Journal of Heredity 2003:94(5)
© 2003 The American Genetic Association 94:381-385
Genetic Diversity Present Within the Near-Complete mtDNA Genome of 17 Breeds of Indigenous Chinese Pigs
From the Laboratory of Molecular Biology and Animal Breeding, School of Animal Husbandry and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China (J. Yang, Liu, Yu, Zhao, and Li); Beijing Genomics Institute/Genomics and Bioinformatics Center, Chinese Academy of Sciences (CAS), Beijing 101300, China (Wang, Han, and H. Yang); CSIRO Division of Livestock Industry, Brisbane 4068, Australia (Kijas); and James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Li).
Address correspondence to Kui Li, Laboratory of Molecular Biology and Animal Breeding, School of Animal Husbandry and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, P R China, or e-mail: lkxblghi{at}public.wh.hb.cn.
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Abstract |
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The genetic diversity present within the near-complete mitochondrial genome (15,982 bp) was determined from 17 indigenous Chinese pig breeds and 3 European breeds. Animals were selected from 17 Chinese breeds that reflect the large phenotypic diversity of Chinese pigs and represent each of the six breed types, which are grouped based on morphological characteristics. Analysis of nucleotide diversity confirmed a high level of divergence between animals of European versus Asian origin; however, much more limited variation was observed between the 17 indigenous Chinese breeds. Each had a unique haplotype, but the lowest pairwise sequence divergence was only 0.01 ± 0.01%, observed between the Tongcheng and Yushan Black. Comparison of control region sequence diversity revealed the 17 Chinese breeds contain a lower average pairwise distance (0.61 ± 0.19%) than a group of European commercial breeds (0.91 ± 0.21%). The dendrogram constructed from the near-complete mtDNA sequences showed the Chinese sequences loosely clustering into two groups. Although some correspondence with geographic origin was present, notable differences between the dendrogram and the traditional pig breed grouping system were observed.
The pig is an extremely important domestic species within China, where the extremes of climate and geography have contributed to the development of more than 100 indigenous breeds, many of which have special and unique characteristics. Zhang (1986) described 48 breeds and classified them into six types according to their geographic distribution and morphological and physiological characteristics. The breeds of northern China (group I) are widely distributed in areas north of the Huai River and Qin Mountains and are almost totally black, with a special tolerance of cold. South China breeds (II) are found distributed in Yunnan, Guangxi, and Guangdong provinces; they are usually white with black spots and are bred for high fat content. Central Chinese types (III) are associated with high intramuscular fat and are distributed in the area between the middle-lower Changjiang River and the Zhujiang River. They are characterized by their marking, which is black with a large white belt covering the middle of the animal. Lower Changjiang River basin breeds (IV) are spread between north and central China and generally are extremely hyperprolific, such as Meishan. Southwestern China (V) and western plateau types (VI) are characterized on the basis of their distribution, surrounding the southwestern mountains and the western plateau, respectively. Together these groups represent a significant proportion of the existing phenotypic diversity in the pig. Many breeds are considered rare, have a small population size, and are under increasing pressure from the introgression of modern commercial breeds. This makes investigations of both population structure and genetic diversity increasingly important.
The genetic variability, which underpins the phenotypic diversity of Chinese indigenous breeds, remains largely uncharacterized. Studies of porcine diversity have often considered only one or a small number of Chinese indigenous breeds (Giuffra et al. 2000; Kijas and Andersson 2001). Conversely, where sampling of Chinese breeds has been more extensive, studies are based on either restriction fragment length polymorphism (RFLP) analysis of mtDNA (Huang and Qui 1998; Lan and Shi 1993) or focus on a relatively small region of the mtDNA control region (Kim et al. 2002; Okumura et al. 2001; Watanobe et al. 1999). The attributes that make the mitochondrial genome attractive as a tool in phylogenetic reconstruction are well documented; still, caution is required when only a small portion is considered. Variable substitution rates both between mtDNA components (Zardoya and Meyer 1996) and between lineages (Ingman and Gyllensten 2001) mean that an increasing number of studies are based on the entire mtDNA genome (Kijas and Andersson 2001; Lin et al. 1999; Nikaido et al. 2001; Ursing and Arnason 1998). The goal of our study was to determine the near-complete mtDNA genomic sequences and genetic relationship existing between 17 Chinese native pig breeds selected to represent each of the six indigenous groups.
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Materials and Methods |
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One individual from each of 17 Chinese breeds was examined: Yimeng Black (group I), Min (I), Diannan small-ear (II), Wuzhishan (II), Xiang (II), Qingping (III), Dahuabai (III), Tongcheng (III), Jinhua (III), Ningxiang (III), Yushanhei (III), Wannanhua (III), Zhong Meishan (IV), Jiangquhai (IV), Erhualian (IV), Rongchang (V), and Zang (VI). Samples were collected as part of a national project to measure genetic distance among Chinese indigenous breeds organized by the Chinese Ministry of Agriculture. Samples were taken from participating farms where farmers collect a subsidy to maintain indigenous breeds. In addition to these Chinese samples, the genomes of three European pigs were examined: Landrace, Large White, and Duroc. A set of 18 primer pairs was designed to span the entire 16,679-bp porcine mtDNA genome in overlapping segments of approximately 1 kb (Kijas and Andersson 2001). Following polymerase chain reaction (PCR), products were purified using the multiscreen 384-PCR-plate system (Millipore) and sequenced using BigDye chemistry and an ABI377 Prism DNA Sequencer (Applied Biosystems). Overlapping fragments from each animal were compiled into a single contiguous 15,982-bp sequence using DNAstar (DNASTAR, Inc.). Ambiguous positions were resolved using a second round of PCR and sequencing, and finished data were deposited with accession numbers AF486855–AF486874. Calculation of genetic distance and its standard error was performed using Kimura's two-parameter algorithm, as implemented in MEGA (Kumar et al. 2001). The same software was used to calculate the average pairwise distances as well as both nonsynonymous (Kn) and synonymous (Ks) substitution rates. A 583-bp portion of the control region [positions 15,435–16,017 as given by Ursing and Arnason (1998)] was used to directly compare the sequence diversity within domestic animals of Asian origin (this study) with those from Europe and the United States [generated by Okumura et al. (2001)].
To ensure that the analysis was not compromised by inclusion of haplotypes present within European animals as a result of introgression from Asia, three criteria were used in selecting sequences deposited by Okumura et al. (2001). For inclusion into the European data set, haplotypes (1) must have been obtained from a European breed, (2) must not have been observed in any animal of Asiatic origin, and (3) must not have grouped with Asiatic sequences following test phylogenetic reconstruction. This reduced data set comprised sequences from 86 animals of 5 breeds (Berkshire, Landrace, Large White, Duroc, and Hampshire) in which a total of 17 distinct haplotypes were present [haplotype numbers 39, 41, and 44–58 as described Okumura et al. (2001)]. Sequences were aligned and the average pairwise distance within both the Asian and European data sets were calculated using MEGA.
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Results |
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Sequence Diversity Between Indigenous Chinese Breeds
A total of 15,982 bp were determined from 20 breeds, which represents 97% of the full-length mtDNA genome (positions 66–16,043; Ursing and Arnason 1998). This included each protein coding and tRNA gene, 12S and 16S rRNA genes, and 608 bp of the control region. Alignment and comparison of 20 sequences revealed a total of 267 polymorphic sites. The vast majority (261/267) were substitutions with a small number of identified indels (6/267). Analysis of sequence divergence revealed the largest distance (1.24 ± 0.09%) was present between a European (Landrace) and Chinese breed (Diannan small ear), while the smallest distance was between two indigenous Chinese animals, Tongcheng and Yushan Black (0.01 ± 0.01%). Comparisons within the 17 Chinese breeds revealed a low level of divergence, with the largest distance observed between the Diannan small ear and both the Jiangquhai and Zang breeds (0.21 ± 0.01%). The average pairwise distance calculated between the 17 Chinese breeds across the 15,982 bp of mtDNA was 0.09% (± 0.01%). A direct comparison was made between the intrapopulation diversity present within Asian domestic pig sequences (this study) with that observed in European domestic breed sequences (Okumura et al. 2001) by using 583 bp of the mitochondrial control region common to both data sets. Care was taken to remove from the analysis sequences present within European animals that likely arose via introgression of Asian germplasm. The average pairwise distance within the remaining European control region sequences (0.91 ± 0.21%) was higher than that found within Asian domestic pigs (0.61 ± 0.19%).
Frequency of Nucleotide Substitutions Within the Protein Coding Genes
The frequency of synonymous (Ks) versus nonsynonymous (Kn) substitutions occurring within each of the 13 protein coding genes is shown in Figure 1. Comparisons between the 17 Chinese breeds identified six genes that completely lacked nonsynonymous substitution. Three of these six genes (COI, COII, and NADH6) demonstrated an absence of nonsynonymous substitution when compared between Chinese and European animals (Kijas and Andersson 2001). Correspondence with this previous study was also seen for those genes that had a nonsynonymous substitution rate that was equal to or exceeded the synonymous rate (ATPase 6, ATPase 8, and NADH3).
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Genetic Compared With Traditional Classification of Native Chinese Breeds
A phylogenetic tree constructed from the near-complete mtDNA genomes of 17 Chinese and 3 European animals is shown in Figure 2. Two European animals (Duroc and Landrace) form a distinct outgroup, while a third (Large White) is found to be very closely related to the Chinese breeds. This is entirely consistent with previous findings that describe a large genetic distance separating European and Chinese domestic breeds while documenting the introgression of Chinese lines into European breeding stock (Giuffra et al. 2000; Kijas and Andersson 2001). Within the Chinese animals, Diannan small ear forms a distinct branch. Of the remaining 16 breeds, the tree can be loosely grouped into two clusters; this shows some correspondence with geographical location. Zang, Wuzhishan, Erhualian, Ningxiang, and Dahuabai pigs form a branch that is defined with some statistical support (bootstrap value = 44). The remaining breeds—Tongcheng, Qingping, Yushan Black, Jinhua, Zhong Meishan, Jiangquhai, Min, Wannanhua, Rongchang, Xiang, and Yimeng Black—are mainly from northern China, eastern China, and central China. While this demonstrates that some correlation exists between the genetic and traditional classification systems, clear differences are present. Seven breeds traditionally considered to be closely related were included from central China (group III). The mtDNA sequences obtained from the Tongcheng, Qingping, and Yushan Black animals were very similar but quite distinct from those found in other central Chinese breeds (Ningxiang and Dahuabai).
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Discussion |
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The indigenous breeds of China contain a remarkable collection of phenotypic diversity and the present analysis attempted to measure the level of underlying genetic variation present within the near-complete mtDNA genome. Selection of 17 breeds aimed to reflect this diversity and include members from each of the six traditional groups of Chinese pigs. The average level of pairwise sequence diversity within these animals was less than 0.1% when assayed across more than 15 kb of sequence. The finding that these genomes shared only limited sequence variation adds to an emerging picture concerning the origin of domestic pigs in Asia. A recent study based on the mtDNA D-loop found lower genetic variation within Chinese breeds than within European breeds (Okumura et al. 2001). This likely reflects the additional variation present in European breeds following the well-documented introgression of Asian animals. When we removed such introgressed sequences and performed a direct comparison of the 583 bp control region (15,379–15,962; Ursing and Arnason 1998), we found the level of variation among Asian domestic pig sequences (0.61 ± 0.19%) was still lower than that among European domestic animals (0.91 ± 0.21%), but the difference was not statistically significant (P =.96). Indeed, a unique haplotype was observed in every Asian animal sequenced, suggesting more haplotypes are likely to be revealed upon analysis of more Chinese animals. From these initial studies, it appears likely that the variability present within Asian and European domestic breeds prior to any systematic introgression was not significantly different. This is supported by an expanded analysis that included indigenous breeds from the surrounding Asian countries of Korea and Japan, which found low levels of Asian diversity that was approximately equal to that observed within European breeds (Kim et al. 2002).
Examination of the phylogenetic tree created from the near-complete mtDNA sequence revealed limited correspondence with the traditional grouping system of Zhang (1986). The latter weights morphological characteristics such as coat color to define groupings; however, as such traits are not encoded by the mtDNA, the two systems are based on variation arising from independent sources. Nevertheless, it was clear that some correspondence was present reflecting the geographic location of the specific breeds. Confirmation of these breed relationships will require additional analysis, as only one animal was used to represent each indigenous Chinese population. Sampling of many more animals is required to estimate the level of genetic diversity within each breed. This study does indicate that analysis of more animals using mtDNA will be informative in understanding the relationships between the breeds. The fast development of technology that has facilitated large-scale DNA sequencing means more near-complete mtDNA data sets can be generated economically. An immediate goal is analysis of individuals from more of the estimated 48 local and indigenous breeds and wild boars from across both China and neighboring Asian countries in an attempt to identify the progenitor population of the Chinese domestic pig.
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Acknowledgments |
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The sequencing work for this research was conducted at the Beijing Genomics Institute/Genomics and Bioinformatics Center, Chinese Academy of Science. This work was supported by National Key Projects for Basic Research and Development Plans of China (G2000016103), the National High Technology and Science Development Plan of China (863), the National Outstanding Youth Science Foundation of China (39925027), and the National Natural Science Foundation of China. Kui Li would like to express his thanks to the Tang Family Foundation for the financial support for his visit to Cornell University, and to Gustavo Aguirre and Norman Scott of Cornell University for their encouragement. Finally, we especially wish to thank the scientific editor of this article, Leif Andersson, and two anonymous referees for their time reviewing the manuscript and for their valuable suggestions and comments.
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Footnotes |
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Corresponding Editor: Leif Andersson
Received August 12, 2002
Accepted May 29, 2003
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References |
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