایگاه ریزماهواره جدید (IBRC01، IBRC02، IBRC03 و IBRC04) شناسایی و برای آنها پرایمر طراحی گردید. تنوع آللی جایگاه‌های فوق در تعداد 40 نمونه شتر دوکوهانه بررسی شد. تعداد آلل‌های مشاهده شده از 5-3 آلل متغیر بود. هتروزایگوسیتی مشاهده شده بین 52/0 تا 71/0 و هتروزایگوسیتی مورد انتظار بین 53/0 تا 80/0 و شاخص شانون بین 89/0 تا 6/1 متغیر بود. درخت فیلوژنی بر اساس توالی‌های به دست آمده و توالی‌های ثبت شده از گونه شتر رسم گردید و تفاوت گونه‌ها را بر مبنای پراکنش جغرافیایی تأیید کرد. نتایج این تحقیق نشان می‌دهد که روش PIMA برای جداسازی و شناسایی نشانگرهای ریزماهواره کارایی خوبی داشته است. همچنین تنوع جایگاه‌های ریزماهواره جدید شناسایی شده خوب بوده و از آنها می‌توان به عنوان ابزاری مفید برای انجام مطالعات ژنتیک جمعیت، تعیین هویت و حفاظت از ذخایر ژنتیکی شتر دوکوهانه استفاده نمود.
Abdul-Muneer P.M. (2014). Application of microsatellite markers in conservation genetics and fisheries management: recent advances in population structure analysis and conservation strategies. Genet. Res. Int. 1, 1-11.
Bulliet Richard W. (1975). The Camel and the Wheel. Harvard University Press, Cambridge, Massachusetts.
Chiang T.Y., Lee T.W., Lin F.J., Huang K.H. and Lin H.D. (2008). Isolation and characterization of microsatellite loci in the endangered freshwater fish cyprinidae(Varicorhinus alticorpus). Conserv. Genet. 9, 1399-1401.
Chuluunbat B., Charruau P., Silbermayr K., Khorloojav T. and Burger P.A. (2014). Genetic diversity and population structure of Mongolian domestic bactrian camels (Camelus bactrianus). Anim. Genet. 45(4), 550-558.
Chung-Jian L.J.P., Wang J.P., Lin H.D. and Chiang T.Y. (2007). Isolation and characterization of microsatellite loci in hemibarbus labeo (cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA). Mol. Ecol. 7, 788-790.
Creste S., Neto A. and Figueira A. (2001). Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining. Mol. Biol. 19, 299-306.
Daneshvar Amoli A., Mohebali N., Farzaneh P., Shahzadeh Fazeli S.A., Nikfarjam L., Ashouri Movasagh S., Moradmand Z., Ganjibakhsh M., Nasimisn A., Izadpanah M., Vakhshiteh F., Gohari N.S., Masoudi N.S., Farghadan M., Mohammadi Moghanhughi S., Khalili M. and Khaledi K.J. (2017). Establishment and characterization of Caspian horse fibroblast cell bank in Iran. In Vitro Cell. Dev. Biol. Anim. 53(4), 337-343.
Ellegren H. (2004). Microsatellites: simple sequences with complex evolution. Nat. Rev. Genet. 5(6), 435-445.
Eltanany M., Elfaroug Sidahmed O. and Distl O. (2015). Assessment of genetic diversity and differentiation of two major camel ecotypes (Camelus dromedarius) in Sudan using microsatellite markers. Arch. Anim. Breed. 58(2), 269-275.
Elyasi Gorji Z., Khaledi K.J., Daneshvar Amoli A., Ganjibakhsh M., Nasimian A., Sadat Gohari N., Vakhshiteh F., Farghadan M., Izadpanah M., Mohammadi Moghanhughi S., Rahmati H., Shahzadeh Fazeli S.A. and Farzaneh P. (2017). Establishment and characteristics of Iranian Sistani cattle fibroblast bank: a way to genetic conservation. Conserv. Genet. Res. 9(2), 305-312.
Fitak R.R., Mohandesan E., Corander J. and Burger P.A. (2016). The de novo genome assembly and annotation of a female domestic dromedary of North African origin. Mol. Ecol. Res.16, 314-324.
Hatanaka T. (2002). A polymorphic, telomeric-like sequence microsatellite in the Neotropical fish Prochilodus. Cytogen. Gen. Res. 98, 308-310.
Lin H.D., Lee T.W., Lin F.J., Lin C.J. and Chiang T.Y. (2008). Isolation and characterization of microsatellite loci in the endangered freshwater fish pararasbora moltrechti (cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA). Conserv. Genet. 9, 945-974.
Lunt D.H., Hutchinson W.F. and Carvalho G.R. (1999). An efficient method for PCR-based isolation of microsatellite arrays (PIMA). Mol. Ecol. 8, 891-893.
Ma H.Y., Bi J.Z., Shao C.W., Chen Y., Miao G.D. and Chen S.L. (2009). Development of 40 microsatellite markers in spotted halibut (Verasper variegatus) and the cross-species amplification in barfin flounder (Verasper moseri). Anim. Genet. 40, 576-578.
Nei M. and Takezaki N. (1996). The root of the phylogenetic tree of human populations. Mol. Biol. Evol. 13, 170-177.
Paredes M.M., Membrillo A., Gutiérrez J.P., Cervantes I., Azor P.J., Morante R., Alonso-Moraga A., Molina A. and Muñoz-Serrano A. (2014). Association of microsatellite markers with fiber diameter trait in peruvian alpacas (Vicugna pacos). Livest. Sci. 161, 6-16.
Potts D. (2005). Bactrian camels and bactrian-dromedaryhybrids Pp. 62-84 in The Silk Road. D. Waugh, Ed. Saratoga: The Silk Road Foundation, Stillwater, New York.
Rousset F. (2008). Genepop'007: a complete reimplementation of the Genepop software for Windows and Linux. Mol. Ecol. Res. 8, 103-106.
Rout Pramod K.J., Manjunath B., Ajoy M., Denis L., Lalji S. and Kumarasamy T. (2008). Microsatellite-based phylogeny of Indian domestic goats. BMC Genet. 9(1), 1-11.
Rozen S. and Skaletsky H.J. (2000). Primer3 on the www for general users and for biologist programmers. Pp. 365-386 in Bioinformatics Methods and Protocols: Methods in Molecular Biology. S. Krawetz and S. Misener, Eds. Humana Press, Totowa, New Jersey.
Sadder M., Migdadi H., Al-Haidary A.I. and Okab A. (2015). Identification of simple sequence repeat markers in the dromedary (Camelus dromedarius) genome by next-generation sequencing. Turkish J. Vet. Anim. Sci. 39, 218-228.
Sanches A. and Galetti Jr P.M. (2006). Microsatellites loci isolated in the freshwater fish Brycon hilarii. Mol. Ecol. Notes. 6, 1045-1046.
Shahkarami S., Afraz F., Mirhoseini S., Banabazi H., Asadzadeh N., Asadi N., Hemmati B., Ghanbari A. and Razavi K. (2012). Genetic diversity in iranian bactrian camels (Camelus batrianus) using, microsatellite markers. Mod. Genet. J. 7, 249-258.
Tamura K., Peterson D., Peterson N., Stecher G., Nei M. and Kumar S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum. Mol. Biol. Evol. 28(10), 2731-2739.
Toth G., Gaspari Z. and Jurka J. (2000). Microsatellites in different eukaryotic genomes. Survey and analysis. Genom. Res. 10, 967-981.
Wu H., Guang X., Al-Fageeh M.B., Cao J., Pan S., Zhou H. and Xie Z. (2014). Camelid genomes reveal evolution and adaptation to desert environments. Nat. Commun. 5, 5188.
Yeh F.C., Boyle T. and Rongcai Y. (1999). POPGENE Version 1.31. Microsoft Window Based Freeware for Population Genetic Analysis. University of Alberta Press, Edmonton, Canada.
Zane L., Bargelloni L. and Patarnello T. (2000). Strategies for microsatellite isolation: a review. Mol. Ecol. 11(1), 1-6.
