Extraction of gDNA from mucosal layer of scat samples using combined Zhang and phenyl-chloroform method ( Cobellis et al., 2004 Glynn et al., 2004 Bernevig, Hughes & Zhang, 2006) yielded significant quantity and quality of DNA, which was sufficient for successful amplification of COI gene/mini-barcode, microsatellite and sex determination primers ( Thornton et al., 2008 Valledor et al., 2014).
In current study DNA was extracted from 50 putative snow leopard scat samples following three protocols ( Taberlet, Waits & Luikart, 1999 Frantzen et al., 1998 Murphy et al., 2002 Wasser et al., 1997). The main objective of this study was the optimization of genomic DNA (gDNA) extraction/amplification protocols and use of SSR markers for genetic analysis using field collected scats samples of snow leopard from GB, Pakistan. Keeping in view the conservation status and smaller size of the population of snow leopard, this study aimed at assessing genetic diversity to evaluate the adaptive potentials of the species under environmental changes.
Studies conducted on snow leopard populations in central and south–east Asian region including Kyrgyzstan and Tajikistan, India, Nepal, Bhutan, China and Mongolia, show a decreased population size, low genetic diversity and mitochondrial DNA variation making the animals vulnerable to certain environmental conditions ( Janečka et al., 2008, 2011, 2017). Retributive killing, natural prey reduction, human-snow leopard conflicts and illegal trade of bones, pelt and other body parts are the causes of population decline in snow leopards ( McCarthy et al., 2017 Maheshwari & Niraj, 2018). Inbreeding depression, loss of genetic variability and increased homozygosity are associated with smaller populations and reduced chances of species to adapt to the environmental changes ( Laurenson & Caro, 1994 Eisen et al., 1998 Keller & Waller, 2002 Chisci, Rossiter & Zappa, 2001 Franklin & Frankham, 1998 Lynch, Conery & Burger, 1995 Slate et al., 2000).Īccording to a recent estimate, the number of snow leopard individuals is 7,463–7,980 worldwide ( McCarthy et al., 2016). These markers can be easily automated for high throughput screening and information on mutation rate, heterozygosity, homozygosity, number of alleles per locus and Shannon index among different populations ( Nei, 1978), while the sex ratio can be determined by using sex chromosome-specific molecular markers. The DNA quantity via scat samples is usually low, but to amplify this low quantity for genetic assessment, simple sequence repeats (SSR) markers are considered excellent, being co-dominant, highly polymorphic and easily transferable between populations ( Jarne & Lagoda, 1996 Luo, Hu & Li, 2003). Researchers have found a solution by introducing noninvasive sampling which provides a gateway for molecular and genetic analysis, aiming at conservation of wildlife species ( Marra et al., 2003 Wolff et al., 2007). These factors contribute toward the difficulties faced by researchers during field sampling for conservation genetic studies ( Nowell & Jackson, 1996). According to International Union for Conservation of Nature Red List of Threatened Species, it is listed as “Vulnerable” worldwide ( McCarthy et al., 2017).Īnimals like snow leopards live in difficult field conditions, have small and scattered populations, nocturnal habitat, excellent camouflaging in the wild and human shy nature. Need for studies on its population genetics is emphasized to protect this threatened species ( Network, 2014 McCarthy & Chapron, 2003). According to recent survey reports, its population may have decreased in the country ( Nawaz & Hameed, 2015). Though its population in Pakistan has remained constant for the last few years it still needs conservational efforts for its future survival ( Sheikh & Molur, 2005). It is considered an indicator of the health of the ecosystem ( O’Brien et al., 1985) and an icon of conservation ( Bonin et al., 2004). Pakistan’s northern mountains are also among its natural habitat, specifically the Gilgit-Baltistan (GB) and Chitral (Khyber Pakhtunkhwa). Snow leopard or ounce ( Panthera uncia) has evolved in the mountainous ranges of Asia ( Bonin et al., 2004).
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