 Index | |||||||||
|
| Edited by Yoko Satta. Haseeb A. Khan: Corresponding author. E-mail: khan_haseeb@yahoo.com |
|
About a century back, there was a wide inhabitancy of Arabian oryx (Oryx leucoryx) throughout the Arabian Peninsula. However, massive hunting and scarcity of habitat caused extirpation of this antelope from the wild in early 1970s (Henderson, 1974). The foresighted determination and sincere efforts of certain groups saved the last few animals from extinction by nurturing them in captivity and then successfully reintroducing them into the protected areas (Mesochina et al., 2003; Spalton et al., 1999; Ostrowski et al., 1998). Although the savior of Arabian oryx in captivity had become an international symbol of conservation success (Dixon and Jones, 1988) some untoward events in Oman indicated that the reintroduction programs might be prone to partial or total collapse despite their initial success (Spalton et al., 1999). Thus, a global perspective about developing more effective captive breeding programs is necessary to maintain the genetic diversity of this endangered species (Iyengar et al., 2007). Vassart et al. (1991) have emphasized the need of carrying a large scale screening of different herds of Arabian oryx maintained in the Middle East and elsewhere in the world for genuine selection of candidates for introduction in wild or captive breeding programs.
Microsatellites or simple sequence repeats (SSRs) are the widely used markers for molecular fingerprinting. The popularity of these markers is due to their ease of amplification by polymerase chain reaction, their co-dominant nature and their typically high levels of allelic diversity at different loci. In this investigation, we have used microsatellite markers to determine the current level of heterozygosity and allelic diversity in a specified population of Arabian oryx.
The blood samples were collected from 24 Arabian oryx; 21 (13 males, 8 females) of these samples were obtained from Mahazat As-Sayd Protected Area (MSPA) and 3 (1 male, 2 females) from National Wildlife Research Center (NWRC), Saudi Arabia.
MSPA is a 2245 km2 protected wild shelter located on the arid plains of Western Saudi Arabia, 170 km North-East of Taif in Makkah Province. It is the second largest fenced reserve in the world. Besides the reintroduced populations of Arabian oryx, reem gazelle, houbara bustard and red-necked ostrich, this reserve also holds large natural populations of red and Ruppell’s fox and significant numbers of sand cat, wild cat and ratel, and the spiny-tailed lizard. It is a major breeding area for the threatened lappet-faced vulture and an important stopover site for migrating birds.
NWRC is located 35 km South of Taif, and possesses a fenced area of 650 ha (6.5 km2). It was founded with the aim to start captive breeding of endangered species of Saudi Arabia. The Center is divided into numerous enclosures varying from 0.5 to 100 ha where the animals are kept in semi-captive conditions. About 70 ha area has been assigned as a botanical reserve for the purpose of studying the evolution of vegetation protected from grazing.
Over the time, the herds of Arabian oryx have been reintroduced into MSPA from seven groups (NWRC at Saudi Arabia, Jordan, Bahrain, Qatar, Swaziland, Germany and USA). The majority of founding animals in NWRC came from Thumamah (a private collection in Saudi Arabia) and some from Gulf and USA. Currently, the number of individuals in MSPA and NWRC is approximately 200 and 100 respectively.
The DNA was extracted from 200 μl blood sample using DNeasy Blood and Tissue Kit (Qiagen GmbH, Germany) according to manufacturer’s instructions. The extracted DNA was finally dissolved in 200 μl of elution buffer and stored at –20°C.
We tested a total of 10 microsatellite markers, of which 3 markers (TGLA68, BM1329 and BMS1341) produced ambiguous peaks and were omitted. The remaining 7 loci (RBP3, MCM38, MNS64, IOBT395, MCMAI, BM3501 and MB066) were successfully amplified in all the samples. The characteristics of these loci are summarized in Table 1. The primer sequences of these markers have been reported earlier (Zhou et al., 2007; MacHugh et al., 1997). The forward primer for each marker was labeled with FAM whereas the reverse primer was unlabelled. The PCR reactions were performed in a total volume of 20 μl containing 2 μl 10 × PCR Buffer, 2.5 mM MgCl2, 200 μM each dNTP, 25 nM of each primer, 25 ng template DNA and 0.5 U Taq DNA polymerase. After initial denaturation at 94°C for 4 min, 25 cycles of 93°C for 45 s, 55°C for 45 s and 72°C for 45 s were repeated followed by the final extension at 72°C for 4 min. The aliquots of PCR products (0.25 μl) were mixed with 9.25 μl formamide and 0.25 μl ROX-500 standard. The contents were heated at 95°C for 2 min and then rapidly cooled on ice before being electrophorsed on 3130XL genetic analyzer for allele identification.
 View Details | Table 1 General characteristics of various microsatellite loci used in this study |
For statistical evaluation, we considered all the 24 samples as a single population due to three main reasons; (i) few samples from NWRC, (ii) NWRC being one of the sources of reintroduction in MSPA and (iii) genetic overlapping because of common founders at both the locations.
The allele frequencies, observed (Na) and effective (Ne) number of alleles, observed (Ho) and expected (He) heterozygosities, test of genotypic frequencies to Hardy-Weinberg equilibrium for each locus and the F-statistics were computed using the POPGENE 1.31 software (Yeh and Boyle, 1997). Mean d2 (relatedness of parental genomes) was calculated using the following equation as reported earlier (Coulson et al., 1998).
Where ni1 and ni2 are the number of repeats of the two alleles of the ith locus and N is the total number of microsatellite loci. Mean d2 is based on the difference in the length of two alleles representing the length of time since the two alleles shared a common ancestor (Coulson et al., 1998). High mean d2 is taken as an indicator of outbreeding whereas a low individual Ho is used as an indicator of inbreeding. Among the parameters of F-statistics, FIT denotes the inbreeding coefficient and FST shows the gene differentiation. These parameters describe the reduction of heterozygosity within individuals relative to the total population either due to non-random mating within subpopulations (FIT) or due to selection or drift (FST). FST is inversely proportional to gene flow (Nm); the larger the FST the smaller is the Nm. As a general rule, Nm < 1 (or FST > 0.2) indicates low gene flow whereas Nm > 10 (or FST < 0.02) indicates high gene flow (Mills, 2006).
Among the 7 microsatellite markers studies, RBP3 (140 and 142 bp) and BM3501 (168 and 170 bp) had two alleles; MCM38 (108, 110 and 120 bp), MNS64 (188, 198 and 200 bp) and MB066 (128, 130 and 132 bp) had three alleles; IOBT395 (90, 106, 110 and 174) and MCMAI (185, 187, 189 and 191) had four alleles each. None of the loci showed significant deviation from Hardy-Weinberg equilibrium (Table 2). The allele frequencies of different microsatellite markers are given in Table 3. Some alleles were comparatively less prevalent including allele B of MCM38 which was present in 2 animals, allele C of MNS64 in 3, allele B of IOBT395 in 2, alleles D of MCMAI in 3 animals, respectively.
 View Details | Table 2 χ2 test for Hardy-Weinberg equilibrium |
 View Details | Table 3 Allele frequency for different microsatellite loci in Arabian oryx |
The mean observed heterozygosity (Ho = 0.601) was comparatively higher than the mean expected heterozygosity (He = 0.565). The locus-wise heterozygosity statistics revealed maximum heterozygosity for MB066 (Ho = 0.750) and minimum for BM3501 (Ho = 0.458) markers (Table 4). The inbreeding coefficient (FIT) for all the loci appeared in negative (FIT < 0) except for MNS64 (FIT = 0.101); FIT values ranged between –0.019 and 0.101 with a mean value of –0.087. The fixation index (FST) ranged from 0.371 to 0.551 with the average FST = 0.456 (Table 4). The average gene flow (Nm) was 0.298, ranging between 0.204 (MNS64) and 0.424 (MCMAI). The Shannon’s information index (I) for a particular locus was directly proportional to the number of alleles (Table 4). The specimen-wise Ho ranged from 0.143 to 1.00 with an average of 0.60 whereas the mean d2 varied from 0.57 to 1023.428 with an average value of 223.357 (Fig. 1).
 View Details | Table 4 Genetic information for 7 microsatellite loci in 24 Arabian oryx samples |
 View Details | Fig. 1 Individual values of observed heterozygosity and mean d2 for 7 microsatellite loci in 24 Arabian oryx samples. Samples 2, 14, 19, 20 and 21 exhibited very high value of mean d2 due to the presence of allele D of IOBT395 marker. |
Despite their inherent high resolution superiority and extensive use in population genetic analyses, microsatellite markers have not been fully explored for molecular conservation of Arabian oryx. The two groups of investigators have used the same set of 6 microsatellite loci, 5 of them specific to sheep and 1 to cattle, for measuring the molecular diversity of Arabian oryx (Marshall et al. 1999) and scimitar-horned oryx (Iyengar et al. 2007). In the present study, we have applied 7 microsatellite markers, one of them (RBP3) is common to the previous studies on oryx (Marshall et al., 1999; Iyengar et al., 2007) whereas the remaining 6 loci have recently been utilized for measuring the genetic diversity of Tibetan antelope (Zhou et al., 2007). None of the loci in our study showed significant deviation from Hardy-Weinberg equilibrium (Table 2). In the previous studies as well, the common locus (RBP3) did not show any significant departure from Hardy-Weinberg equilibrium, though some other microsatellite loci exhibited significant deviations (Marshall et al., 1999; Iyengar et al., 2007).
An array of 7 microsatellite markers used in this study clearly differentiated the individual animals. Earlier, a set of 3 polymorphic loci of a protein-based fingerprinting protocol had been applied for reliable identification of sires in the Arabian oryx herd at MSPA (Vassart et al., 1991). The mean allelic polymorphism in our study was comparatively higher (3.00 versus 2.10 or 1.17, respectively) than the mean number of alleles reported earlier in microsatellite (Marshall et al., 1999) or alloenzyme (Vassart et al., 1991) based genotyping of Arabian oryx. Marshall et al. (1999) reported that the set of microsatellite markers they used had limited power for parentage inference due to their low allelic polymorphism however the same markers were found to be suitable for forensic use and confirmed the identity of a poached animal. On the other hand, a comparatively high level of polymorphism of microsatellite loci reported by us could provide a better resolving power for individual identity. Some of the alleles in our study showed very low frequency (Table 3). Marshall et al. (1999) have also reported deficiencies of rare alleles in two of the four contemporary populations of Arabian oryx. During the event of a bottleneck, the so called rare alleles are lost easily than the common alleles rendering the population to be deficient in rare alleles. In case of Arabian oryx, the demographic bottlenecks have been reported to be too short a duration to exert a significant impact on heterozygosity (Vassart et al., 1991). Furthermore, because of the loss of rare alleles, the heterozygosity in the bottlenecked population turns to be higher than expected for the remaining alleles (Marshall, 2007).
We have noticed a comparatively high level of observed heterozygosity (Ho = 0.601) than the expected heterozygosity (He = 0.565) in this population of Arabian oryx (Table 4). The levels of observed heterozygosities of MSPA (Ho = 0.622) and Thumamah (Ho = 0.624) populations of Arabian oryx have been reported to be slightly higher, a decade ago (Marshall et al., 1999). Marshall (2007) have pointed out that although some loss of genetic variation from Arabian oryx has occurred, a substantial amount of genetic variation still remains due to timely exchange of animals among various breeding programs. High values of Shannon information index (I = 0.898) also indicated a rich molecular diversity in the representative population of Arabian oryx of our study. We have also observed an excess of heterozygotes (FIT < 0) for all the microsatellite loci except MNS64 (FIT = 0.101). All the loci demonstrated a high degree of differentiation (FST = 0.456), while the population differed most about the locus MNS64 (FST = 0.551) and least for MB066 (FST = 0.371) (Table 4). Marshall et al. (1999) have also reported comparatively low but significant population differentiation in Arabian oryx due to substantial genetic mixing between populations as a management strategy.
Microsatellite markers are known to offer an opportunity to detect genetic variation both during and after foundation. Both, heterozygosity and mean d2 are suitable parameters for detecting inbreeding and outbreeding depressions respectively (Coulson et al., 1998; Neff, 2004). Low individual heterozygosity is taken as an indicator of inbreeding whereas a high value of mean d2 reflects an outbreeding. Moreover, mean d2 is also considered as a good predictor of divergence times of parental lineages over large time scales provided there is no gene flow between the lineages (Neff, 2004). Since the heterozygosity and allelic diversity are the reliable predictors of both the survival and adaptation abilities of populations it is important to maintain a high level of heterozygosity and allelic diversity to ensure the success of captive breeding programs (Allendorf, 1986; Templeton et al., 1987). The results of mean d2 not only exhibited the allelic diversity but also revealed some sort of outbreeding mainly due to the presence of allele D of IOBT395 locus (Fig. 1). Some evidence for outbreeding depression in Arabian oryx has also been reported earlier (Marshall, 2007). Recently, Da Silva et al. (2009) have observed a significant correlation between the fitness of roe deer offspring with the mean d2, but not with the individual heterozygosity, suggesting a possible outbreeding advantage. Although the microsatellite loci used in this study originate from different chromosomes of sheep and cattle (Table 1) they have been found to be quite beneficial for molecular differentiation of Arabian oryx using the information of individual heterozygosity and mean d2. Factually, both these indices (heterozygosity and mean d2) of inbreeding-outbreeding continuum are computed from the data generated from the same experiment. Hence their combined utilization would be more informative for determining the suitability of individuals for captive breeding programs. Furthermore, identification of the genetic lineage of Arabian oryx representing the allele D of IOBT395 locus would be quite advantageous in maintaining an optimum inbreeding-outbreeding balance in a captive breeding program.
In conclusion, the findings of this study demonstrate a fair degree of molecular diversity in captive bred and reintroduced population of Arabian oryx. The protocol described in this study is simple to apply and of practical application for the management of captive breeding as well as the reintroduction of genetically healthy groups of Arabian oryx back into the wild. The inbreeding can be significantly minimized by taking the advantage of microsatellite profiles of the potential couples. Moreover, a prudent application of microsatellite information can also be used for the preservation of rare alleles in this endangered animal. Further studies are warranted to search for more informative microsatellite loci for Arabian oryx, especially for determining the outbreeding in combination with IOBT395 locus.
This study was supported by a grant from HRH Prince Sultan Bin Abdulaziz Research Chair Program for Environment and Wildlife, Riyadh, Saudi Arabia. We sincerely thank the Secretary General of the National Commission for Wildlife Conservation and Development and the Director of NWRC for supporting our research and making arrangements for sample collection. The technical assistance of Dr. Saud Anagariyah and Mr. Anis Ahamed for blood sampling and DNA extraction, respectively, is highly appreciated.