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ANIMAL GENETICS |
* Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan;
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Taitung Animal Propagation Station, Livestock Research Institute, Taitung, Taiwan;
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Kaohsiung Animal Propagation Station, Livestock Research Institute, Pingtung, Taiwan;and
Department of Life Science, National Taiwan Normal University, Taipei, Taiwan
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: control region • cytochrome b • genetic diversity • Lanyu pig • mitochondrial DNA • phylogenetic relationship
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Two herds of Lanyu pigs were transferred to Taiwan for conservation purposes before 1980 (Chyr et al., 2001). On the basis of the polymorphism of the mitochondrial DNA (mtDNA) control region, only 2 haplotypes of the control region have been identified in all the conserved Lanyu pigs. The Lanyu pig, with one haplotype (Type I), possesses a unique maternal genetic lineage distinct from Asian and European breeds, whereas Lanyu pigs of the other haplotype (Type II) are clustered in the major Asian pig clade (Wu et al., 2007), indicating that the original Lanyu pig possesses a unique genetic background.
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). Some traits of Formosan wild pigs show striking phenotypic differences from Lanyu pigs, but their body conformation and small erect ears are similar. The extent of the genetic relationship between the Lanyu and Formosan wild pig is unknown. In addition, the degree and pattern of introgression of genetic material from the exotic breeds introduced into Lanyu pigs is also currently unknown.
Because the population of conserved Lanyu pigs stood at only 44 animals in 2006 (Wu et al., 2007), this study was undertaken to assist in the future genetic conservation and recovery of this unusual breed. The phylogenetic relationship among conserved Lanyu, Asian, and European pig breeds was determined by analysis of the polymorphism of their cytochrome b (Cytb) sequences. The genetic diversity within the conserved population, and the presence of any genetic affinity among conserved Lanyu pigs, Formosan wild pigs, and exotic breeds in Taiwan, and among pigs currently extant in the Lanyu Islet were investigated by the diversity of mtDNA. We were also interested in whether any maternal mtDNA genetic introgression had occurred previously between the conserved Lanyu pig and the Formosan wild pig.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Sample Collection and Preparation of mtDNA
Conserved herd Lanyu pig blood samples were collected from all 39 pigs from the Taitung Animal Propagation Station (TAPS) and from all 5 pigs from the National Taiwan University (NTU) teaching farm. Blood samples of exotic pigs were obtained from 4 Meishan, 12 Taoyuan, 10 Berkshire, 14 Landrace, 10 Yorkshire, and 5 Duroc pigs from reference stocks at the Taiwan Livestock Research Institute (TLRI). Twelve current Lanyu pig blood samples were collected separately from pigs of 6 aboriginal tribes on the Lanyu Islet. Five Formosan wild pig blood samples were obtained from Taitung and Hualien counties (Figure 1
). The mtDNA were extracted and purified from platelet-rich plasma by using Qiagen’s QIAamp DNA mini kit (Qiagen, Valencia, CA). The quality of purified mtDNA was evaluated via electrophoresis on a 1% agarose gel.
Primer Design and Amplification of mtDNA Fragments by PCR
Entire sequences of the mtDNA control region were amplified by PCR in an MJ thermal cycler (MJ Research, Waltham, MA) using the following primers: L1, 5'-CCAAGACTCAAGGAAGGAGA- 3' (sequence of position 16,542 to 16,561 of pig mtDNA, GenBank accession number AF034253) and H1, 5'-GGCGCGGATACTTGCATGTG-3' (position 1,290 to 1,309). The entire sequence of Cytb was amplified by using the following primers: CbR1, 5'-GTCCTGCCCTGAG-GACAA-3' (position 15,735 to 15,752); CbL1, 5'-GGTGCTG ATGGCGGAGTT-3' (position 16,581 to 16,564); CbR2, 5'-CCAAGACTCAAGGAA GGAGA-3' (position 15,363 to 15,382); and CbL2, 5'-GGCGCGGATACTTGCATGTG-3' (position 115 to 134). Thermal cycling was conducted in 50-µL volumes using the FastStart High Fidelity PCR system (Roche, Penzberg, Germany), each containing 1 ng of mtDNA, 10-mM Tris-HCl (pH 8.3), 1.8-mM MgCl2, 0.4-µM each primer, 200-µM each dNTP, and 2 units of FastStart polymerase. Thermal cycling parameters were as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 80 s, with a final extension at 72°C for 4 min. The resultant PCR products were then purified by using the PCR-M cleanup system (Viogene, Taipei, Taiwan). The complete control regions were sequenced in both directions by using the following primers: L1; H1; L2, 5'-CCTATGTACGTCGTGCATTA-3' (position 160 to 179); L3, 5'-TACTTCAGGACC ATCTCACC-3' (position 434 to 453); H2, 5'-AGTGTAAGTTAGGCTTATTG-3' (position 963 to 982); and H3, 5'-TTGTGGTAGATTGGCGTAAA-3' (position 1,072 to 1,091). The Cytb fragments were bidirectionally sequenced by using the following primers: CbR1, CbL1, CbR2, and CbL2.
All sequences were determined by using an Applied Biosystems 3730 DNA sequencer and analyzed with SeqEd software (Perkin-Elmer, Applied Biosystems, Foster City, CA). Full sequences of the control region and Cytb were generated by overlapping forward and reverse sequences with SeqEdit software (DNASTAR, Madison, WI; see Hein and Støvlbaek, 1996). The following reference mtDNA control region sequences were obtained from NCBI GenBank: 6 Yorkshire (AM040633 to AM040638), 4 Meishan (I, AY230821; II, AY230827; III, D17739; and IV, AB041474), 6 Taoyuan (I, AM040641 to AM040645; and II, AM040646), 1 Hampshire (AY574046), 1 Berkshire (AM040639), 4 Landrace (AM040613 to AM040616), and 6 Duroc (AM040623 to AM040628) pigs, a Japanese wild pig (AB015085), a Ryukyu wild pig (AB015087), and an Italian wild pig (AB015094). All of the above sequences (except Meishan and Hampshire pigs, and Japanese, Ryukyu, and Italian wild pigs) came from pigs reared in isolation at the TLRI. The Cytb sequences were also obtained from the National Center for Biotechnology Information (NCBI) GenBank, comprising 5 Ryukyu wild pigs (AB015071 to AB015075), 6 Japanese wild pigs (AB015065 to AB015070), Satsuma (AB015076), Erhualian (AF486861), Tongcheng (AF486862), Wannanhua (AF486873), Taoyuan (DQ534707), Meishan (AB015077), Diannan (AF486869), Large White (AF486874), Berkshire (AY574045), Landrace (AF034253), Duroc (AY337045), Hampshire (AY574046), Yucatan miniature (AB015081), and 2 Italian wild pigs (AB015082 to AB015083; Watanobe et al., 1999; Kim et al., 2002; Yang et al., 2003).
Analysis of the Full-Length mtDNA Genome
The Type I and II mtDNA control regions of Lanyu pigs were identified after their mtDNA was obtained. Twenty pairs of primers were designed according to the Landrace mitochondrial genomic sequence AF034253, and the annealing conditions of PCR were as listed in Table 1. The locations of primers for the full mitochondrial genome sequencing are listed in Supplemental Table 1 (available online at http://jas.fass.org; doi:10.2527/ jas.2007-0049). The PCR was performed by using the Long PCR Enzyme Mix (Fermentas, Hanover, MD) in 50-µL volumes with the following parameters: 94°C for 5 min; 32 cycles of 94°C for 30 s, annealing for 30 s, and 68°C for 90 s; with a final extension at 68°C for 10 min. The resultant PCR products were then purified by using the PCR-M cleanup system (Viogene) before direct sequencing was performed. All the amplified sequences were confirmed by bidirectional sequencing.
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In the analysis of pairwise distance of the control regions, the tandem repeat motif CGTGCGTACA, with a variable number of repeats in individuals, and the Type I and II Lanyu-specific repeat motifs (ACACAAACC and TAAAACACTTA, respectively) in the mtDNA control region were excluded from the analysis (Wu et al., 2007). Sequence alignment of the control region and Cytb was performed by using MegAlign multiple alignment software (DNASTAR; Hein and Støvlbaek, 1996). Haplotype and nucleotide diversities within the conserved Lanyu pigs, exotic pigs, and extant pigs on the Lanyu Islet were obtained with DNA Sequence Polymorphism (DnaSP) software version 4.10.9 (Rozas et al., 2003). The PHYLIP program package (version 3.66) was used to obtain the maximum likelihood (ML) phylogenetic tree (Felsenstein, 2006). Tree-Puzzle (version 5.0) software, based on the quartet puzzling method, was used to analyze confidence intervals of the phylogeny and likelihood-mapping analyses (Schmidt et al., 2002). The significance of the difference among pig groups was tested by using 10,000 permutations in the quartet puzzling algorithm (Strimmer and von Haeseler, 1996). For the ML analysis, Modeltest version 3.6 software (Posada and Crandall, 1998) was used to determine the best fit model of the data, including nucleotide composition, substitution matrix among nucleotides, and proportion of invariant sites. Nodal supports of the ML tree were evaluated by bootstrap resampling (1,000 replications) using the fast heuristic search algorithm implemented in software PAUP4.0β10 (Swofford, 2002). Fu and Li’s D* neutrality test values were determined with DnaSP software.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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We had previously studied the phylogenetic relationship among conserved Lanyu pigs and 43 Asian and European pig breeds based on the pairwise distance of the mtDNA control region (Wu et al., 2007). Only 2 control region haplotypes were identified in all conserved Lanyu pigs in Taiwan. The Lanyu pigs reared at TAPS had a mixture of Type I (28 head) and Type II (11 head) mtDNA haplotypes, whereas all pigs (5 head) at the NTU teaching farm had the Type I haplotype. Here, to determine whether the sequence polymorphism of the Cytb coding gene matched that seen in the non-coding control region, the variation in Cytb sequences among the conserved Lanyu pigs was analyzed. Mitochondrial DNA Cytb sequences were obtained from all 44 Lanyu pigs kept at TAPS and NTU. Consistent with the control region analyses, all mtDNA Cytb sequences of the Lanyu pigs from TAPS and from the teaching farm at NTU could be categorized into 2 types (Figure 2). The phenotypic conformation of both types of Lanyu pigs was indistinguishable. All 5 of the Lanyu pigs at the NTU teaching farm had the same Cytb haplotype (Type I), whereas 2 haplotypes were found in the Lanyu herd at TAPS (Figure 2). In the mtDNA control region, 18 polymorphic sites (2 haplotypes), including 17 transitions and 1 transversion, were found by Wu et al. (2007) in the conserved Lanyu pigs. An additional 10 transitions and 1 transversion in Cytb were found in the same populations in the present analysis (Figure 2; Supplemental Figure S1, available at http://jas.fass.org). Based on the diversity of Cytb sequences, Fu and Li’s D* test for neutrality was used to estimate the genetic variation within the population of conserved Lanyu pigs, and the test value was determined to be 1.44055 (P < 0.05), indicating significant deviation from neutrality. Among all 44 conserved Lanyu pigs, the low haplotype numbers and nucleotide diversities shown by DnaSP software analysis (h = 0.384 ± 0.066; p = 0.0037 ± 0.00064) indicated severe genetic drift. This confirmed that there has indeed been a significant loss of heterozygosity of mtDNA haplotypes in the population of all conserved Lanyu pigs, probably as a result of the small population size and uncontrolled mating in the past. Additionally, studies of gene frequency and genotypes of 19 microsatellite markers revealed that severe nuclear gene drift has occurred in the conserved Lanyu pig population (Cheng et al., in press).
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We had shown earlier that the Lanyu Type I mtDNA control region sequence was genetically remote from Asian and European breeds (Wu et al., 2007), and we were interested in whether the Cytb coding region was similar in distance to the noncoding control region, and whether gene introgression had occurred between Lanyu and Formosan wild pigs. The Cytb sequences of Asian and European pig breeds were obtained from the NCBI database (see the Materials and Methods section for details). In addition, the Cytb sequences of 5 Formosan wild and 44 conserved Lanyu pigs were determined. Fifty nucleotide substitutions were identified in Cytb sequences from the aforementioned pig breeds (Figure 2). The nucleotide substitutions in positions 15,422, 15,425, 15,623, and 16,323 were specifically found in Type I Lanyu Cytb sequences. A unique transversion in position 15,425 was also identified in Lanyu Type I (thymine) compared with the consensus sequence (adenine). The pairwise genetic distance among the Cytb sequences was determined and an ML phylogenetic tree was constructed. The most appropriate model for this data set was found to be HKY + I (–ln = 1,935.7043; K = 5; AIC = 3,881.4087, where AIC refers to Akaike’s information criterion). The ML estimates of base frequencies were A: 0.3174; C: 0.2887; G: 0.1292; and T: 0.2647. The estimated proportion of invariable sites was 0.7964. A transition:transversion ratio of 10.4402 was used to obtain the ML tree.
Two major clades (Asian and European clades) were recognized in the ML tree (Figure 3). The treelike topology and phylogenetic signal were obtained by the quartet puzzling method (quartet puzzling support value, 80.2%) supporting the branch assignments in this phylogenetic tree (Supplemental Figure S2, available at http://jas.fass.org). In the Asian clade, Type I Lanyu and Ryukyu wild pig sequences formed 2 separate subclades distinct from the Asian major subclade. Sequences from Japanese and Formosan wild pigs and the Satsuma, Meishan, Taoyuan, Diannan, Wannahua, Tongcheng, Erhualian, Large White, and Berkshire breeds clustered with the Type II Lanyu haplotype in the major Asian subclade.
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The Lanyu Type I control region and Cytb haplotypes were unique sequences. We next compared entire mitochondrial genome sequences to find variations in entire mtDNA genomes of both Lanyu haplotypes. After excluding the tandem repeat motifs (5'-CGTGCGTACA), the length of the Type I Lanyu mitochondrial genome was 16,491 nucleotides (nt; accession number: EF375877), and the length of the Type II Lanyu genome was 16,494 nt (accession number: DQ972936). The total lengths of the 2 types of mtDNA control regions differed because of the varying numbers of ACA-CAAACC and TAAAACACTTA repeat motifs in their control regions (Wu et al., 2007). With the 5' end of the control region assigned as the first nucleotide of the Type II sequence, the L-strand replication origin was located at positions 6,223 to 6,269. Both types of Lanyu mitochondrial genome encoded 37 genes, including 2 ribosomal RNA (rRNA; 12S and 16S), 22 transfer RNA (tRNA), and 13 protein-coding genes, as listed in Table 2. A total of 124 nucleotide substitutions (107 transitions; 17 transversions) were identified between the Lanyu sequences. All protein-coding genes in the mitochondrial genome used identical start and termination codons in both Lanyu sequences.
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). These 2 repeated motifs may be due to heteroplasmy and may have resulted in different lengths of full mitochondrial genomes in the 2 haplotypes of Lanyu pigs.
A typical animal mitochondrial genome encodes 36 to 37 genes, which are powerful markers for inferring phylogenetic relationships (Saccone et al., 2000; Gerber et al., 2001). The numbers of tRNA (22), rRNA (2), and protein-coding genes (13) in both haplotypes of Lanyu mtDNA were identical to the numbers in domestic pigs (Ursing and Arnason, 1998; Lin et al., 1999). All of the initiation codons (9 ATG, 3 ATA, and 1 GTG) of the 13 protein-coding genes were ATG, except the codons for NADH dehydrogenase subunit 2 (NADH2), NADH3, and NADH5, which were ATA, and for NADH4L, which was the rare GTG. The ATA initiation codon for the NADH2 gene was found in both types of Lanyu mtD-NA, which differed from the rare ATT initiation codon in Landrace and Swedish domestic pigs (Ursing and Arnason, 1998; Lin et al., 1999). Some other slight differences noted were the termination codons of 6 and 2 of 13 protein-coding genes used TAA and TAG, respectively, except those for NADH3, NADH4, cytochrome c oxidase subunit (CO) II, COIII, and Cytb, which were TAT, TAC, TCA, TAC, and AGA, respectively (Table 2); and 4 genes (COII, COIII, NADH3, and NADH4) terminated with an incomplete TNN stop codon, where NN was the 5' terminus of the adjacent tRNA gene. The incomplete stop codon (TNN) forms a stop codon by posttranscriptional polyadenylation (Anderson et al., 1981; Ojala et al., 1981; Wolstenholme, 1992).
The vertebrate control region is subdivided into 3 domains. The central domain of the control region, containing the replication origin of the heavy strand, is relatively well conserved. The 2 regions (domains I and II) flanking the central domain are hypervariable in base substitution, insertion, and deletion (Saunders and Edwards, 2000). The complete domestic pig mtDNA has been sequenced and the control region is located between genes encoding tRNA-Pro and tRNA-Phe, containing approximately 1,245 nucleotides (Ursing and Arnason, 1998; Lin et al., 1999).
Phylogenetic Comparison of Exotic, Formosan Wild, and Conserved Lanyu Pigs
Many exotic pig breeds (including Taoyuan, Meishan, Berkshire, Yorkshire, Landrace, and Durocbreeds)were introduced into Taiwan to improve the production performance of local pigs, first by colonizers from 120 to 50 yr ago and then by the Taiwanese government in more recent times (Chyr et al., 2001). To explore whether the genes of exotic breeds and Formosan wild pigs had introgressed into the conserved population of Lanyu pigs, the pairwise distance and ML methods were used to investigate the phylogenetic relationship among the conserved Lanyu pigs, the exotic breeds, and Formosan wild pigs. Some of the mtDNA control region sequences of exotic breeds were obtained from the NCBI Web site, and mtDNA samples of further individuals, including 12 Taoyuan, 4 Meishan, 10 Berkshire, 5 Duroc, 14 Landrace, and 10 Yorkshire breeds, were obtained from the TLRI (Supplemental Figure S3, available at http://jas.fass.org/content/vol86/issue7/). The variable sites of the mtDNA control region of conserved Lanyu, Formosan wild, and exotic pigs are listed in the supplemental data (Supplemental Figure S3, available at http://jas.fass.org/content/vol86/issue7/). Unique nucleotide substitutions, including transitions at nucleotide positions 302, 391, 535, 542, and 657, and a transversion at position 871 (thymine in Type I Lanyu and adenosine in consensus sequence), were found in the control region of Type I Lanyu mtDNA, but not in other sequences used in this study. Pairwise distance analysis of the mtDNA haplotypes was again performed by using DnaSP. A phylogenetic ML tree was constructed by using the PHYLIP program package (Figure 4), which showed that all pig sequences clustered into 3 major clades. Sequences from many European pig breeds, including the Hampshire, Landrace, Duroc, and Italian wild pig breeds, were categorized as one major clade (referred to here as the European pig clade). Most other sequences, including those from the Formosan wild, Japanese wild, Ryukyu wild, Taoyuan, Meishan, Yorkshire (Large White), and Berkshire breeds, and the Type II Lanyu sequence, were assigned to another major clade (referred to here as the Asian pig clade). The Type I Lanyu sequence clustered as a unique clade distinct from the 2 major clades mentioned above, indicating that the maternal lineage of pigs containing the Type I Lanyu sequence had never crossbred with the Formosan wild and the exotic breeds. The pairwise distance showed that the 2 Lanyu sequences were very different from each other in their maternal lineages.
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Distinct Genetic Lineage of Lanyu, Taoyuan, and Formosan Wild Pigs
We previously showed that the Taoyuan (accession no. AM040645, AM040646) and Lanyu pigs possess distinct control region haplotypes (Wu et al., 2007). Here, we obtained an identical result after we increased the number of individual Taoyuan pigs in our analysis, indicating no mtDNA gene introgression between Taoyuan and Lanyu pigs (Figure 4). In the present study, we found a remote pairwise distance (0.01882 ± 0.00755 and 0.00936 ± 0.00102) of both the control region and Cytb coding sequences between Type I Lanyu and Formosan wild pigs. On the basis of the pairwise distance of Cytb, the Formosan wild pigs were clustered together with Japanese wild pigs, with 0.00361 ± 0.00120 pairwise distance, whereas the Type II Lanyu sequence clustered with the major Asian breed subclade, with 0.00328 ± 0.00072 pairwise distance. This result indicates no mtDNA introgression between the Formosan wild pig and Lanyu pigs. The Diannan breed has a phenotype similar to that of the Lanyu pig, but its Cytb sequence was identical to the Cytb sequence in Formosan and Japanese wild pigs, suggesting that the mtDNA of East Asian wild pigs might have introgressed into the Diannan breed.
Gene Introgression from Exotic Pig Breeds into Pigs Extant on the Lanyu Islet
The Lanyu Islet was isolated by the Taiwanese government during the aboriginal culture protection period. When it was opened to tourism and unrestricted travel after 1960, because most of the Lanyu Islet pigs were bred in free-range piggeries, there was a significantly increased opportunity for the introduction of and crossbreeding with exotic pig breeds from Taiwan. To understand the current diversity of mtDNA in pigs distributed throughout the Lanyu Islet, we obtained mtD-NA from 12 individual Lanyu pigs reared by 6 tribes on the Lanyu Islet during February 2005. Their mtDNA control region sequences were subjected to phylogenetic analysis and compared with the sequences of Lanyu pigs conserved in Taiwanese, Formosan wild pigs, and exotic pigs in Taiwan. An ML tree was constructed and sequences from the modern pigs from the Lanyu Islet were clustered into 3 groups (Figure 4). Four extant pigs on the Lanyu Islet had control region sequences identical to the Type I Lanyu haplotype. Two pigs had sequences that clustered together with the Meishan I and II, Yorkshire, Japanese wild pig, Formosan wild pig, and Type II Lanyu sequences, but these 2 sequences had lost one of the repeated TAAAACACTTA motifs present in duplicate in the Type II Lanyu haplotype. The remaining 6 extant Lanyu pigs had mtDNA sequences that were grouped together with Taoyuan, Berkshire, and Meishan III and IV sequences (Figure 4). This confirmed that most extant Lanyu pigs on the Lanyu Islet had hybridized with the Taoyuan, Berkshire, Meishan, or Yorkshire exotic pig breeds.
Most pigs extant on the Lanyu Islet have a dark-pigmented coat color, and some of them present phenotypes of exotic domestic breeds that are now or were previously found in Taiwan, such as the Landrace, Yorkshire, Hampshire, Duroc, Taoyuan, Meishan, and Berkshire breeds. The present study supports the hypothesis of recent introgression of Meishan, Taoyuan, Berkshire, and Yorkshire genes into extant pigs on the Lanyu Islet. Although 4 extant pigs on the Lanyu Islet had mtDNA control region sequences identical to the Type I Lanyu, we observed that those pigs possessed physical characteristics typical of Hampshire, Landrace, and Duroc phenotypes, suggesting that the introgression of exotic pig genes was more serious than could be detected by a simple comparison of the variation in maternal-linked mtDNA. No extant pigs on the Lanyu Islet were found to possess the duplicate TAAAACACTTA motif in their control regions, suggesting that the Type II Lanyu sequence might be becoming extinct on the Lanyu Islet, the original habitat of the Lanyu. One pig from the Lanyu Islet actually possessed 4 ACACAAACC repeat motifs, indicating either a recent mutation or that 4 ACACAAACC repeat motifs might have existed in Lanyu pigs previously. These results demonstrated significant genetic introgression from exotic pig breeds into the pigs extant on the Lanyu Islet, and showed that gene drift is currently occurring on the Lanyu Islet, resulting in the loss of the Type II Lanyu sequence.
Evolution of Lanyu Pigs
On the basis of the mtDNA sequences and morphometric data from museum specimens and tissue samples, Lucchini et al. (2005) presented a possible scenario for pig speciation in Southeast Asia (SEA): the SEA ancestral pig species (genus Sus) might have crossed from Sundaland to the Philippines during the Pliocene (5.3 to 1.8 million years ago). Larson et al. (2005) constructed a consensus tree based on mtDNA obtained from 686 wild and domestic pig specimens from museums worldwide, and showed that basal lineages (origin) of S. scrofa occurred on the western island of SEA (ISEA) and dispersed into the Indian subcontinent, then dispersed northward into the Asian continent, followed by subsequent westward radiations into mainland Asia, and a final, progressive dispersal across Eurasia into Western Europe. More than 1 domestication event [2 in China (Gansu and Hunan provinces), 1 in India, 1 in Burma and Thailand, and another in Cape York of Northern Australia], followed by rapid radiative expansion in Asia, was identified by median-joining network analysis of Asian domestic and wild pig mtDNA haplotypes (Larson et al., 2005). Later on, Larson et al. (2007) investigated human-mediated Sus dispersal in ISEA based on the polymorphism of 781 mtDNA sequences from modern and ancient Sus specimens. They concluded that the endemic pigs in Taiwan had a genetic link with mainland East Asian, Micronesian, and Philippine pigs, but not with pigs in Brunei, Sumatra, Oceania, and Polynesia.
Based on variation of the control region, the pairwise distances of the Type I Lanyu haplotype versus Asian and European pigs were 0.01726 ± 0.00275 and 0.02216 ± 0.00889, respectively (Wu et al., 2007). The Type I sequence formed a unique clade distinct from the major Asian clade and the European clade in the constructed ML tree. In addition, the pairwise distances between the Type II Lanyu control region versus the Type I Lanyu control region (0.01744 ± 0.00125), the Type II Lanyu sequence versus the Asian clade (0.00471 ± 0.00054), and the Type II Lanyu versus the European clade (0.01941 ± 0.00356) revealed apparent divergence of Type I and Type II Lanyu mtDNA control region sequences (Wu et al., 2007). In the present study, the pairwise distances of both haplotypes of Lanyu Cytb sequences versus Asian and European breeds were consistent with the pairwise distances of the control region variants. Our results, together with those of others, suggest that the Type I and II mtDNA haplotypes had quite different origins, as evidenced by the large calculated genetic distance between the 2 types, and the fact that the 2 types specifically possess different numbers of the repeated ACACAAACC and TAAAACACTTA motifs in their control region. The formation of unique Type I haplotype mtDNA on the Lanyu Islet happened earlier than the formation of the Type II haplotype and the haplotypes of Formosan wild pigs. The ancestor of Type I Lanyu pigs may have crossed through Sundaland into Taiwan and the Lanyu Islet before the last glacial maximum (Meijaard, 2003) and may have evolved in isolation in Taiwan and the Lanyu Islet after the last glacial period ended. To address the origin of the Type II haplotype, the Type II mtDNA haplotype was aligned with 172 Sus mtDNA control region sequences published by Larson et al. (2005, 2007). The Type II DNA sequence was identical to the Sarawak (Malaya, DQ779294) specimen and differed by 1 base pair from the Guizhou Xiang (China, AY486118) and Guam D (Northern Mariana Island, AY884677), and differed by 2 base pairs from the Andaman (India, AY884705), Large Black (Australia, AY463075), Kune Kune (New Zealand, AY463076), and Satuma (Japan, AB015091) breeds. Our results revealed that the Type II Lanyu pig in Taiwan shared genetic lineage with pigs distributed in ISEA and Oceania, which was missed in the sampling by Larson et al. (2005, 2007). The short pairwise distance between Type II and Asian breed mtDNA (0.00471 ± 0.00054 in the control region and 0.00328 ± 0.00072 in Cytb) shows that the Type II Lanyu mtDNA is genetically almost identical to that of Asian breeds, suggesting that the formation of Type II mtDNA might have occurred in recent times. These results promote 3 hypotheses about the formation of the Type II mtDNA of the Lanyu pig. The first hypothesis is that the Type II mtDNA originated in ISEA and spread northward into the main Asian continent and Japan, and then eastward into Australia and New Zealand through Taiwan and the Lanyu Islet, mediated by human migrations. The second hypothesis is that the Type II Lanyu sequence originated during domestication in mainland Asia and then crossed through Taiwan and the Lanyu Islet and spread into Japan, ISEA, and Oceania. The third hypothesis is that the Type II Lanyu might have originated in Taiwan and the Lanyu Islet and then spread into the Asian mainland, Japan, ISEA, and Oceania. To evaluate the actual origin of the 2 mtDNA haplotypes of Lanyu pigs in Asia, a further combination of genetic, biogeographic, and zooarcheological data is needed.
The conserved Lanyu pigs possesses a distinct maternal lineage to Asian and European type breeds, with no evidence of mtDNA gene introgression from exotic and Formosan wild pigs during recent times. The results of this study further emphasize their unique maternal genetic characterization and the importance of understanding their genetic origin in assessing the trajectories of prehistoric human migration from mainland East Asia into ISEA. The significant loss of mtDNA diversity in the conserved Lanyu pigs may be due to the small population size and exotic gene introgressions. The discovery that the majority of pigs extant on the Lanyu Islet are hybrids indicates that more effort is needed for the recovery of this native breed. For further conservation or restoration of the Lanyu pig as a distinct breed, the nuclear phylogenetic relationship of the remaining Lanyu pigs, Asian pigs, and other breeds throughout the world will require further analysis. Future population management will also require deeper analysis of global nuclear genetic characteristics within the population of conserved Lanyu pigs by using microsatellite markers or coding genes.
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Footnotes |
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2 Corresponding author: ytju{at}ntu.edu.tw
Received for publication January 19, 2007. Accepted for publication March 10, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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