903 Genetic Variability of mtDNA Sequences in Chinese Native Chicken Breeds Z. G. Liu*, C. Z. Lei1, J. Luo1, C. Ding, G. H. Chen2, H. Chang2, K. H. Wang, X. X. Liu, X. Y. Zhang X. J. Xiao2 and S. L. Wu2 Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou, Jiangsu 225003, P. R. China ABSTRACT : The variability of mtDNA hypervariable segment I (HVS I) sequences was investigated in a total of 48 birds belonging to 12 Chinese native chicken breeds. Sixteen haplotypes were identified from 35 polymorphic nucleotide sites which accounted for 6.4% of a sequenced 544 bp fragment. Diversity analysis of the haplotypes showed that Tibetan, Langshan and Henan cockfight chicken had only one haplotype, while ancient haplotypes existed in Taihe silky and Chahua chicken. Phylogenetic analysis of the haplotypes suggested that Chinese native chicken breeds shared 5 maternal lineages and some breeds would share the same maternal lineage, regardless of their external features and ecological types. Both divergent and phylogenetic analysis of the haplotypes indicated the close genetic relationships between the Chinese native chicken breeds and G. g. gallus and G. g. spadiceus from different areas, which implied that G. g. gallus and G. g. spadiceus were the original ancestors of the Chinese native chicken breeds. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 7 : 903-909) Key Words : Chinese, Native Chicken, Jungle Fowl, mtDNA, Haplotype, Original Ancestor INTRODUCTION rate of mtDNA evolution was about 5 to 10 times faster than nuclear DNA, and its genes did not recombine. So Domestic chicken taxonomically belongs to Galliformes, mtDNA analysis has been used to investigate the genetic Pharsianidae, Gallus. Chicken breeds are considered to backgrounds of both closely related species and individuals have originated either from G. gallus or from G. sonnerati within species. The D-loop region of mtDNA is known to (grey jungle fowl), G. lafayettei (Ceylonese jungle fowl) or be more variable in sequence than in other regions and thus G. varius (green jungle fowl), respectively (Mason, 1987). has been frequently used by geneticists for phylogenetic Domestic chicken is the earliest domesticated fowl and analysis of closely related breeds within species. there are 60 native chicken breeds in China (Chang, 1995). Phylogenetic relationships among Chinese native chicken At present, researchers have analyzed the genetic breeds had been determined before using RFLP of mtDNA characteristics of Chinese native chicken breeds using (Wang et al., 1994; Zhou et al., 1997). However, results of methods such as cytogenetics, biochemical genetics and these studies provided only limited information. Sequencing DNA fingerprinting (Zeng, 1987; Cheng et al., 1991; Wang of mtDNA allowed for more powerful phylogenetic et al., 2003). inference because it can detect all the polymorphic sites Mitochondria, one of the important organelles in present. Fu et al. (2001) were the first to use D-loop eukaryotic cells, are presumed to represent bacteria-like sequence polymorphism to determine phylogenetic organisms incorporated into eukaryotic cells over 700 relationships among 5 native chicken breeds in Zhejiang million years ago (perhaps even as far as 1.5 billion years province. In our study, we used the D-loop hypervariable ago) and function as the sites of energy metabolism. In segment ⅠⅠ (HVS ) of mtDNA to determine the genetic higher vertebrates, mitochondria strictly follow the path of differentiation backgrounds and to probe into the origin of maternal transmission, i.e., the descendents of the same Chinese native chicken breeds. maternal ancestor have almost identical mitochondria. Mitochondrial DNA (mtDNA) is the genetic material in MATERIALS AND METHODS mitochondrion. Generally, evolution of mtDNA occurred primarily as single base pair substitutions, with only Specimen collection infrequent major sequence rearrangements. Moreover, the Based on related documents (Chang, 1995; Qiu et al., 1988; Compilation Committee of Annals of Domestic * Corresponding Author: Liu, Zhang-guo. Tel: +86-514-7233488, Animal and Poultry Breeds in Yunnan Province; Guizhou Fax: +86-514-7254416, E-mail: [email protected] Provincial Development station of Animal Breeds, 1997; 1 College of Animal Science and Technology, Northwest Sci-Tech Lai et al., 2001), information on the native chicken breeds University of Agriculture and Forestry, Yangling, Shaanxi we examined are listed in Table 1. We typically collected 12 712100, P. R. China. relatively ancient native chicken breeds from different 2 College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225001, P. R. China. animal husbandry culture areas in China. Each breed was Received October 31, 2003; Accepted March 22, 2004 represented by 4 specimens from four different lines. 904 LIU ET AL. Table 1. List of native breeds examined Breed Animal husbandry Time of breed Breed Ecological type Collection site Primary location code culture area origin Tibetan chicken ZJ Other a Tibet Qing Zang area b 1,000 years ago Qingyuan blotted chicken QY Meat a Qingyuan county, Min Yue area c 900 years ago Guangdong Henan cockfight DJ Recreation a Kaifeng city, Henan Southeast of China 2,000 years ago Chahua chicken CH other a Dehong county, Southwest of China Many years ago Yunnan Big bone chicken DG Meat and egg concurrent a Zhuanghe county, Northeast of China 200 years ago Liaoning Beijing youkei BY Meat and egg concurrent a An’ding area, Beijing North of China 250 years ago Langshan chicken RS Meat and egg concurrent a Rudong county, Southeast of China Very ancient breed Jiangsu Yugan Wugu YG Medicine Yugan breed farm Yugan county, Jiangxi Xiang’er’Gan area d Han dynasty chicken (2,000 years ago) Souguang SG Meat and egg concurrent Cilun breed farm Souguang city, North of China 1,500 years ago chicken Shandong Taihe Silky SY Medicine Taihe breed farm Taihe county, Xiang’er’Gan area d 1,000 years ago chicken Jiangxi Wumeng Wugu WM Medicine Bijie city, Bijie city, Southwest of China Many years ago chicken Guizhou Guizhou Yanjing Wugu YJ Medicine Yanjing county, Yanjing county, Southwest of China Han dynasty chicken Yunnan Yunnan (2,000 years ago) a represents Poultry Institute, Chinese Academy of Agri. Sci., b contains Qinghai, Tibet and adjacent areas. c contains Fujian, Guangdong and adjacent areas, d contains Hunan, Hubei, Jiangxi and adjacent areas. DNA extraction according to the manufacturer’s instructions. Sequencing The blood specimens were preserved by 75% ethanol at was performed by using an ABI model 377 automated the volume rate of 1:4 (blood:ethanol) and stored at room sequencer (PE). The sequencing primers were the forward temperature. DNA was extracted from these specimens primer L16750 and the reverse primer H543. A consensus using phenol/chloroform. sequence of approximately 544 bp for each bird resulted from assembling of sequence reads in both strands. PCR amplification The primers used were those of Dejardins et al. (1990) Data analysis and Randi et al. (1998) which amplified a 544 bp fragment All mtDNA nucleotide sequences obtained in this work between sites 16,750 and 543 (GenBank NC-001323), the were aligned by using the Clustal X software (Thompson et forward primer was L16750: 5’-AGGACACGGCTTGAAA al., 1997), and identical sequences were considered as the AGC-3’, and the reverse primer was H543: 5’-ATGTGCC same haplotype. Referring to the D-loop sequences of TGACCGAGGAACCAG-3’. The reagent kits used for several chicken breeds in GenBank, haplotypes dissimilar PCR were from Shanghai Biologic Engineering Company. to those found in the present study were selected to enrich The PCR reaction was carried out in a total volume of 50 µl, our data. Using MEGA software (Kumar et al., 1993), and the final concentration or content of each component Kimura 2-parameter distance matrix of all haplotypes were calculated to construct the unrooted NJ phylogenetic tree was as follows, 1×PCR buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 1.7 U DNA polymerase, 0.3 µM of each primer with G. lafayettei and G. variu s as outgroups. Bootstrap and 150 ng DNA template. PCR was performed in a confidence levels (BCL) of the phylogenetic tree were Progene thermal cycler (PE 9600). The reaction profiles estimated by 1,000 random bootstrap resampling of the data. included an initial denaturation at 95°C for 3 min, followed The haplotypes of the D-loop sequences for all of the species of Gallus in GenBank were used to draw the by 33 cycles, each consisting of 30 sec denaturation at 94°C, unrooted NJ cladogram of haplotypes for both Chinese 36 sec primer annealing at 63°C, 56 sec extension at 72°C, native chicken breeds and Gallus. Bootstrap confidence and then a final 5 min extension at 72°C. The amplified levels were evaluated by 1,000 random bootstrap products were all electrophoresed by 2.0% (wt/vol) agarose resampling of the data. gel in 1×TBE buffer, with 5 v/cm voltage for 1 h. After the run, the gel was stained with ethidium bromide. RESULTS PCR products sequencing Sequence and genetic variation of mtDNA D-loop The amplified products were purified by using the TM The sequences we studied have submitted to GenBank Wizard PCR Preps DNA purification kit (Promega) (from AY465960 to AY466007). The nucleotide GENETIC VARIABILITY OF mtDNA SEQUENCES IN CHINESE NATIVE CHICKEN BREEDS 905 Table 2. Haplotypes of studied breeds obtained from GenBank Breed Code of haplotype Accession No. in GenBank Author Collection site Chahua chicken CH5-6 AF512085, AF512089 Y. P. Liu, et al. Yunnan, China Yanjing Wugu chicken YJ5-7 AF512324, AF512326, AF512327 Y. P. Liu, et al. Yunnan, China Qinyuan blotted chicken QY5 AF512260 Y. P. Liu, et al. Guangdong, China Gushi chicken GuS5-8 AF512144, AF512145, AF512146, AF512150 Y. P. Liu, et al. Henan, China Bai’er yellow chicken BeH5 AF128322 Y.