genetic variation of mhc class ii dra of two...

วารสารสัตว์ป่าเมืองไทย ปีที่ 22 ฉบับที่ 1 พ.ศ. 2558 Journal of Wildlife in Thailand Vol. 22 No. 1, 2015

GENETIC VARIATION OF MHC CLASS II DRA OF TWO ENDANGERED

CERVIDAE SPECIES, Cervus eldii AND Cervus porcinus

Suthathip Dejchaisri1,2,3, Yuttamol Muangkram4, Waradee Buddhakosai4,5, Nongnid

Kaolim6, Nikorn Thongtip1,2,7, Naris Bhumpakphan8, Ronglarp Sukmasuang8,

Tarasak Nipanunt9, Boripat Siriaroonrat10, Sumate Kamolnorranath10, Worawidh

Wajjwalku1,2,5,11,*, Manakorn Sukmak6,11, Hugo Volkaert1,2 & Anuchai Pinyopummin1,2,7

ABSTRACT

DRA, the monomorphological loci of major compatibility complex class II (MHC class II) in the most mammals, was first

analyzed from two endangered cervids in Thailand, Eld’s deer Cervus eldii and hog deer Cervus porcinus. (It is also listed

under Axis procinus) The nearly-complete sequence (244 bp) of DRA loci at exon 2 found 8 haplotypes of Eld’s deer, (Cedi-

DRA*0101 to Cedi-DRA*0108) and 2 haplotypes of hog deer (Cepo-DRA*0101 and Cepo-DRA*0102) with 6 and 2

polymorphisms, respectively. Of interest was the finding that both haplotypes of hog deer shared the same nucleotides with

Eld’s deer. One haplotype was, additionally, similar to the red deer. These results demonstrate the inter-genus relationship

within Cervus spp they were. Furthermore, the phylogenetic relationship of this fragment within the Artiodactyla clearly

shows the separation between the Cervidae and Bovidae. The comparative phylogenetic analysis with available mammal

species sequences complied from GenBank (9 orders; 22 families; 64 species) could support more their phylogenetic

relationship information. In both species, this region could encode the same 81 amino acids except one haplotype of Eld’s

deer showed little different (47R to 47C). Nevertheless, this substitution was not predicted as functional significance but

adjacent to the putative antigen binding site.

Key words: Eld’s deer, hog deer, major histocompatibility complex, genetic diversity, polymorphism

1Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand2Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO-CHE), Bangkok, 10900, Thailand3The Graduate School, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand.4The Graduate School, Kasetsart University, Bangkhen Campus, Bangkok, 10900, Thailand5Interdisciplinary Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkhen Campus, Bangkok, 10900,Thailand6Kamphaeng Saen Veterinary Diagnosis Center, Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng Saen Campus, Nakhon

Pathom, 73140, Thailand7Department of Large Animal and Wildlife Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng Saen

Campus, Nakhon Pathom, 73140, Thailand8Department of Forest Biology, Faculty of Forestry, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand9Department of National Park, Wildlife and Plant Conservation, Bangkok, 10900,Thailand10Bureau Conservation Research and Education, Zoological Park Organization, 10300, Thailand11Department of Farm Resources and Production Medicine, Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng Saen Campus,

Nakhon Pathom, 73140, Thailand.*Corresponding author, E-mail: [email protected]


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INTRODUCTION

The major histocompatibility complex (MHC) is a large family of gene-encoding proteins that play a role in the immune-

response system. It includes the most polymorphic loci in the vertebrates. In humans MHC exhibits twice the level of nucleotide

diversity compared with the genomic average (Hughes & Nei, 1989). This characteristic is maintained and driven by the

pathogen exposure. There is an association between specific alleles and infection levels (Buitkamp et al., 1996) and between

MHC diversity and disease resistance or susceptibility, reported in the both avian and ungulate populations (Briles et al., 1977;

Paterson et al., 1998; Spurgin & Richardson, 2010). Thus reduction of variability in MHC may cause a reduced fitness (O'Brien

et al., 1985; Yuhki & O'Brien, 1990) and low diversity of the MHC loci may impact the survival of some threatened species.

In humans, the complex is called the human leukocyte antigen (HLA) and is located on the short arm of chromosome

6. The complex may be situated on different chromosomes in different species. For example, MHC in the goat (OLA), the

horse (ELA) and the buffalo (Bubu) is located on chromosomes 23, 20, and 2, respectively. MHC is divided into 3 classes;

class I, II, and III. Class I and II MHC are more commonly referred to as the antigen-presenting molecules. Class I loci express

the protein on all nucleated cell surfaces whereas class II genes encoding for the protein are exhibited only on the antigen-

presenting cell surface. The class I MHC molecule is composed of the three external domain of α chain (α1, α2 and α3),

coded by the MHC class I region and the lighter β chain (expressed from other loci outside the MHC). The class II molecule

is comprised of two domains, one from each of the α (α1, α2) and β (β1, β2) chains encoded inside the complex. Both

class I and II MHC molecules form the peptide-binding site as a groove between the α1 and α2 domain of the class I molecule

or between the α1 and β1 domain of the class II molecule. With the vital role of the peptide-binding site being the response

to various pathogens it therefore demonstrates a high polymorphism. Moreover, the MHC is co-dominantly expressed. Thus,

the probability that any two individuals will express the same MHC molecules is quite low. The MHC genes are closely linked

and inherited together as a package, the haplotype. Combinations of both the phenotypic and genotypic levels are incorporated

to define the appropriate nomenclature of the alleles.

To date, the MHC genes had been sequenced for genotypic study in several taxa including humans, non-human primates,

rodents, ungulates, carnivores, birds, and fish. Some wild species have been subjects for MHC diversity assessment. The

genetic diversity on MHC is important for both ex-situ and in-situ population management. The Artiodactyla, especially deer,

are comprised of numerous species and are key species in an ecosystem, acting as prey for predators.

The characteristic and diversity of MHC class II genes has been reported in various species of deer, such as red deer

Cervus elaphas (Swarbrick et al., 1995; Swarbrick & Crawford, 1997), fallow deer Cervus dama (Mikko et al. 1999), roe deer

Capreolus capreolus (Mikko et al. 1999), muskoxen Ovibos moschatus (Mikko et al. 1999), white tail deer Odocoileus

virginianus (Van Den Bussche et al., 1999), moose Alces alces (Mikko & Andersson, 1995; Ellegren et al., 1996; Wilson et

al., 2003), caribou Rangifer tarandus (Kennedy et al., 2010; Taylor et al., 2012), Père David's deer Elaphurus davidianus (Wan

et al., 2011; Zhang et al., 2012), black muntjac Muntiacus crinifrons (Wu et al., 2012), etc. Most of these studies focus on the

genetic variation on the β loci. The number of alleles reported in those studies varies between among populations and species.

The polymorphic pattern reported indicates the trans-species persistence of alleles (Piertney & Oliver, 2006). Different species

may contain alleles that are more closely related than some of the alleles found within species (Nei & Rooney, 2005). The

polymorphism between alleles is mostly found in the second exon which codes for the peptide binding site of the molecule.

In this exon the ratio of non-synonymous to synonymous substitutions (dN/d

S) is higher than would be expected under neutral


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evolution. This phenomenon therefore indicates evidence that the MHC is under strong positive evolutionary selection.

While the products of non-classical class II genes (DM, DN, DO) play a role in antigen processing, the classical MHC

class II molecules (DP, DQ, DR) are involved in the antigen presenting process. Among the classical MHC class II molecules

DR is expressed at the highest level on the antigen presenting cells, including B lymphocytes, macrophages and dendritic

cells. The α locus of DR region, the so-called DRA, is described as being mono-morphological in most mammals. The

number of DRA alleles, similar to other loci, differ between species and is usually lower than the number of DRB alleles. For

example, only three DRA alleles were identified in 129 Alaskan caribou whereas 21 DRB alleles were detected within the

same herd (Kennedy et al., 2010). In human, the data from IPMG/HLA database (www.ebi.ac.uk/imgt/hla/index.html) shows

that there are only seven HLA-DRA alleles whereas 1,740 HLA-DRB alleles are reported (data updated to February, 2015).

Although the DRA region contains low polymorphism and only a couple alleles may be identified in each species its

combination with the highly polymorphic DRB products can generate great protein diversity.

Five species of deer are found in Thailand, playing a vital role in balanced ecosystems and maintaining the diversity of

the predator species. However, there have been few reports examining the genetic diversity, especially on MHC loci, of these

deer.

The objectives of this study were 1) to characterize the DRA region at exon 2 of the captive Eld’s deer and hog deer in

Thailand and 2) to investigate molecular evolution and clarify whether the region harbors trans-species polymorphism. The

obtained data would be fundamental for captive population management. New data obtained will provide preliminary

knowledge to facilitate further study of the genetic diversity and the natural adaptation of these species.

MATERIALS AND METHODS

Sample collection

We collected blood samples from captive Eld’s deer and hog deer held at Huai Kha Khaeng Wildlife Sanctuary, Salak-

pra Wildlife Sanctuary and one zoo registered by the Zoological Park Organization of Thailand, namely Khao Kheow Open

Zoo.

DNA analysis

These samples had their DNA extracted with phenol/chloroform (Nelson & Krawetz, 1992; Tavitchasri et al., 2011)

with minor modification. The exon 2 of DRA segments were amplified using a pair of primers (Sena et al., 2003) via a

polymerase chain reaction (PCR) using Phusion Hot Start II High-Fidelity DNA polymerase (Fermentas Finnzyme) according

to the manufacturer's instructions. The forward primer on intron 1 was 5'- CCC CCT TTC TTG TCT TTT CAG AG -3' and

the reverse primer on intron 2 was 5'- CAA TTC CCA AGT CTA GGA GGA CTG -3'. The total PCR reaction volume was

50 μl, which included 0.5 μM of each primer, 0.2 mM of each deoxyribonucleoside triphosphate (dNTP), 1 U of Phusion

Hot start II DNA polymerase, 10 μM 5×Phusion HF buffer, and RNase-free water added to take it to the 50 μl. Each cycle

profile of the PCR for 35 cycles consisted of denaturation for 30 s at 98oC, annealing for 30 s at 53oC, and extension for 30 s

at 72oC. The final extension was held at 72oC for 5 min. The PCR products sized 307 bp were confirmed using 2% agarose


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gel electrophoresis and purified using the modified method from Boom et al. (1990). The results were then sequenced by the

1st BASE Laboratories, Malaysia. Heterozygous samples were recognized by the present of double peak at specific positions

in the chromatogram. These were processed by cloning using the InsTAclone PCR cloning kit (Fermentas) according to the

manufacturer's instruction, thereby identifying each individual allele. At least three individual clones were selected and

sequenced to determine each allele.

Phylogenetic analysis

The PCR product sized 260 bp (excluded the pair of primers) was showed the highest maximum identity with MHC-

DRA sequences using BLAST program (www.ncbi.nlm.nih.gov/BLAST). It was composed of a nearly complete sequence

of exon 2 (244 bp) and a partial sequence of the second intron. The second exon sequences of our study were aligned using

CLUSTALW algorithm (Thompson et al., 1994) to indicate each individual loci. The calculation of phylogenetic relationship

was performed via MEGA5 (Tamura et al., 2011). The phylogenetic tree were constructed via the Kimura 2-parameter model

with 5 rate categories of discrete gamma distribution using the best fit DNA model with Bayesian information criterion (BIC).

The tree was computed by a maximum likelihood statistical method (Saitou and Nei, 1987; Nei and Kumar, 2000) with

confidence values established by 10,000 bootstrapping (Felsenstein, 1985). Comparative phylogenetic analysis from this study

and utilizing availably representative sequences of mammal species (9 orders; 22 families; 64 species) complied from GenBank

based on DRA loci at exon 2 were performed (Table 1). The relative frequencies of non-synonymous (dN) and synonymous

substitutions (dS) were calculated via MEGA5, following the method of Nei and Gojobori (1986).

Nomenclature

We designated the alleles of both species by following the MHC nomenclature system (Klein et al., 1990; Ellis et al.,

2006). Herein, the Eld’s deer (Cervus eldii), consists of the identical first two letters with red deer (Cervus elaphus) which

recently was a subject to evaluate the polymorphism on DRA gene (Ballingall et al., 2010). We used the next two letters of

species (Cervus eldii) to name the alleles of Eld’s deer in this study (e.g. Cedi-DRA*0101). All alleles have been deposited

in the GenBank database (accession number KT215527 to KT215536).

Taxonomy Allele Accession no. L1 L2 L3

Order: Primate

Family: Hominidae

Gorilla gorilla Gogo EU877226 •

Homo sapiens Hosa NM019111 •

Pan troglodytes Patr EU877224 •

Pongo pygmaeus Popy EU877227 •

Table 1 The length of sequences in this study and complied sequences from GenBank were shown follow by order and

family of each species with allele and accession number. Length 1 (L1): 246 bp; Length 2 (L2): 243 to 245 bp;

Length 3 (L3): 180 to 189. The “*”referred to accession number KT215527 to KT215534 and the “**”referred

to accession number KT215535 and KT215536.


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Family: Hylobatidae

Nomascua gabriellae Noga EU877229 •

Nomascua siki Nosi EU877230 •

Family: Cercopithecinae

Macaca fascicularis Mafa FR717416 •

Macaca nemestrina Mane GQ214407 •

Macaca sylvanus Masy EU877221 •

Order: Artiodactyla

Family: Bovidae

Bison bonasus Bibo AF385485 •

Bos gaurus Boga AF385486 •

Bos grunniens Bogr JF298905 •

Bos indicus Boin FM986339 •

Bos javanicus Boja AF385487 •

Bos taurus Bota DQ821713 •

Bubalus bubalis Bubu AF385489 •

Bubalus depressicornis Bude AF385484 •

Budorcas taxicolor Buta FM986345 •

Capra falconeri Cafa FM986346 •

Capra hircus Cahi AB008755 •

Connochaetes taurinus Cota FM986343 •

Oryx dammah Orda FM986342 •

Ovis aries Ovar FM986336 •

Ovis canadensis Ovca FM986340 •


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Ovis dalli Ovda FM986341 •

Ovibos moschatus Ovmo AF227193 •

Rupicapra rupicapra Ruru FM986344 •

Syncerus caffer Syca AF385490 •

Family: Camelidae

Lama pacos Lapa FM986349 •

Family: Cervidae

Cervus elaphus Ceel FM986347 •

Cervus eldii Cedi This study* •

Cervus porcinus Cepo This study** •

Rangifer tarandus Rata FM986348 •

Family: Suidae

Sus scrofa Susc AB215119 •

Order: Lagomorpha

Family: Leporidae

Oryctolagus cuniculus Orcu NM001171118 •

Order: Cetacea

Family: Physeteridae

Physeter catodon Phca FM986352 •

Family: Delphinidae

Delphinus capensis Deca EF375604 •

Grampus griseus Grgr EF375602 •

Stenella attenuata Stat EF375591 •

Stenella coeruleoalba Stco EF375585 •

Tursiops aduncus Tuad EF375594 •


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Family: Phocoenidae •

Phocoena phocoena Phph EF375598 •

Phocoenoides dalli Phda EF375600 •

Family: Platanista

Platanista gangetica Plga EF375596 •

Family: Balaenoptera

Balaenoptera omurai Baom EF375605 •

Family: Pontoporia

Pontoporia blainvillei Pobl EF375595 •

Family: Lipotidae

Lipotes vexillifer Live DQ851844 •

Order: Perissodactyla

Family: Equidae

Equus asinus Eqas FJ487912 •

Equus burchellii Eqbu HQ637395 •

Equus caballus Eqca M60100 •

Equus grevyi Eqgr EU930116 •

Equus hemionus Eqhe EU930128 •

Equus kiang Eqki FJ657514 •

Equus zebra Eqze EU930124 •

Family: Rhinocerotidae

Ceratotherium simum Cesi AF113552 •

Diceros bicornis Dibi AF113550 •

Rhinoceros unicornis Rhun AF113554 •


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RESULTS

In this study the DRA loci at exon 2 in Thai captive Eld’s deer and hog deer were well characterized using a PCR based

method. The nearly complete sequences of both species were contained 244 bp that could encode 81 amino acids of α1

domain of DR molecule. The G+C content was 0.497.

Eight haplotypes of Eld's deer with 6 polymorphic sites (2 variants of 3 singleton variable sites and 3 parsimony

informative sites) were discovered (Table 2). Nucleotide diversity (π) was 0.00929. The maximum likelihood estimated

transition/transversion bias was 3.01. Most of these sites are synonymous substitutions. Only one non-synnonymous

substitution was found. The results of the estimated diversity and selection parameters are shown in table 3. Among captive

Eld’s deer in Thailand eight DRA alleles (Cedi-DRA*0101 to Cedi-DRA*0108) were identified. The most common allele

was Cedi-DRA*0101. Seven alleles expressed 100% identity amino acid residues. The putative antigen binding sites were

determined based on the HLA equivalents (Reche and Reinherz, 2003) and noted as the crosses (+) in the figure 3. R47C

amino acid substitution was found in Cedi-DRA*0108. The impact of this amino acid substitution was evaluated by the

Sorting Intolerant from Tolerant (SIFT) Blink software to predict its functional significance when compared to the reference

sequence (Ceel-DRA*0101). The predicted score was 0.15, higher than the threshold cut off score (0.05). This result defined

the R47C of Cedi-DRA*0108 as a tolerated substitution. It was not likely to have a significant effect on the structure and

function in antigen presenting (data not shown).


Family: Tapiridae

Tapirus bairdii Taba AF113547 •

Tapirus indicus Tain AF113548 •

Order: Carnivora

Family: Ursidae

Ailuropoda melanoleuca Aime DQ019989 •

Family: Canidae

Canis lupus Calu NM001011723 •

Family: Felidae

Felis catus Feca NM001128071 •

Order: Rodentia

Family: Sciurus

Sciurus aberti Scab M97620 •


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Hog deer in this study carried 2 alleles (Cepo-DRA*0101 and Cepo-DRA*0102). These two alleles differed by two

transitional mutation sites (A>G) from those which were found in the Eld’s deer. The transition/transversion bias, was estimated

by the same method as that performed in the Eld’s deer, equaled 2.53 (Table 2). No non-synonymous substitution was found

among hog deer population. The translated amino acid sequences were identified within the population found the similarity

to the Cedi-DRA*0101 to Cedi-DRA*0107 alleles and were related with the Ceel-DRA*0101.

The phylogenetic tree of nucleotide sequence and amino acid sequence were computed under the 10,000 bootstrapping

tests. Both constructed phylogenetic trees showed a similar topology. Eld’s deer alleles and hog deer alleles were allocated

to the same cluster with red deer (Cervus elaphus) allele Ceel-DRA*0101, suggesting that trans-species evolution has occurred

in the Cervidae, as has been previously speculated in several other species (Ballingal et al., 2010; Klein et al., 1993; Van Den

Bussche et al., 1999).

Table 2 The multiple alignment of nucleotide sequence of DRA at exon 2 (244 bp) has showed eight haplotypes of Eld’s

deer with six polymorphisms; four transitional and two transversional substitution and two haplotypes of hog

deer diverged two polymorphisms of transitional substitution type. A dot indicates the same nucleotide as Cedi-

CRA*0101 at the same column. The nucleotide sequence polymorphic site begin at the first base of exon 2.

Nucleotide sequence polymorphic sites

Allele Accession number 80 116 131 140 141 161

Cedi-DRA*0101 KT215527 T G A A C T

Cedi-DRA*0102 KT215528 C . G G . .

Cedi-DRA*0103 KT215529 . . G G T .

Cedi-DRA*0104 KT215530 . . G G . .

Cedi-DRA*0105 KT215531 . . . G . .

Cedi-DRA*0106 KT215532 . . G . . .

Cedi-DRA*0107 KT215533 . . G . . G

Cedi-DRA*0108 KT215534 . T . . . G

Cepo-DRA*0101 KT215535 . . . . . .

Cepo-DRA*0102 KT215536 . . G G . .

Species A Length (bp) R p dN

dS

dN/d

S

Eld’s deer (Cervus eldii) 8 244 3 0.01 0.006 0.113 0.054

Hog deer (Cervus porcinus) 2 244 2.5 n/a 0.016 0.076 0.214

Table 3 The diversity and selection indices at DRA loci of two cervids; Eld’s deer and hog deer.

A=number of alleles found in this study, R = transition/transversion bias, π = nucleotide diversity, dN = non-synonymous

substitution rate, dS = synonymous substitution rate


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Figure 1 The comparative phylogenetic analysis of DRA loci at exon 2 among our study and available sequences complied from GenBank in table 1. The eight haplotype samples of Eld’s deer and two haplotype samples of hog deer are belonged to Cervidae family as same group of Cervus elephus (FM986347) and Rangifer tarandus (FM986348). Overall, DRA loci of mammal species showed their distinguish inter-families relationship. The consensus phylogenetic tree is constructed using Kimura 2-parameter model with gamma distribution rate categories (rate=5).


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Figure 3 The phylogenetic relationship within order Artiodactyla between Cervidae family (subfamily cervinae) and Bovidae family (subfamily Bovinae-Caprinae-Alcelaphinae) showed the inter-families relationship. This results also could served as molecular tools to recognized the different of phylogenetic relationship between antler species and antelope species. The phylogenetic tree was constructed using Jukes-Cantor model with evolutionary invariable (JC+I). Delphinus capensis was representative as outgroup.


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DISCUSSION

This report describes the first characterization of DRA at exon 2 of Eld’s deer and hog deer. No previous studies have

considered the genetic diversity on this locus for these two species. The annotated Cedi-DRA sequences were highly similar

to the ortholog Ceel-DRA*0101. Most variants found in Eld’s deer and hog deer (this study) and the red deer (previous report)

showed the sharing allele (Cedi-DRA*0101, Cepo-DRA*0101, and Ceel-DRA*0101, respectively). This may reflect that

the trans-species allelic lineage has been maintained over a long evolution, as has been stated in many reports. More

comprehensive data from other cervids in Thailand and other regions would be important for clarifying trans-species evolution

in Cervidae and related species. Sharing allelic variants of MHC may be associated with the common pathogens that are

shared among the whole population. Although there were eight nucleotide variants of DRA found in this study, most variants

shared the same phenotypes. In addition, most alleles were homozygous. These characteristics may lead the reduced fitness

of the population. Based on the basic knowledge, arginine (R) is the positive charge amino acid while cysteine (C) is nonpolar

amino acids. R47C was probable to affect to the structure or function of molecule. Nevertheless, this substitution was not

predicted as functional significance but adjacent to the putative antigen binding site. We also found the evidence from the

dN/d

S of this population that support the purifying selection in non antigen binding sites.

Figure 3 The multiple alignment of translated 81 amino acid sequences in comparison to the alleles previously reported in other species in order Artiodactyla. The putative peptide binding sites were noted as the crosses (+) above the sequence, according to the orthologous sequence HLA-DRA (Reche and Reinherz, 2003).


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In this study, we reported only the homozygous alleles. Heterozygous population (distinguished by the heteroplasmy of

chromatogram) was separately recorded. If we assume that the populations are from the same founder group then the possible

pattern of heterozygosity can be implied from the number of homozygous alleles. The more homozygous alleles that were

identified, the more possible heterozygous patterns could be found. Based on the “heterozygosity advantage hypothesis” (Penn

& Potts, 1999; Sommer, 2005; Spurgin & Richardson, 2010), low heterozygosity might lead the reduced fitness of the

population. To maintain heterozygosity, a homozygous population should be concurrently maintained. Accordingly, a

structured breeding plan for the captive population is essential in order to prepare for any reintroduction. Genetic data, especially

for functional loci such as the MHC gene, is critical to ensure there is the capacity for natural adaptation of the reintroduced

population in the future.

ACKNOWLEDGEMENT

We would like to acknowledge Andrew Routh from Head of Veterinary Services, Durrell Wildlife Conservation Trust

for the useful comments on the original manuscript. This research is supported by the Center of Excellence on Agricultural

Biotechnology, Science and Technology Postgraduate Education and Research Development Office of Higher Education

Commission, Ministry of Education (AG-BIO/PERDO-CHE).

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genetic variation of mhc class ii dra of two...

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