Molecular Assessment of Genetic Diversity in Dromedaries and Bactrian Camel Using Microsatellite Markers

Dromedary and Bactrian camels are two species of camel in Iran that have ecological adaptation to cold and hot desert area, respectively. They play an important role in the life and food security of nomadic tribes. The present study was conducted to investigate genetic diversity of 180 Iranian camels using microsatellite markers. In a panel of 20 microsatellite markers, we observed 214 alleles with a mean number of 10.7 alleles per locus. All loci exhibited PIC values more than 0.7. The genetic differentiation values (FST) per locus was different from 0.01 to 0.039 with an average of 0.021 across all loci. The estimate of genetic differentiation level between all Iranian camel populations in this study was low (FST: 0.008-0.021). High gene flow between populations was also observed. Phylogenetic tree illustrated that the highest genetic distance was between Bactrian and dromedary camel from YaD. However, the results of the present microsatellite analyses showed close genetic relationship in the studied populations. All of the population-locus combinations showed significant deviations (P< 0.01) from Hardy-Weinberg equilibrium. Keyword: Genetic conservation, Gene flow, Hardy-Weinberg equilibrium, Phylogenetic tree _____________________________________________________________________________ 1 Department of Animal Science, Faculty of Agricultural and Natural Resource, University of Mohaghegh Ardabili, Ardabil, Islamic Republic of Iran.  Corresponding author; e‐mail: nhedayat@uma.ac. ir 2 Department of Animal Science, Faculty of Agriculture and Natural Resources, University of Tehran, Karaj, Islamic Republic of Iran. 3 Department of Animal Science, Sari Agricultural Sciences and Natural Resources University, Sari, Mazandaran, Islamic Republic of Iran. INTRODUCTION Camel is one of the most successful animals in the evolution process. During their evolution, camels have acquired features that made them the best choice for living in harsh environments. Camels are cheap for keeping due to their ability to eat low-quality food and turn it into meat and milk in desert regions. Based on FAO definition, food security depends on the quantity and quality of food produced, as well as on its availability all the times (FAO, 2015). Camels are responsible for supporting a significant part of food security in arid climates. Iran is located in one of the most arid regions in the world and over 84 percent of the country is arid and semi-arid (Lehane, 2014). Accordingly, it seems that in the near future, camel could be the dominant domestic animal in Iran. However, camel’s breeding and husbandry in Iran is traditional, and little efforts have been made to convert them as farm animals. Genetic diversity is a basic factor in carrying out breeding schemes and genetic conservation programs (Sharma et al., 2015). Reduced populations of some species have raised conservation concerns in many regions in the world. Camel is one of the species that are faced with worldwide unkindness in recent years. Sharp decrease in camel population in D ow nl oa de d fr om ja st . m od ar es . a c. ir at 1 :1 8 IR D T o n S un da y A pr il 5t h 20 20 ________________________________________________________________ Hedayat-Evrigh et al. 1138 some countries such as India (Prasad et al., 2015), Mongolia (Chuluunbat et al., 2014) and Iran is not the good sign for future of this unique species. It is worth to mention that there are less than 150 Bactrian camels in Iran and this valuable animal is known as endangered species (Khademi, 2017). Modernization and changes in the lifestyle of people, as well as categorizing camels as “animals of primitive societies” (Yagil et al., 1994) lead to decline in camel’s product acceptance and, consequently, lower camel husbandry. Small populations lose genetic diversity as a result of genetic drift and inbreeding. Conservation of genetic variation is important due to its role in empowering the populations for response to environmental changes as well as its correlation with reduced population fitness (Reed et al., 2003). Assessment of genetic variation within and between species is necessary to understand population structure, local adaptation, and differentiation between populations (Bos et al., 2008). Since some countries, especially Iran, have both species of camels, it might be expected to observe hybrid animals. Therefore, it is possible to investigate the hybridization between the mentioned species using genetic markers. Microsatellite marker due to its random distribution across the genome, codominance and highly polymorphic (Putman et al., 2014) are ideal for estimation of genetic diversity and the relationship among the livestock breeds and species (Hampton et al., 2004). They are also useful for evaluation of genetic distance and detection of population bottlenecks (Putman et al., 2014). The aim of this study was to assess the genetic relationships between Iranian camels population using microsatellite markers polymorphisms. MATERIALS AND METHODS Sampling and Genomic DNA Extraction We used four geographic subpopulations of camels including one area for Bactrian camels (ArB) from Ardabil Province in the northwest of Iran (cold area) and three for dromedary camels in the center of Iran (hot area) including: (1) Trod Station (TrD) (camels collected from all over Iran and this station is the gene bank of dromedary camels in country); (2) Semnan Province (SeD) (that was a local herd in north of Dashte-e Kavir), and (3) Yazd Station (YaD) (in the center of Iran). Blood samples were collected from Jugular vein using 4 mL vacutainer tubes containing EDTA (1 mg mL -1 ) of 180 camels including 140 Camelus dromedarius (50, 55, and 35 samples from YaD, TrD, and SeD, respectively) and 40 Camelus bactrianus (from Ardabil Province) (Figure 1) according to guidelines of the Iranian Council of Animal Care (1995). Genomic DNA was purified using Genomic DNA Mini Kit (Real Biotech Corporation, RBC, South Korea). Microsatellite Markers, PCR Aplification, and Genotyping All test animals were genotyped using 20 microsatellite markers (FAO, 2011) (Table 1). Amplification was carried out in a total volume of 25 μL containing 1.2 mM MgCl2, 0.2 mM dNTP, 1.5 UTaq DNA polymerase, 0.15 μM of forward and reverse primers (Invitrogen) and 100 ng genomic DNA. Thermo cycling profile consisted of initial denaturation at 94 o C for 5 minutes, followed by 35 cycles of 94 o C for 40 seconds, annealing at 51-64 o C for 40 seconds and extension at 72 o C for 40 seconds, with a final extension step at 72 o C for 5 minutes. The PCR amplified products (microsatellite loci) were genotyped by 8% PolyAcrylamide Gel Electrophoresis (PAGE) using the manual sequencer (Bio-Rad: Sequi-Gen@ GT), and colored with silver nitrate (Benbouza et al., 2006). Statistical Analyses The basic parameters for each locus and population including allele frequencies, effective Number of alleles (Ne), observed D ow nl oa de d fr om ja st . m od ar es . a c. ir at 1 :1 8 IR D T o n S un da y A pr il 5t h 20 20 Molecular Assessment of Genetic Diversity in Camel _______________________________ 1139 Figure 1. Map of Iran and sampling areas. Bactrian camels (ArB), Trod Station (TrD), Semnan Province (SeD) and Yazd Station (YaD). Table 1. Characteristics of primers used in current study. Annealing Tm Accession No. N Backward 5’ to 3’ Forward 5’ to 3’ Loci 60 AF329157 GGAGTTTATTTGCTTCCAACACTT ATTTTGCAATTTGTTCGTTCTTTC CMS16 58 AF329149 TGTAGGGTTCATTGTAACA TTTATAGTCAGAGAGAGTGCTG CMS50 57 AF217601 CAGGCAGATATCCATTGAA GAAGAGGTTGGGGCACTAC CVRL01 58 AF217602 AGTGTACGTAGCAGCATTATTT TGTCACAAATGGCAAGAT CVRL02 62 AF217606 CATAATAGCCAAAACATGGAAACAAC TTTTAAAAATTCTGACCAGGAGTC