ReseaRch aRticle Journal of Agricultural and Marine Sciences 2021, 26(2): 35–41 DOI: 10.24200/jams.vol26iss2pp35-41 Received 15 Dec 2020 Accepted 13 Mar 2021 Characterization of Genetic Diversity in Dhofari Wild Gazelles Ahmed Jashool1, Alya Al Ansari2, Waleed Al Marzooqi2, Othman Alqaisi3, Mansour Al Gahdhami3, Mohammed A Al-Abri2,* Mohammed A Al-Abri2,*( ) abri1st@squ.edu.om,2Department of Animal and Veterinary Sciences, College of Agriculture and Marine Sciences, Sultan Qaboos Universit y, 1Office of the Conservation of Environment, Royal Court Affairs, 3Department of Biology, College of Science, Sultan Qaboos Universit y Introduction The genus gazelle belongs to family Bovidae. Ac-cording to the International Union of Conser-vation of Nature, IUCN, there are eleven differ- ent gazelle species (Mallon and Kingswood, 2001). In Oman, two known Gazelle species have been document- ed. Namely, Gazella arabica and Gazella subgutturosa marica. Gazella arabica is a vulnerable gazelles species found in the Arabian peninsula (IUCN, 2017; Massolo et al., 2008). In Oman, gazelles are found in protected areas and are scattered in various the wild areas around the country. The numbers of gazelles in the wild are es- timated to be 1737 animals in Jabal Samhan and Nejed areas of Dhofar governorate (Al Hikmani et al., 2015). توصيف التنوع اجليين للغزال الربي مبحافظة ظفار أمحد جشعول، علياء األنصاري، وليد املرزوقي، عثمان القعيسي، منصور اجلهضمي وحممد العربي Abstract. Wild gazelles are scattered in most arid and semi-arid areas in the Sultanate of Oman particularly in val- leys, mountains and sandy zones of Rub’ al Khali desert. Recently, however, gazelles’ numbers have been declining in Oman mainly due to the loss of habitat. Consequently, a gradual loss of their genetic diversity is inevitable. However, little is known about the status of the genetic diversity of the Omani wild gazelles. This study aimed to determine the extent of inbreeding, population structure and genetic diversity in the Omani gazelles’ populations in Dhofar region. Samples from four different locations namely Gara, Stom, Solot and Ayon were collected. DNA belonging to 74 gazelles’ fecal samples was extracted using the human stool DNA extraction protocol. Following extraction, four microsatellite nuclear markers were used to calculate the levels of inbreeding, population differentiation and genetic diversity. PCR inhibitors were significantly removed using Bovine Serum Albumin (BSA) and dimethyl sulfoxide (DMSO). The mean inbreeding for the population was 0.228 for all loci with a standard error of 0.09. It is therefore postulated that Dhofari gazelles are generally undergoing gradual inbreeding, which may lead to lack of fitness in future generations. The ge- netic differentiation (Fst) ranged between 0.071 (between Gara and Stom) and 0.231 (between Gara and Ayon). On the other hand, the Fst estimate between Solot (most distant) versus other Dhofari gazelles populations (pooled together) was 3.7%. Principle Components Analysis (PCA) clustered Ayon and Gara populations apart from one another and closer to Stom while placing Solot further than all other populations, which is in agreement with the Fst results and the geographical distribution. In conclusion, the results of this preliminary study provides an insight towards the conserva- tion of wild gazelles in Dhofar in Oman. It provides an initial report on the status of the genetic diversity of Dhofari wild Gazelles and serves as a reference point for future studies assessing their genetic diversity and variability. Keywords: noninvasive samples, microsatellite, inbreeding, genetic diversity, population structure. امللخص:تنتشــر الغــزالن الربيــة يف معظــم املناطــق القاحلــة وشــبه القاحلــة مــن ســلطنة عمــان وخاصــة يف الــوداين واجلبــال واملناطــق الرمليــة مــن صحــراء الربــع اخلــايل، ويف اآلونــة األخــرة ، تواجــه الغــزالن إخنفاًضــا يف أعدادهــا، األمــر الــذي قــد يــؤدي اىل فقــدان التنــوع اجليــي تدرجييــا ممــا قــد يتســبب يف انقراضهــا مــن الربيــة، ويعتــرب الفقــدان التدرجيــي للتنــوع اجليــي خســارة ألحــد املقومــات الرئيســية الــي قــد حتمــي مــن األنقــراض، وحــى االن ال ُ يعــرف ســوى القليــل عــن حالــة التنــوع الوراثــي للغــزالن العربيــة الربيــة يف حمافظــة ظفــار. هــدف هــذا املشــروع إىل حتديــد أفضــل الطــرق ألخــذ عينــات روث الغــزال وحتليلهــا والتعــرف علــى أفضــل بروتوكــول إلســتخاص احلمــض النــووي الريبــوزي )DNA( والتحســن مــن أداء تفاعــات سلســلة البوليمــرز )PCR(. ويعتــرب إســتخاص احلمــض النــووي مــن الــروث مــن أفضــل البدائــل للحيــواانت الربيــة وهــو بديــل عــن إســتخاصه مــن الــدم والــذي قــد يتطلــب ختديــر احليــوان ممــا يعــد عمليــة مضنيــة وخطــرة علــى احليــواانت الربيــة، أمــا األهــداف الثانويــة فهــي تطويــر فهــم مســتوايت زواج األقــارب و نوعيــة الرتكيبــة اجلينيــة والتنــوع الوراثــي لــدى جمموعــات الغــزالن الربيــة، وقــد إســتنتج أن أفضــل بروتوكــول إلســتخراج احلمــض النــووي مــن عينــات الــروث هــو الربوتوكــول الــذي يســتخدم يف اســتخراج احلمــض النــووي للــرباز البشــري، وبعــد اســتخراج احلمــض النــووي مــن هــذه العينــات مت اســتخدام أربعــة عامــات وراثيــة مــن نــوى اخلــااي اجلســمية يف حتديــد مســتوى زواج األقــارب والتنــوع اجليــي يف الغــزالن، ولقــد حتســنت هــذه )DMSO( وثنائــي ميثيــل سلفوكســيد )BSA( وذلــك عــن طريــق إضافــة مــادة املصــل البقــري الــزااليل PCR القياســات بعــد أن متــت إزالــة مثبطــات تفاعــل سلســلة البوليمريــز وأظهــرت النتائــج أبن متوســط زواج األقــارب هــو 0.228 جلميــع عينــات حمافظــة ظفــار وبنســبة خطــأ قياســي )SE( قــدره 0.09، حيــث يشــر ذلــك أبن الغــزالن متــر مبرحلــة مبكــرة مــن التــزاوج الداخلــي التدرجيــي، أمــا فيمــا يتعلــق ابلتنــوع الوراثــي اجليــي فقــد إعتمــد علــى حســاابت Fst املتبعــة بــن جمموعتــن مــن الغــزالن مثــل نتائــج Fst الــي تصــل اىل 3.7٪ بــن جمموعــة غــزالن منطقــة صولــوت Solot و ابقــي الغــزالن املضمنــه يف الدراســة، وأمــا قياســات ال Fst لباقــي الغــزالن الربيــة فهــي تــرتاوح مــا بــن 0.071 )بــن وادي غــارة ووادي ســتوم( و 0.231 )بــن وادي غــارة ووادي عيــون(، ولقــد حللــت اخللفيــة اجلينيــة بطريقــة حتليــل املكــوانت الرئيســية للمجموعــات األربــع مــن الغــزالن الربيــة املوجــودة يف أربــع وداين رئيســية وأشــارت النتائــج عــن بعــد غــزالن عيــون عــن غــارة جينيــا وتبعــد عنهــا بقليــل غــزالن وادي ســتوم وأمــا غــزالن منطقــة صولــوت فقــد كانــت األبعــد عــن غــزالن املناطــق األخــرى، وتتفــق هــذه النتائــج الFst وواقــع التوزيــع اجلغــرايف ملواقــع مجــع العينــات، ويف اخلتــام ، تعــد نتائــج هــذه الدراســة هــي األوىل مــن نوعهــا يف دراســة إمكانيــة إســتخاص احلمــض النــووي مــن عينــات الــروث يف الغــزال يف الســلطنه وهــي ذات أمهيــة يف معرفــة الرتكيبــة اجلينيــة للغــزالن الربيــة يف ســلطنة عمــان وميكــن اســتخدام نتائجهــا كمرجــع للدراســات املســتقبلية املعنيــة ابلتنــوع اجليــي يف الغــزال العــريب يف الســلطنة. 36 SQU Journal of Agricultural and Marine Sciences, 2021, Volume 26, Issue 2 Characterization of Genetic Diversity in Dhofari Wild Gazelles The first action taken by the Omani government to pro- tect wild gazelles was to set up several sanctuaries that encompassed gazelles. These sanctuaries include Ras Al Shagar protected Area, Al Wusta Wildlife Reserve, and Jabal Samhan protected area, established in 1982, 1994 and 1997 respectively. Together, these sanctuaries have greatly attributed to the protection of gazelles’ popula- tion in Oman although several populations are still strug- gling to survive due to habitat destruction (as a result of highways construction and urban sprawl), pouching and reduction of pastures due to lack of rainfall (Ministry of Environment and Climate Affairs, Sultanate of Oman, personal communication). Other areas where wild ga- zelles have been reported including Al Saleel Natural Park, Al Hajar Mountain, and southern coastal plain of Mirbat and Sadah. Additionally, few separated individ- uals have been reported throughout Dhofar Nejd areas (Ministry of Environment and Climate Affairs, Sultanate of Oman, personal communication). Unfortunately, gazelles population are continuously declining due to illegal hunting and animal capturing (Al Hikmani et al., 2015; Massolo et al., 2008). Gazelles are a main dietary component for some wild species, such as the Arabian leopard Panthera pardus nimr (com- monly found in Dhofar mountains) which (Judas et al., 2006). In addition, gazelle’s juveniles are considered one of the opportunistic mammalian preys for the scaven- ger white vulture’s (considered an endangered species by the IUCN, 2017) which relies on gazelles juveniles as feed for its hatchlings (Margalida et al., 2012). In addition, habitat degradation and population fragmen- tation also threaten Arabian gazelles. The presence of various kinds of flora in many valleys in Oman is a pri- mary source of feed for many wildlife species. However, plants destruction along valleys due draught or weather conditions is common. An example of conditions is the floods caused by the tropical cyclone Gonu in northern Oman in 2007, and more recently, the destructive cy- clone (Mekunu) and the cyclone (Luban), which impact- ed Dhofar Governorate in 2008. These cyclones could diminish the numbers of wild gazelles and the types of plants that they feed on in addition to threatening their livelihood as gazelles become unable to adapt to sudden habitat and environmental catastrophes (Ministry of Environment and Climate Affairs, Sultanate of Oman, personal communication). Another challenge facing the gazelle populations is the construction of road networks across gazelles habitats separating gazelles herds small- er herds, which could increase inbreeding and reduce fitness. For instance, the road between Qurayyat and Sur Wilatats splits the wild gazelles population into two herds (groups) of gazelles with little interbreeding be- tween both populations. The pouching of adult gazelles in various parts of Oman remains a continuous threat the gazelles’ population. Non-invasive sampling is advantageous and it is easier and cheaper than invasive sampling (Taberlet et al., 1999) and is also in line with ethics and conserva- tion strategies for wild animals. Nevertheless, there are some drawbacks associated with in non-invasive sam- pling. For instance, in fecal samples, there is a chance of cross contamination of feces belonging to different individuals. This can be avoided by properly selecting the pellets exactly from the top of the fecal colony to get fecal pellets belonging to only one individual. The challenge associated with fecal DNA is its degradation because of the sun’s UV light (King et al., 2018). The fecal moisture is yet another concern as it enhances the attachment of the soil to the feces and increase the inhibitors from the soil, which in turn prevents prop- er amplification of DNA. Thus, gathering fresh fecal samples and preserving them in very dry and low tem- perature is essential for successful DNA amplification (Murphy et al., 2007). The feces of both Nubian ibex and domestic goats is occasionally confused with the gazelles scats and this could affect the accuracy of the results of genetic diversity studies. In such cross-spe- cies contamination of fecal samples, the alleles obtained for the analysis could give false genotyping results re- sulting in allelic dropout or multiple alleles. In order to avoid such complications, developing basic knowl- edge of differentiation between species fecal samples becomes essential. The separation between individual gazelles fecal samples is also important in assessing the level of genetic diversity of the species. Collecting scat samples from a spot scat of different gazelles are found and it is assumed that it belongs to one individual could result in higher estimates of heterozygosity. Although the threats of gazelle populations in Oman are continuous, the impacts of these threats on the fit- ness and genetic variation of these populations had not been assessed. It is therefore a matter of importance to assess the status of the genetic variation in the Omani Gazelle population. Such assessment is not only import- ant to guide policy makers to take appropriate actions today, but can be a reference point helping future re- searchers compare today’s genetic variation with their future findings. Monitoring the genetic diversity of Omani gazelles is crucial as it helps us to predict their future fitness, disease susceptibility and the levels of inbreeding (Hedrick and Garcia-Dorado, 2016; Szulkin et al., 2013). Therefore, genetic diversity assessment is required for shaping policies in gazelles’ protection. The aim of this project was to determine population struc- ture and genetic diversity of the Arabian gazelles in Dhofar using microsatellite DNA markers. We utilized a non-invasive sampling approach in which we extracted DNA from gazelles’ scat samples. Our approach is the initial of its kind in assessment of genetic diversity in ga- zelles’ populations in Oman. 37Research Article Jashool, Al-Ansari, Al-Marzooqi, Alqaisi, Al-Gahdhami, Al-Abri Materials and Methods Collection and Grading of Faecal Samples for DNA Extraction In this project, 110 gazelles’ feces specimens were col- lected from 69 locations from four different valleys in Dhofar Governorate Stom, Gara, Ayon and Solot (Ta- ble 1). Gazelles scat samples were located by tracing ga- zelles toe prints. Gazelle toes prints are small footprints shaped like a longitudinal symmetrical cross section of an apple with clear med-line as shown in Figure 1. The diameter of a scat pellet’s area is roughly 30 cm. In collection zones, different pellets from different gazelles usually exist in the same spot. Therefore, in a cluster of fe- ces, old or fresh samples can be found. However, only fresh scat pellets were sampled carefully from the top of any scat colony in order to best ensure they belong to the same indi- vidual. Although we took this measure, it is still imperative to indicate that the samples number is not always reflective of the individuals’ number in a certain site since an individ- ual can defecate at more than one spots within a location. We graded the gazelle’s faecal sample based on their color on a scale of A (fresh gazelle’s feces) to D (at least 5 days old feces). The colors of grades A to D samples ranged from dark black to white respectively as shown in Figure 2. The difference in color is attributed to evaporation of moisture Table 1. Collection of scat samples in gazelles habitats for Dhofar governorate. Population Valleys Locations Samples 1 Solot 25 38 2 Wadi Ayon 11 14 3 Wadi Gara 18 29 4 Wadi Stom 15 29 Total 69 110 and physicochemical changes the difference of plants spe- cies that gazelles graze on in different locations. The phys- ical characteristics and grading of scat samples of various colors are given in Table 2. Samples collection was conducted in the same day and in any given location to limit the collection of scat from the same herd twice. This mitigated the chance of double sampling of the same individual as gazelles could migrate from one location to another especially during lack of water resources and competitions for pasture. The fecal samples were collected in plastic bags, labeled and preserved in a cool box and later frozen at -80°C until DNA extraction. DNA Extraction Pre-DNA extraction, crusting of fecal samples was per- formed using sterile and disposable blades into 4 mL Ep- pendorf tubes. For the extraction protocols in this proj- ect, we crusted the outer layer of 11 pellets from each sample for a total weight of 0.18 g to 0.22 g. In total, 913 pellets belonging to 83 fecal samples were crusted for DNA extractions. DNA extraction was performed using the QIAamp® human stool DNA extraction protocol fol- lowing the manufacturer’s procedure. DNA concentra- tion and purity were assessed using a nano-drop. Polymerase Chain Reaction (PCR) Fecal samples carry some compounds that inhibit PCR reactions. These compounds came from the soil, bile salts, complex polysaccharides, collagen, heme, hu- mic acid, and urea. To overcome PCR inhibitors, 50% DMSO and 10% BSA were used in an amount 2.5µL of total PCR volume. The composition of the master mix was used for amplifying various gazelle molecular mark- ers. These were performed in 25 µL of total volume (1X) containing on average 25 ng/µL of DNA, 200 µM of each dNTP, 2 µM MgCl2, 5 pmol of each primer, 1-unit hot start polymerase, 2.5 µL of the same amount of both 50% DMSO and 10% BSA. Finally, 7.8 µL nuclease free water were added to complete the total PCR volume. The phases of PCR cycles started with 94°C for 7 min and ended with 72°C for 7 min and in-between cycles were as follows: (i) DNA denaturation at 94°C for 30 s, (ii) primers annealing phase for 30 s (at a temperature chosen to be the lowest annealing temperature amongst both primers), and (iii) DNA extension took place at 72°C for 30 s in presence of (Taq) polymerase enzyme. Fragment Analysis A size standard “ROX 400” (ABI) (Internal Lean Stan- dard) was run concurrently in each capillary to create the standard curve. Three markers (FAM, HEX and TA- MARA) labeled with different dyes, were used to label the product of the 9 Microsatallite markers shown in Table 4. The markers were run in groups of three (A 3´) Forward Reverse Dye Tm C° A BM302 F-GAATTCCCATCACTCTCTCAGC R-GTTCTCCATTGAACCAACTTCA 5´ HEX 58.4 SR-CRSP6 F-CATAGTTCATTCACAATATGGCA R-CATGGAGTCACAAAGAGTTGAA 5´ FAM 57.5 ETH10 F-GTTCAGGACTGGCCCTGCTAACA R-CCTCCAGCCCACTTTCTCTTCTC 5´ TAMRA 66.4 B TEXAN6 F-AGGCAGTTACCATGAACCTACC R-ATCCTGGTGGGCTACAGTCTAC 5´ FAM 62.1 BM4505 F-TTATCTTGGCTTCTGGGTGC R-ATCTTCACTTGGGATGCAGG 5´ HEX 58.4 TEXAN19 F-CTGAAACCCTCTTATTCAAATTGTG R-TGCAGAGTCAGATAAAAATCCC 5´ TAMRA 58.4 C OarFCB304 F-CCCTAGGAGCTTTCAATAAAGAATCGG R-CGCTGCTGTCAACTGGGTCAGGG 5´ FAM 66.8 INRA40 F-TCAGTCTCCAGGAGAGAAAAC R-CTCTGCCCTGGGGATGATTG 5´ TAMRA 59.4 D BM415 F-GCTACAGCCCTTCTGGTTTG R-GAGCTAATCACCAACAGCAAG 5´ TAMRA 59.4 Table 5. Summary of Chi-Square Tests for Hardy-Weinberg Equilibrium. The markers names, degree of freedom (DF), Chi-Square value (ChiSq) and its probability are shown. Population Locus DF ChiSq Prob. Gara BM4505 15 20.160 0.166 Gara TEXAN19 28 39.020 0.081 Gara INRA40 1 0.194 0.659 Gara BM415 1 0.000 1.000 Solot BM4505 3 6.000 0.112 Solot TEXAN19 36 56.525 0.016 Solot INRA40 21 67.089 0.000 Solot BM415 3 20.989 0.000 Solot BM4505 15 30.000 0.012 Solot TEXAN19 36 73.229 0.000 Solot INRA40 21 30.238 0.087 Solot BM415 6 11.194 0.083 Ayon BM4505 3 4.160 0.245 Ayon TEXAN19 28 48.333 0.010 Ayon INRA40 15 22.440 0.097 Ayon BM415 1 0.141 0.708 Table 6. Mean and standard error (SE) of sample size (N), no. alleles (Na), no. effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and fixation index (F) for the populations. Pop Mean/ SE N Na Ne Ho He F Gara Mean 7.250 4.500 2.863 0.519 0.563 0.030 SE 0.479 1.500 0.708 0.086 0.122 0.075 Solot Mean 21.250 5.500 3.127 0.248 0.617 0.595 SE 6.223 1.500 0.651 0.118 0.106 0.152 Stom Mean 13.000 6.500 4.172 0.663 0.740 0.067 SE 2.415 1.041 0.611 0.111 0.048 0.222 Ayon Mean 8.500 4.750 2.494 0.357 0.505 0.219 SE 1.555 1.377 0.713 0.070 0.119 0.143 Table 7. Gazelle populations pairwise population Fst values. Gara Solot Stom Ayon Gara Solot 0.000 Stom 0.073 0.000 Ayon 0.104 0.133 0.000 Fst Values below diagonal. Fst=0 (panmixis), Fst=0.01 (moder- ate diversity), Fst<0.2 (High diversity). 40 SQU Journal of Agricultural and Marine Sciences, 2021, Volume 26, Issue 2 Characterization of Genetic Diversity in Dhofari Wild Gazelles Discussion The largest allele number obtained was in Solot (21.25) followed by Stom (13) and lowest sample size observed was in Gara (7.25). However, the number of alleles only increased slightly as the sample size increased. The ob- served heterozygsity did not depart largely from the observed heterzygosity except for Solot and Ayon. The results of the fixation index (Table 6) showed some de- gree of inbreeding in all the locations. However, it was the highest in Solot (0.595) and lowest in Gara (0.030). The Fst values showed the presence of a clear population differentiation in our data with no panmixic populations (lower that 1%). The Fst value between Solot and Stom populations was 7.3%, which indicates that the two pop- ulations are genetically close. This is also an indication that the two populations are near panmixia and might be undergoing random mating. The Fst value between Gara and Ayon (23.1%) is a relatively higher genetic dif- ferentiation compared to that between other popula- tions. This suggests that there was little interbreeding or migration between the two populations. The Fst value between Gara and Stom was 7.1%, which was moderate and similar to the value between Solot and Stom (7.3%), while that between Ayon and Solot was 10.4%. However, the genetic diversity between Gara and Solot, was 14.5%, which was higher than Ayon and Solot. Altogether, our results indicated a moderate to high genetic diversity in wild Dhofar gazelle’s populations. In contrast, computing Fst pairwise between two gazelles population (Solot versus remaining populations) gives 3.7% of genetic diversity. Taken together, our results show that all populations had a moderate genetic differ- entiation from one another. However, the levels of ge- netics differentiation found in this study are considered within the range reported for gazelle populations. In a previous study on wild gazelles of the southern Levant, the pairwise Fst between Dorcas gazelles (Arava) and Acacia gazelles was found to be 30.9 % which is a rel- atively high genetic differentiation (Hadas et al., 2015). Principle components analysis of PC 1 vs PC 2 shown in Figure 3a agreed with Fst results and showed that Gara and Stom (lowest pairwise Fst) were closer to each other compared to the rest of the populations. It also placed Gara and Ayon distantly from one another (Highest pairwise Fst). Conclusion There was a low to moderate inbreeding levels and moderate to high genetic diversity in wild gazelles pop- ulations included in this study, which indicated higher within population mating and lower between popula- tions mating. Our genetic differentiation (Fst) analysis showed that the highest differentiation was between Gara and Ayon (23.1%). The PCA was in agreement with the genetic differentiation analysis and clustered Gara and Ayon further away from one another. In ad- dition to that, the PCA analysis clustered the popula- tions according to their geographical distribution in the map with Solot being further away from the other sampling locations. Our study illustrates the successful utilization of noninvasive sampling in assessment of genetic diversity in wild gazelle populations in Oman. Nevertheless, additional markers and a larger sample size are required in order to get accurate estimation of population genetics parameters in future studies. Alternatively, utilization of modern genotyping tech- niques such as genotyping by SNPs would ultimately yield more markers and increase the confidence and reliability of the results compared to microsatellites. Figure 3. Clustering of gazelle populations using principal coordinates analysis a (PC1 vs PC2), b (PC2 vs PC3) and c (PC1 vs PC3). 41Research Article Jashool, Al-Ansari, Al-Marzooqi, Alqaisi, Al-Gahdhami, Al-Abri Acknowledgment We would like to thank the Office for Conservation of the Environment, Sultanate of Oman for funding this study. 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