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Genetic Diversity of Mitochondrial DNA of Chinese Black-bone Chicken

ABSTRACT

The black-bone chicken has special economic value in Chinese poultry breeds, which also are valued for the medicinal properties of their meat in traditional Chinese medicine. In order to protect the genetic resources of native black-bone chicken breeds, we analyzed the genetic diversity and matrilineal components of 64 mtDNA D-loop partial sequences from three native black-bone chicken breeds, together with reported 596 black-bone chicken mtDNA sequences from China, Japan, and Korea. A total of 108 haplotypes were observed from 73 variable sites. These domestic chicken mtDNA sequences could be assigned into seven clades (A-G). The results indicated that 71.97% of the black-bone haplotypes were related to the reference sequence that may originate from Eurasia, while the minor part of mtDNA sequences presumably derive from Southeast Asia, China, and Japan. Three clades were shared by Korean, Japanese, and Chinese black-bone chickens. These results provide basic data useful for making new breeding and conservation strategies for the black-bone chicken in China.

Keywords:
Chinese Black-bone Chicken; MtDNA; D-loop region; Haplotype; Maternal origin

INTRODUCTION

Domestic chicken (Gallus domesticus) may overwhelmingly originate from the Red Jungle Fowl of Asia (Fumihito & others 1994Fumihito A, Miyake T, Sumi S, Takada M, Ohno S, Kondo N. One subspecies of the red junglefowl (Gallus gallus gallus) suffices as the matriarchic ancestor of all domestic breeds. Proceedings of the National Academy of Sciences 1994;91:12505-12509.; Kanginakudru and others 2008Kanginakudru S, Metta M, Jakati RD, Nagaraju J. Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken. BMC Evolution Biology 2008;8:174.). After a long-time of domestication, domestic chicken is one of the major sources of proteins in human’s food. Beyond that, the domestic chickens have some other roles in entertainment, religion and ornamentation (Liu and others 2006). However, the black-bone chicken is different from the domestic chicken, and it is the unique special medical property, which was recorded in the Chinese traditional medicine dictionary about 700 years ago (Xie.,1995). The black-bone chicken, with distinct medical usage from domestic chicken attracts scientist’s curiosities to explore their genetic evolution and origins.

Due to a vast territory, diverse environment, different selection targets and rearing conditions, and geographic proximity, a large variety of black-bone breeds have been developed in East Asia (Xie.,1995). To date, there are eight Chinese Silkie breeds registered in the Domestic Animal Diversity Information System (DAD-IS) of the Food and Agriculture Organization of the United Nations (http://www.dad.fao.org/). In Korea, Ogol chicken has been registered as a natural monument, which also has black features (Lee and others 2007Lee YJ, Bhuiyan MSA, Chung HJ, Jung WY, Choi KD, Jang BG,et al. Mitochondrial DNA diversity of korean ogol chicken. Asian-Australasian Journal Animal Science 2007;20:477-481.). To fully utilize this valuable resource, we investigated these genetic resources with the aim to protect precious black-bone chicken breeds and develop new breeds.

Mitochondrial DNA (mtDNA) is maternally inherited, which provides abundantly genetic information for breed structure and maternal origins (Miao & others 2013Miao YW, Peng MS, Wu GS, Ouyang YN, Yang ZY, Yu N, et al. Chicken domestication: an updated perspective based on mitochondrial genomes. Heredity 2013;110:277-82.). mtDNA D-loop region with highly polymorphic information is a major genetic resource research tool to unveil the phylogenetic relationship, investigate the maternal origin, evaluate the population diversity, and determine the phylogeographic structure (Groeneveld & others 2010Groeneveld LF, Lenstra JA, Eding H, Toro MA, Scherf B, Pilling D, et al. Genetic diversity in farm animals--a review. Animal Genetics 2010;41(Suppl 1):6-31.; Wilkinson & others 2012Wilkinson S, Wiener P, Teverson D, Haley CS, Hocking PM. Characterization of the genetic diversity, structure and admixture of British chicken breeds. Animal Genetics 2012;43:552-563.).

China has rich black-bone chicken genetic resources (Niu & others 2002Niu D, Fu Y, Luo J, Ruan H, Yu X-P, Chen G, et al. The origin and genetic diversity of chinese native chicken breeds. Biochemical Genetics 2002;40:163-174.). In previous reports, researchers demonstrated the genetic diversity, phylogenetic relationship and origins by mitochondrial D-loop regions or microsatellite markers in some Chinese Silkie and Japanese fowls (Rowshan & others 2011Rowshan J, Kumagae M, Nishibori M, Yasue H, Wada Y. Japanese silkie fowls are widely distributed in the phylogenetic tree derived from mitochondrial complete d-loop nucleotide sequences. The Journal of Poultry Science 2011;48:176-180.; Zhou & others 2010Zhou B, Chen S-Y, Zhu Q, Yao Y-G, Liu Y-P. Matrilineal Components and genetic relationship of silkies from China and Japan. The Journal of Poultry Science 2010;47:22-27.). However, these studies displayed the evaluation of biodiversity only based on a few black-bone chicken breeds, without systematic analysis on genetic diversity and maternal origin of black-bone chicken in China. Therefore, more extensive samples of black-bone chicken are required to systematically describe the pattern of mtDNA variation, unveil maternal origin, phylogenetic relationship and phylogeographic structure of black-bone chicken breeds. In this study, we gained a large amount of available Chinese black-bone mtDNA sequences to assess the variations of 26 native breeds, including the Korean black-bone chicken breed, one Japanese black-bone chicken breed and 24 Chinese black-bone chicken breeds. We aim to determine the degree of shared maternal mtDNA haplotypes among different populations of black-bone chicken breeds in East Asian, reveal the phylogenetic relationship and maternal lineages of origin.

MATERIAL AND METHODS

Sampling and Reported Data

A total of 64 chicken samples were collected from three China native breeds (Nanchuan = 18, Zengfu= 27, and Yanjin = 19). For the comparison of haplotypes, previous D-loop sequence data from 22 Chinese black-bone chicken breeds were included in the analysis, as well as Japanese Silkies and 31 Korean Ogol chicken were retrieved from GenBank. All together, a total of 594 black-bone chicken sequences belonging to 26 breeds: Nanchuan Mountain Wugu (NC), Zengfu Wugu (ZF), Chengdu Black Silkie (CD), Ya’anWugu (YA), Sichuan Mountain Wugu (SCM), Dwarf Wugu (DW), Guizhou Mountain Wugu (GZM), Wumeng Wugu (WM), Gushi Wugu (GS), Hubei Black Silkie (HB), Yunxian Wugu (YX), Xuefeng Wugu (XF), Jiangsu Silkie (JS), Taihe Silkie (TH), Yuganwugu (YG), Luke egg Wugu (LK), Wangfeng Wugu (WF), Wuding Wugu (WD), YanjingWugu (YJ), Tengchong (TC), Guangdong Silkie (GD), Zhejiang Wugu (ZJ), Jiangshan Wugu (JSH), Bairong Silkie (BR), Japan Silkie (JAS) and Korean Ogol (KO). More details about the 594 mtDNA D-loop partial sequences from 26 black-bone chicken breeds are shown in Table 1 and Table S1.

Table 1
Sample information and genetic diversity of black-bone chicken

Table S1
Sample information of NCBI Accession number

DNA extraction and PCR amplification

Chicken blood samples were obtained from the wing veins and collected in 5mL vacuum blood collection tubes, which were stored at -20°C until use. DNA was isolated from blood samples using the phenol-chloroform procedure (Zhang & others 2012Zhang T, Lu H, Chen C, Jiang H, Wu S. Genetic diversity of mtDNA D-loop and maternal origin of three chinese native horse breeds. Asian-Australasian Journal of Animal Sciences 2012;25:921-926.). PCR was used to amplify a 518bp fragment in the D-loop hypervariable region in mtDNA by the following primer pair (mtF:5’-AGGACTACGGCTTGAAAAGC-3’ and mtR:5’-ATGTGCCTGACCGAGGAACCAG-3’) as previously described (Randi & Lucchini 1998Randi E, Lucchini V. Organization and evolution of the mitochondrial dna control region in the avian genus alectoris. Journal of Molecular Evolution 1998;47:449-462.) (Table 2). PCR amplifications were performed in a 50 μL volume containing 5 μL of 10×buffer, 1.5 mM MgCl2, 0.25 mM dNTPs, 0.2 mM each primer, 1.5 U Taq DNA polymerase (TaKaRaBiosystems, Dalian, China) and approximately 20ng genomic DNA. Thermal cycling was carried out on a BIO-RAD T100 Thermal cycler. The PCR was carried out using a standard program with 4 min denaturation at 95 ºC, 35 cycles for 30 s at 94 ºC, 60 s at 55 ºC, and 90 s at 72 ºC, and final extension for 10 min at 72 ºC (Han & others 2015Han H, Zhang Q, Gao K, Yue X, Zhang T, Dang R, et al. Y-single nucleotide polymorphisms diversity in chinese indigenous horse. Asian-Australasian Journal of Animal Sciences 2015;28:1066.). PCR products were purified on agarose gel according to the manufacturer’s instructions, and DNA sequencing was performed using an ABI 377 automated sequencer (PE Applied Biosystems). The original PCR primers could be used as sequencing primers. All sample sequences were deposited in GenBank under accession numbers (MG554050-MG554113).

Table 2
Primers for PCR analysis of mtDNA D-loop target region

Data analysis

Sequence alignments were performed using DNAman (version 6.0.40). Haplotype numbers (K), nucleotide variable sites (Ps), haplotype diversity (Hd), nucleotide diversity (π) and average number of nucleotide differences (K) were determined with DnaSP 5.10.01 software (Librado & Rozas 2009Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 2009;25:1451-1452.). The same sequences were considered to be one haplotype. Software Network 4.6 (www.fluxus-engineering.com) was used to construct a median-joining network to evaluate relationships among different samples. The network also included nine haplotypes as references representing the main clades (A to I), which were found in populations originating from the Chinese and Eurasian region (Liu & others 2006).

RESULT

Genetic diversity of mtDNA D-loop region of black-bone chicken breeds

Mitochondrial D-loop sequence variations were used to calculate Ps, K, Hd and π among the chicken population (Table 1). A total of 73 polymorphic sites were detected, which represented 14.09% of the total D-loop sequence analyzed (518bp), which were far more than the 27 sites in the research of silky by Zhou et al. (Zhou & others 2010Zhou B, Chen S-Y, Zhu Q, Yao Y-G, Liu Y-P. Matrilineal Components and genetic relationship of silkies from China and Japan. The Journal of Poultry Science 2010;47:22-27.). All these variations were either transitions or trans­versions, and no insertions or deletions were detected (Table S2). A total of 108 haplotypes were defined (Table 3). Hd and π of populations are the main indices for evaluating mtDNA variation and genetic diversity of a breed or a population. Nucleotide diversity of all black-bone chicken was estimated at 0.01479 ± 0.00021 (Table 1). Nucleotide diversity of all black-bone chicken breeds ranged from 0.00232 ± 0.00137 in Wangfeng black-bone chicken to 0.01566 ± 0.00271 in Zhejiang black-bone chicken. One hundred and eight haplotypes were identified in 25 black-bone chicken populations. In our study, haplotype diversity was relatively quite high (Hd = 0.936 ± 0.005). Compared with different black-bone populations, Yugan black-bone chicken had the highest haplotype diversity (Hd = 1.000), followed by Jiangshan black-bone chicken (0.973 ± 0.024), and the lowest haplotype diversity was detected in Zhejiang black-bone chicken. Our research found that the haplotype diversity and nucleotide diversity of China black-bone chicken were respectively 0.933 ± 0.005 and 0.01460 ± 0.00025, and slightly exceeded that of Korean and Japanese black-bone chicken.

Table S2
Variable sites for 108 haplotypes observed in the black bone chicken populations

Table 3
Haplogroup distribution frequency of black-bone chicken

Figure 1
The network graph of the mtDNA haplotypes in black bone chicken samples. Circle areas are proportional to haplotype frequencies. White, grey, and black colors were denoted for Chinese, Korean, and Japanese Silkies, respectively. Red points are the oretical intermediate nodes, indicates potential mutations but not found in this study. Haplotypes A1, B1, C1, D1, E1, F1 and G1 were clarified as a reference sequence.

Additionally, these data illustrated that the genetic diversity was significantly different among some Chinese native black-bone chicken populations. Collectively, these results indicated that Chinese black-bone chicken breeds harbored rich genetic resources, however, a few native breeds are on the verge of extinction.

Phylogeny of the haplotypes and Network analysis

The 108 haplotypes could be classified into seven divergent clades (A-G) (Table 3). The haplotypes A1, B1, C1, D1, E1, F1, and G1 in this study were the same as the partial sequence of each haplotype from the clades described by Liu et al. (2006), while we did not detect the H1 and I1 described by Liu et al. (Liu & others 2006). Briefly, the clade A, B and E were the most widely distributed clades, and contained 25, 23 and 21 haplotypes, respectively. The Clades D, F, C and G shared other haplotypes ranging from 3 to 18. In each of the seven clades (A, B, C, E, F, and G), there was a dominant haplotype, and that, haplotypes A1, B1, C1, E1, E2, F1, and G1 were dominant and shared by 92, 71, 57, 60, 31, 11 and 16 samples, respectively. In addition, fifty-five haplotypes are unique. Meanwhile, 12 unique haplotypes (12/108 = 11.11%) belong to Yanjing breeds, and 13 unique (13/104 = 12.03 %) haplotypes belong to Jiangshan breed, and for other breeds, the number of unique haplotypes varies from one to four (Table 1). In general, all the Chinese Black-bone Chicken samples were present in clades A, B, C, E, F, and G, while Japanese Black-bone Chicken samples were distributed in clades A, B, C, E, and Korean Black-bone Chicken were only found in clades B, C and E. The number of black-bone individuals in each clade was listed in Table 2. Further more, these data elucidated obvious differences among different Chinese breeds according to the clades distribution, such as five Zhejiang Wugu samples that were only classified into B clade, and Chengdu Black Silkie, Hubei Black Silkie, Yunxian Wugu, Jiangsu Silkie, Luke egg Wugu, Wangfeng Wugu and Wuding Wugu that were not detected in the dominant clade E. The results indicated that the genetic diversity of Chinese black-bone chicken was far abundant, and suggested different that geographic structures were detected in chicken populations. Thus, we could make a conclusion that Chinese black-bone chicken had different origins through the geographic structure of the clades.

DISCUSSION

The formation of the reconstructed network profile of Chinese black-bone chicken was in accord with previous report (Liu & others 2006). The wide clades distribution of black-bone fowl suggests that the present black-bone chicken has high genetic divergence and has different origins of black-bone chicken. Some scholars studied the genetic relationship between Silkies and other chicken breeds using mtDNA sequence variations, and detected no obvious differentiation (Niu & others 2002Niu D, Fu Y, Luo J, Ruan H, Yu X-P, Chen G, et al. The origin and genetic diversity of chinese native chicken breeds. Biochemical Genetics 2002;40:163-174.). In previous study, the distribution of main mtDNA haplogroups in chicken presented geographic pattern:clades A, B, and E were distributed ubiquitously in Eurasia; clade C was prevalent in Japan and Southeast China; clades F and G were exclusive to Yunnan, China (Liu & others 2006). In this study, the majority of Chinese chickens could be classified into clades A, B, and E. A small number of black-bone chickens were distributed in clades F and G. The phenomenon indicates that F and G clades have little contribution to black-bone chicken. But a large portion of Yanjin Silky (37.3%) was observed in clade G, and Tengchong black-bone chicken (79.31%) was in clade F, respectively, both of which originated from Yunnan, China. The results indicated that the two clades had great contributions to Yanjin Silky and Tengchong black-bone chicken (Liu et al., 2006). In this study, 14 haplotypes that were identified in Japanese chickens belonged to five clades (A-E), and nine haplotypes identified in Korean Ogol chickens belonged to three clades (B, C, and E), while clade D was only found in Japanese chickens. Clade D contained gamecocks, which is a latest domestic population, and originates from Southwest China and/or surrounding areas. The results disguised itself clearly from the Chinese and Korean black-bone chickens, which were consistent with previous studies (Zhu & others 2014Zhu WQ, Li HF, Wang JY, Shu JT, Zhu CH, Song WT, et al. Molecular genetic diversity and maternal origin of Chinese black-bone chicken breeds. Genetic Molecular Research 2014;13:3275-3282.). The wide distribution of black-bone chicken suggests that the present Chinese black-bone chicken has high genetic divergence, and different clades may originate from different regions.

In the present study, we investigated the D-loop SNPs and haplotypes for the identification of black-bone chicken populations in East Asia. The data indicated that the genetic resource and genetic diversity of Chinese black-bone chicken were abundant. A total of 108 haplotypes were observed from 73 variable sites, which could be assigned into seven clades. Three clades were shared by Korean, Japanese and Chinese black-bone chickens. In summary, our results provided evidences for the genetic connection across Chinese, Japanese, and Korean chicken. The wide distribution of black-bone chicken in different clades suggested that black-bone chicken was a special and unique domestic chicken population, and the two populations are closely related, which are multiple origins from different regions in Asia.

ACKNOWLEDGEMENTS

This research was supported by the Shaanxi province agricultural science and technology innovation and technological project (2016NY-084), National Natural Science Foundation of China (NSFC) (31402071).

REFERENCE

  • Fumihito A, Miyake T, Sumi S, Takada M, Ohno S, Kondo N. One subspecies of the red junglefowl (Gallus gallus gallus) suffices as the matriarchic ancestor of all domestic breeds. Proceedings of the National Academy of Sciences 1994;91:12505-12509.
  • Groeneveld LF, Lenstra JA, Eding H, Toro MA, Scherf B, Pilling D, et al. Genetic diversity in farm animals--a review. Animal Genetics 2010;41(Suppl 1):6-31.
  • Han H, Zhang Q, Gao K, Yue X, Zhang T, Dang R, et al. Y-single nucleotide polymorphisms diversity in chinese indigenous horse. Asian-Australasian Journal of Animal Sciences 2015;28:1066.
  • Kanginakudru S, Metta M, Jakati RD, Nagaraju J. Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken. BMC Evolution Biology 2008;8:174.
  • Lee YJ, Bhuiyan MSA, Chung HJ, Jung WY, Choi KD, Jang BG,et al. Mitochondrial DNA diversity of korean ogol chicken. Asian-Australasian Journal Animal Science 2007;20:477-481.
  • Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 2009;25:1451-1452.
  • Liu Y-P, Wu G-S, Yao Y-G, Miao Y-W, Luikart G, Baig M, et al. Multiple maternal origins of chickens: Out of the Asian jungles. Molecular Phylogenetics and Evolution 2006;38:12-19.
  • Miao YW, Peng MS, Wu GS, Ouyang YN, Yang ZY, Yu N, et al. Chicken domestication: an updated perspective based on mitochondrial genomes. Heredity 2013;110:277-82.
  • Niu D, Fu Y, Luo J, Ruan H, Yu X-P, Chen G, et al. The origin and genetic diversity of chinese native chicken breeds. Biochemical Genetics 2002;40:163-174.
  • Randi E, Lucchini V. Organization and evolution of the mitochondrial dna control region in the avian genus alectoris. Journal of Molecular Evolution 1998;47:449-462.
  • Rowshan J, Kumagae M, Nishibori M, Yasue H, Wada Y. Japanese silkie fowls are widely distributed in the phylogenetic tree derived from mitochondrial complete d-loop nucleotide sequences. The Journal of Poultry Science 2011;48:176-180.
  • Wilkinson S, Wiener P, Teverson D, Haley CS, Hocking PM. Characterization of the genetic diversity, structure and admixture of British chicken breeds. Animal Genetics 2012;43:552-563.
  • Zhang T, Lu H, Chen C, Jiang H, Wu S. Genetic diversity of mtDNA D-loop and maternal origin of three chinese native horse breeds. Asian-Australasian Journal of Animal Sciences 2012;25:921-926.
  • Zhou B, Chen S-Y, Zhu Q, Yao Y-G, Liu Y-P. Matrilineal Components and genetic relationship of silkies from China and Japan. The Journal of Poultry Science 2010;47:22-27.
  • Zhu WQ, Li HF, Wang JY, Shu JT, Zhu CH, Song WT, et al. Molecular genetic diversity and maternal origin of Chinese black-bone chicken breeds. Genetic Molecular Research 2014;13:3275-3282.

Publication Dates

  • Publication in this collection
    Jul-Sep 2018

History

  • Received
    19 Jan 2018
  • Accepted
    16 Mar 2018
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