Characterization of the Mitochondrial Genome of the Vietnamese Central Highland Wild Boar (Sus scrofa)

TL;DR

This study analyzes the mitochondrial genome of Vietnamese Central Highland wild boars to better understand their genetics and aid conservation efforts.

Contribution

The study provides the first complete mitochondrial genome sequence of the Vietnamese Central Highland wild boar and identifies unique genetic features.

Findings

The mitochondrial genome is 16,581 base pairs long and contains 37 genes.

Vietnamese wild boars share a close genetic relationship with Asian pigs based on mitochondrial analysis.

232 nucleotide substitutions were found when comparing this genome to other Sus scrofa genomes.

Abstract

Vietnamese Central Highland wild boars are under serious threat due to illegal poaching, habitat destruction, and uncontrolled breeding with domestic pigs. These issues can damage the unique genetic traits of wild boars, making conservation more difficult. At present, very little is known about the genetics of these animals. This study characterizes the complete genetic material (mtDNA) found in the mitochondria—a small part of the cell responsible for energy production—of the Vietnamese Central Highland wild boar. The molecule is 16,581 base pairs long and contains 37 genes with important functions. We identified unique features in one mitochondrial gene, which help trace the maternal origins of Vietnamese Central Highland wild boar populations. Our findings suggest that Vietnamese wild boars are more closely related to Asian pigs. This study adds value to current knowledge on the genetics of Vietnamese wild boars, contributing to future protection, conservation, and possibly breeding of these animals to prevent further decline.

Hybridization between domestic pigs and wild boars of unknown origins has disrupted the precious gene pool of Vietnamese wild boar (Sus scrofa) populations in the Central Highlands. However, the genetic background of Vietnamese wild boars remains largely unknown. This study describes the complete mitochondrial genome of the Vietnamese Central Highland wild boar, a circular molecule comprising 16,581 base pairs (bp). The mitogenome contains 37 genes, which encode for 2 ribosomal RNAs, 22 transfer RNAs, and 13 mitochondrial proteins. It has a conserved gene order, gene orientation, and similar nucleotide composition indexes to other boars and pig breeds across the world. Notably, 232 nucleotide substitutions were detected when comparing this genome with 19 previously described Sus scrofa genomes. Partial cytochrome b gene analysis revealed the distribution of Asian haplotypes in the Vietnamese Central Highland Sus scrofa. A phylogenetic tree constructed from 32 Sus scrofa’s whole mitogenome sequences demonstrated the close relationship between Vietnamese wild boars and domestic pig breeds. The study provides additional insights into the genetics of Vietnamese wild boars, paving the way for future research in conservation, evolution, and breeding of Vietnamese wild boar populations.

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1. Introduction

Located in the Indo-Burma biodiversity hotspot, which is well known for its largest wild boar diversity in the world, Vietnam is home to two wild boar species: Sus scrofa and Sus bucculentus [1]. While Sus bucculentus was claimed extinct, Sus scrofa has a rather wide distribution across the country, inhabiting wetlands and forests from the North to the Central Highlands [2,3]. Vietnamese Central Highland wild boars can be distinguished from domestic pigs through distinct phenotypic traits such as a small head, long snout, and a compact body shape coated with black or gray fur [2]. Wild boars exhibit remarkable intrinsic properties, ranging from survival resilience, adaptive capabilities, and disease resistance to good-quality meat and a high reproduction rate. Thus, wild boar farming holds significant importance for agricultural development in rural communities. Hybridization between wild boars, especially those of unknown origins, and domestic pig breeds poses a serious threat to the genetic integrity of natural wild boar populations [4,5,6,7]. This consequently leads to mixed genotypes and loss of unique traits, putting the number of genetically pure Vietnamese wild boars on rapid decline and ultimately altering wild boar genetic resources [8]. Until now, the genetic data of Vietnamese wild boars still remain underexplored.

The mitochondrial DNA (mtDNA) is a circular, double-stranded DNA molecule, containing 37 genes and a non-coding control region (D-loop). Mammalian mtDNA is approximately 16.5 kb in size and is characterized by high copy number, maternal inheritance, haploid nature, and absence of recombination [9]. Such characteristics make mtDNA a valuable molecular marker, providing insights into the study of genetic diversity, population history, and evolutionary origins [10]. Of the mitochondrial genome, the cytochrome b gene has been extensively studied to assess the genetic relationship among various pig breeds, serving as an essential tool for phylogenetic analysis and species identification [11,12,13,14]. An evaluation of mtDNA showed that native Vietnamese pigs were genetically diverse, sharing genetic patterns similar to pigs originating from neighboring countries [15,16]. Another study on the sequence variability of cytochrome b among Vietnamese Central Highland pigs revealed the close relationship between Vietnamese wild boars and Asian pig breeds [17]. Recently, the complete mitochondrial genome of several Vietnamese domestic pig breeds, namely I pig, Dong Khe pig, and Ha Lang pig, has been reported, aiding in the efforts to trace the origin of Vietnamese pig populations [18,19,20]. However, the mitochondrial genome of the Vietnamese wild boar and its phylogenetic background are still poorly understood.

This study reports the complete mitochondrial genome of the Vietnamese Central Highland wild boar and compares it with other wild boars and domestic pig breeds. We focus on characterizing mtDNA features and investigating the sequence variability of whole mitogenome sequences, providing valuable information for conservation and genetic research of Vietnamese wild boars.

2. Materials and Methods

2.1. DNA Extraction and Sequencing

A testicular tissue sample of the Vietnamese Central Highland Sus scrofa was provided by the Biological Museum, Tay Nguyen Institute for Scientific Research, Vietnam Academy of Science and Technology. The sample was stored at 4 °C in ethanol until DNA extraction. Total genomic DNA was extracted using the QIAmp DNA Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. The integrity of extracted DNA was checked on agarose gel electrophoresis. DNA quantification and purity determination were performed using a Qubit spectrofluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Samples having a concentration ≥ 2 ng/μL with a total yielded amount ≥ 90 ng and an OD260/OD208 ratio ≥ 1.70 were considered suitable for further experiments. Samples with DNA size <1000 bp were flagged.

Whole-genome sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for the Illumina platform (New England Biolabs, Ipswich, MA, USA). The library concentration was measured fluorometrically, and the average library size was determined using a bioanalyzer (Agilent, Santa Clara, CA, USA) according to Illumina’s library evaluation guidelines. Samples with concentration ≥ 0.50 ng/μL (for genome size < 1 Gb) or ≥2 ng/μL (for genome size > 1 Gb) were considered suitable for sequencing. Sequencing was subsequently performed using an Illumina sequencing system (Illumina, San Diego, CA, USA).

2.2. De Novo Assembly

Fragments of various sizes were assembled using the GetOrganelle (v1.7.7.0) pipeline with optimal parameters to construct a complete mitochondrial genome without the use of a reference genome. De novo assembly results are presented in Supplementary Table S3, showing a total contig length of 16,581 bp. This length is consistent with the size of vertebrate mitochondrial genomes, which typically range from 14 to 20 kb [21].

2.3. Bioinformatics Analysis

The raw sequencing data were preprocessed using fastp v0.23.1 and stored in FASTQ file format (Supplementary Data S1) [22]. Based on the Phred score value recorded for each nucleotide, unreliable or unidentified nucleotides with poor sequencing quality (type N nucleotides) were eliminated. The reading quality results are shown in Supplementary Table S2. The de novo assembly of purified reads was conducted using GetOrganelle v1.7.7.0 software [23]. Assembly quality was evaluated using Quast v5.2.0 [24] and by locally aligning reads to the assembled contigs. This enables the detection of regions with unusually low values of depth coverage compared to neighboring regions, which were noted in the assembly results. The annotation of the assembled mitogenome was completed using Mito FISH version 4.03 [25] and the MITOS2 tool in the Galaxy webserver platform [26], with a specialized database for mitochondrial genomes. The mitogenome sequence of the Vietnamese Central Highland Sus scrofa was deposited in GenBank with an accession number PV693689.

The graphical genetic map of the circular Sus scrofa mitogenome was constructed using Mito Annotator version 4.03 [25]. The amino acid and nucleotide compositions of the entire mitogenome were determined and compared with those of other Sus scrofa using MEGA12 [27]. Strand asymmetry was calculated using the following formulas: AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) [28]. Specific regions of transfer RNA (tRNA), PCGs, ribosomal RNA (rRNA), or control were determined by aligning the Sus scrofa mitogenome with the homologues of similar species. The secondary structures of 22 tRNAs were predicted using tRNAscan-SE software v2.0 [29] and Mitos Webserver [26]. The Relative Synonymous Codon Usage (RSCU) values of Sus scrofa mitogenome were computed using MEGA12 [27].

2.4. Phylogenetic Tree

To determine the molecular location of the Vietnamese Central Highland Sus scrofa in the evolutionary tree and its genetic relationship with other pigs, a total of 32 Sus scrofa whole mitogenomes were subjected to phylogenetic analysis. This includes domestic pig breeds from Asia (8), Europe (4), and Vietnam (4), and wild boars from Europe (5), China (4), Korea (2), Vietnam (2), India (2) and Malaysia, with Phacochoerus africanus used as an outgroup [30]. The mitogenomes were aligned using the ClustalW algorithm of MEGA12 [27]. The Tamura and Nei model was used as a genetic distance model [31]. A Neighbor-Joining tree was constructed using 1000 bootstrap replicates.

3. Results

3.1. Complete Mitochondrial Genome Analysis

The complete mitochondrial genome of the Vietnamese Central Highland Sus scrofa is 16,581 bp in length, which is smaller than the previously published mitogenomes of Sus scrofa from other regions, such as Europe (KP301137, 16,770 bp), China (EF545573, 16,620 bp), and India (MG725630, 16,738 bp). It encodes a total of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a non-coding control region, as typically reported in vertebrates. Apart from nad6 and 8 tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, and tRNA-Pro), which were located on the light (L) strand, the remaining 28 genes of the mitogenome were found on the heavy (H) strand (Figure 1 and Table 1).

3.2. Nucleotide Composition Pattern

The whole Vietnamese Sus scrofa mitogenome shows a similarly high A + T content to other Sus scrofa, accounting for 60.6% (Table 2), with the highest values being recorded in trnF (78.3%) and trnH (76.8%) [32]. mtDNA demonstrates a noticeable bias towards A/T in codon usage, with the third nucleotide in most codons tending to be an A or T base (Figure 2). A significantly similar pattern of nucleotide composition was identified among 15 Sus scrofa from different geographic regions and 2 members of the Suidae family. The length and A + T content did not vary significantly despite their diverse habitats, highlighting the conserved nature in the mitogenome of Sus scrofa. Of these, the amino acid distribution among the wild boars was significantly similar (Figure 3). The most frequently observed amino acids included Leu (15.23–15.28%), Ile (8.85–8.9%), Thr (8.2–8.4%), Ser (7.4–7.5%), and Met (6.9–7.0%), whereas Cys was rare (0.66–0.69%).

3.3. Protein-Coding Genes

Consistent with other vertebrates, the Sus scrofa mitochondrial genome also contains 13 core PCGs. The concatenated PCG sequence was estimated to span 11,342 bp, constituting 68.4% of the complete mitogenome. Of the 13 PCGs, 12 PCGs were located on the H strand (majority strand), whereas nad6 was located on the L strand (minority strand). The overall A + T content of the PCGs was 60.3%, ranging from 56.1% (cox3) to 67.6% (atp8), which is consistent with the nucleotide composition pattern of the whole mitogenome.

The strand asymmetry of the Sus scrofa mitogenome across the PCGs was assessed through AT and GC skews. All the PCGs exhibited a positive AT skew and a negative GC skew, reflecting a particular preference towards adenine and cytosine (Table 3). This pattern is consistently found in Sus scrofa across other regions, where GC skew commonly ranged from −0.402 to −0.356. Among the PCGs, nad6 displayed the highest AT skew (0.349) and the lowest GC skew (−0.562). Interestingly, the adenine and thymine contents in cox1 were nearly equal, represented through an AT skew of 0.001 (Figure 4).

Additionally, all PCGs in Sus scrofa began with the start codon ATN (ATG or ATT), except for nad4l, which started with GTG. Incomplete termination codons (T--) were observed in six PCGs, namely nad1, nad2, cox2, cox3, nad3, and nad4.

3.4. Control Region

The control region (D-loop) is rich in A + T content and located between the tRNA-Pro and tRNA-Phe genes. It is 1145 bp in length (positions 15,781–16,581 and continuing at 1–344), typical for vertebrate mitogenomes [33]. An alignment of 20 Sus scrofa control regions revealed an 11 bp repeat consensus (CGTACACGTG) starting at site 705. The Vietnamese Central Highland wild boar harbors only 9 copies of this repeat, while up to 45 copies were found in the European wild boar (FJ237000). In addition, the A + T content and AT and GC skews of the control region were 59.7%, −0.15, and −0.29, respectively. Interestingly, the D-loop of the Vietnamese Central Highland Sus scrofa also displayed the highest adenine and cytosine bias compared to 19 other pigs.

Further investigation detected 29 polymorphic sites among 20 Sus scrofa samples within the control region, most of which were similarly found in different Sus scrofa individuals (Supplementary Table S4).

3.5. tRNA and rRNA Genes

Notably, 22 genes encoding 22 tRNAs were identified by tRNAscan-SE, each corresponding to a specific amino acid, while serine and leucine were each transferred by two different tRNAs. The predicted tRNA genes could be folded into a typical secondary clover-leaf structure, except for the D-arm lacking trnS1, which could not form a stable structure (Figure 5). The gene length varied for each tRNA, ranging from 59 bp (trnS1) to 75 bp (trnL2 and trnS2). Of the 22 tRNA genes, 8 were located on the L strand, while the remaining were located on the H strand. The average base composition of the tRNA genes was A: 32.2%, T: 30.6%, G: 19.7%, and C: 17.5%, with the highest and lowest GC content observed in trnM (47.1%) and trnR (21.7%), respectively. In addition, a total of seven mismatches were detected in six tRNA genes, located in the amino acid acceptor (AA) stem, the pseudouridine (TΨC) stem, and the anticodon (AC) stem (Table 4). The structure and number of tRNA genes found in this study are similar to previous reports on Sus scrofa [18,32].

Two rRNA genes encoding for the small ribosomal subunit (12 S) and large ribosomal subunit (16 S) were positioned between trnF and trnV genes, and between trnV and trnL2 genes, respectively. The lengths for the 12 S and 16 S rRNAs were 962 bp and 1572 bp, respectively.

3.6. Overlapping and Intergenic Regions

The Vietnamese Central Highland Sus scrofa mitogenome had nine overlapping sequences among all the genes, with a total length of 76 bp, ranging from 1 bp to 43 bp in length (Table 1). The overlaps were located on both the H strand and L strand, four of which resided within the PCGs, and the remaining were observed between the tRNA and rRNA genes. Notably, the overlap between the trnV and rrnL (2 bp) was uniquely identified in the Vietnamese Central Highland Sus scrofa. The 1 bp overlap between trnV and rrnS (1 bp) was also found in I pig [18], whereas the other overlapping regions are commonly present in other Sus scrofa mitogenomes, with the longest observed between atp8 and atp6 (43 bp).

There are 10 intergenic spaces among all genes, ranging from 1 bp to 32 bp and making up a total of 58 bp (Table 1). The longest space (32 bp) was located between trnN and trnC, as typically observed in other wild boars and pig breeds.

3.7. Mutations

A comparative analysis of 13-PCG concatenated sequences of Vietnamese Central Highland wild boar and 19 other Sus scrofa revealed 232 polymorphic sites, resulting in 51 amino acid substitutions (Supplementary Tables S5 and S6). The Vietnamese Central Highland Sus scrofa and Asian Sus scrofa shared an almost similar amino acid profile, while some variations were only present in European Sus scrofa. No amino acid alteration was unique to the Vietnamese Central Highland Sus scrofa, although three mutations at 1461, 4577, and 8367 positions were found only in the Vietnamese Central Highland wild boar, Dong Khe pig breed, and Ha Lang pig breed, which are of Vietnamese origin. The Vietnamese Central Highland wild boar differed from the Vietnamese wild boar in 13 nucleotides, resulting in 2 amino acid changes.

Further evaluation in partial cytochrome b sequences of 8 Vietnamese wild boars (7 of which were collected from the Central Highlands) revealed 15 variable positions, representing 3.11% of the total length of the DNA sequences analyzed (Supplementary Table S7).

3.8. Phylogenetic Analysis

The genetic relationship of the Vietnamese Central Highland wild boar with 31 other wild boars and domestic pig breeds is depicted in the neighbor-joining phylogenetic tree (Figure 6). The tree topology was similar to previous studies in which pigs formed two distinct monophyletic clades corresponding to their Asian and European origins, supported by a bootstrap value of 98% and 89%, respectively. Indian wild boars and Malaysian wild boars fell out of these clades. The two Vietnamese wild boars belonged to two different groups within the Asian clade. The Vietnamese Central Highland wild boar formed a subgroup with Dong Khe pig and Ha Lang pig with a 96% bootstrap support, while the Vietnamese wild boar clustered with the I pig.

4. Discussion

This study reports the molecular characterization of the complete mitochondrial genome of Vietnamese Central Highland wild boars, which comprises 37 genes encoding for 2 RNA subunits, 22 tRNAs, and 13 mitochondrial proteins. The mitogenome was comparable with earlier reports of other wild boars and pig breeds in various perspectives, highlighting the conserved nature and important function of mtDNA. The genetic diversity of the Vietnamese Central Highland Sus scrofa compared with other wild boars and domestic pig breeds’ taxa was reflected through differences in their structural and nucleotide composition patterns. These variations were analyzed based on specific gene sequences, AT content, and skewness indices.

The Vietnamese Central Highland wild boar exhibited incomplete termination codons in 6 out of the 13 PCGs, as similarly observed in Chinese (EF545573, EU333163, EF545569, and EF545572) and Indian wild boars (MG725630, MG725631, and OM162160). In contrast, only four PCGs (nad1, nad2, cox2, and nad3) in European (FJ237000, FJ237001, FJ237002, and FJ237003) and Korean (AY574047 and DQ268530) wild boars lack a complete termination codon. Incomplete termination codons can be post-transcriptionally completed by polyadenylation of mRNA, a common feature in mammalian mitochondrial genomes [34]. This variation may reflect regional differences in mitochondrial genome evolution, suggesting a more conserved mitochondrial structure or shared evolutionary ancestry in different wild boars of Asian lineages compared to European wild boars.

The control region of Vietnamese Central Highland Sus scrofa is relatively small compared to what was observed in other pigs across the world, such as European wild boars (FJ237000, 1505 bp), Korean wild boars (AY574047, 1214 bp), Indian wild boars (OM162160, 1253 bp), Duroc pig (FJ236997, 1505 bp), and Ha Lang pig (KY800118, 1285 bp). This greater size variation is attributed to the presence of multiple tandem repeats and variations in their copy numbers [35]. Additionally, one mutation at site 405 was only found in the Vietnamese Central Highland wild boar, Ha Lang pig, and Dong Khe pig, while an 11 bp duplication (TAAAACACTTA) was notably identified in the two former pigs, suggesting potentially distinctive features of pigs from Vietnam. Despite the difference in tandem repeat copy numbers, the Vietnamese Central Highland wild boar and Vietnamese wild boar exhibited only three nucleotide variations. These nucleotide variations may possibly be influenced by natural selection and genetic drift, whereas tandem repeats are potentially involved in regulating the mitochondrial genome replication process [36,37].

As observed in vertebrates, the rRNA genes are separated by the trnV gene [38]. The A + T content of both rRNA genes matches well with earlier findings about the mitogenome of Sus scrofa, showing a conserved nature of mitochondrial genomes [18].

Intergenic regions may contain functional elements that play an essential role in regulating gene expression [39]. These intergenic spacers are generally found in other Sus scrofa individuals, although their length may slightly vary.

Despite having similar nucleotide variations to other pig breeds, the Vietnamese Central Highland Sus scrofa exhibited several polymorphisms unique to Vietnamese pigs, indicating distinct genetic variation between the Vietnamese Central Highland wild boar and other pig breeds’ taxa. Notably, the mismatched base pairs identified in the tRNA genes may potentially influence the tRNA function, possibly reflecting how the wild boar adapts to its particular living habitat.

The cytochrome b gene has been widely used in phylogenetic and evolutionary studies of many mammalian species due to its high variability [40]. Major pig haplotypes can be classified into region-specific groups based on single-nucleotide polymorphisms at four positions 15,036 (T/C), 15,038 (G/A), 15,041 (C/T), and 15,045 (G/A) [12]. It should be noted that the positions are putative with respect to the reference sequence used. The E1 (TGCG) and E2 (TGTG) haplotypes were of European origin, whereas the A1 (CATA), A2 (CATG), and A3 (TATG) haplotypes were Asian [17,41,42]. The analysis of eight Vietnamese boars revealed that the Vietnamese Central Highland wild boar characterized in this study exhibited A1 haplotypes. While one wild boar (MZ574575.1), also from the Central Highlands, displayed A3 haplotypes, A2 haplotypes were observed in all the remaining boars, indicating the presence of all three Asian lineages within Vietnamese wild boars. The reference mitogenome displayed E1 haplotypes. Notably, a substitution at position 16,379 was found in all Vietnamese boars, thus suggesting a potential marker to differentiate Asian from European pigs.

The phylogenetic tree revealed a clear distinction between Asian and European pigs. Additionally, the tree demonstrated mutual genetic contributions between Asian and European pig breeds, consistent with evidence of introgression reported by Giuffra [41]. The wild boars analyzed in this study do not form a separate clade but are scattered among domestic pig breeds, which could be attributed to the long history of domestication, feralization, introgressive hybridization, and pig breeding [43,44,45,46,47,48].

Phylogenetic analysis showed the close genetic relationship between Vietnamese wild boars and domestic pig breeds. Interestingly, the Vietnamese Central Highland wild boar is genetically close to Vietnamese northern pigs (Ha Lang pig and Dong Khe pig), despite the regional difference. While the Vietnamese wild boar represents a close relationship to Chinese wild boars (Hainan and Yunnan wild boars), the Vietnamese Central Highland wild boar is relatively distant from other wild boars. The two Vietnamese wild boars were positioned distantly on the phylogenetic tree, possibly reflecting their origin from genetically distinct populations. Additionally, the tree indicated that the Vietnamese Central Highland wild boar was closely related to the Xiang and Mashen pigs. This result contradicts the findings of Lan and Shi, who reported a high genetic divergence between Vietnamese wild boars and Chinese domestic pigs [49].

5. Conclusions

The complete mitochondrial genome of the Vietnamese Central Highland Sus scrofa presents characteristics typically observed in other vertebrates. While this characterization serves as an initial molecular analysis of the Vietnamese wild boar, an in-depth investigation is required to further understand its genetic variation and evolutionary history. Combining genetic data with ecological and demographic data may hold great importance for effectively preserving the gene integrity of natural wild boar populations.

This study has several limitations. First, the focus on the mitochondrial genome solely reflects maternal inheritance, excluding the influence of paternal genetic contributions and recombination events, which requires more complex and extensive genomic analyses. Second, the data reported were limited to wild boars from the Central Highlands region and thus were insufficient to assess the genetic diversity across other wild boar populations throughout Vietnam. Notably, phylogenetic analysis demonstrated high genetic variations between the two Vietnamese wild boar individuals, highlighting the need for future research to evaluate the origin and population structure of Vietnamese wild boars.

Given the loss of unique wild boar traits caused by widespread hybridization, this study provides comprehensive insights into the genetic characteristics of native wild boars in the Central Highlands of Vietnam, serving as the basis for future research in conservation, evolutionary biology, and the use of wild boar genetic resources in breeding programs.