Rapid Communication- Primer Design and Sequence Information for Cytochrome B Gene in the Chinese Painted Quail (Excalfactoria C
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-Rapid Communication- Primer Design and Sequence Information for Cytochrome b Gene in the Chinese Painted Quail (Excalfactoria chinensis) Xiang-Jun SHEN, Masaoki TSUDZUKIa, Takao NAKAMURAb The United Graduate School of Agricultural Science, Gifu University, Gifu-shi 501-1193, Japan Faculty of Applied Biological Science, Hiroshima University, Higashi-Hiroshima-shi 739-8528,a Japan bFaculty of Agriculture , Gifu University, Gifu-shi 501-1193, Japan (Received April 21, 1999; Accepted May 15, 1999) Abstract In accordance with the chicken and quail mitochondrialDNA sequences,three pairs of primers encompassing light and heavy strand (L14832, L15269, L15500, H15924, H15497, and H16207) with high specificity were designed, synthesized, and used for amplification and sequencing of cytochrome b (Cyt b) gene. The PCR products were sequenced by the direct-sequencing method. The Chinese painted quail cytochrome b gene consisted of 1,143bp nucleotides (base composition: 327 A; 399 C; 281 T and 136 G), encoded for a 380 amino acid sequence, and showed 89.0%, 86.4%, and 86.2%, homology with nucleotide sequences of Japanese quail, silky fowl, and White Leghorn chicken, respectively. The homology of the amino acid sequence of the cytochrome b gene of Chinese painted quail showed 98.2%, 95.3%, and 95.3% with those of Japanese quail, silky fowl, and White Leghorn chicken, respectively. The sequencesreported in this paper have been depositedin the GenBank database(Accession No. AF109217). Animal Science Journal 70 (4): 240-242,1999 Key words: Chinese painted quail,Excalfactoria chinensis, Cytochromeb, Sequence,Homology Chinese painted quail (Excalfactoria chinensis) belongs the test: to the order Galliformes, family Phasianidae, genus (1) L14832: 5'-TACCTGGGTTCCTTCGCCCT-3' Excalfactoria5),and it is the smallest species in the family (2) H15497: 5'-TGTAGGAAGGTGAGTGGAT-3' Phasianidae. It originated from India, Southeastern area (3) L15269: 5'-GCCACTGCTTTCGTAGGATA-3' of Clrina5),had a very short domestication history, and (4) H15924: 5'-ACTGGTTGGCTTCCGATTCA-3' gradually became a laboratoryresearch animal4). (5) L15500: 5'-GAATCAGGCTCAAATAACCC-3' Chinese painted quail has all of the advantages that (6) H16207: 5'-GCTTGTGCGTGGGTTGTCTC-3' Japanese quail possesses, that is, hardy, small body size, The letters L and H refer to the light and heavy strands, rapid sexual maturation, short generation interval, and and the numbers refer to the positions, for the 3' bases of high laying performance. Even Chinese painted quail is the primer in the complete mtDNA sequence of chicken1). superior to Japanese quail in these aspects. Very limited Three mitochondrial fragments encompassing the Cyt b biological research has been performed on Chinese gene were amplified from the mtDNA preparations via painted quails compared to Japanese quail. No report is PCR with three pairs of designed primers. Samples were available, however, on the complete cytochromeb (Cyt b) amplified through 35 cycles, with each cycle of 1min of denaturation at 94℃, annealing for 45 sec at 54℃ gene sequences in Excalfactoria chinensis. , and extending for 1min at 68℃. The template DNA were 50 Materials and Methods ng of Chinese painted quail mtDNA. DNA sources. Chinese painted quails introduced from Sequencing. Using the same set of primers , the amplified the University of Osaka Prefecture were used in this PCR products were gel purified and sequenced following experiment. Mitochondrial DNA (mtDNA) samples were automated sequencing procedures for the ABI system. prepared from the liver tissue using modified alkaline Strategies for PCR and Sequencing . The primers used in lysis method. the PCR and sequencing , and the corresponding strategies PCR. To design PCR primers for the Cyt b region, we for PCR and sequencing were shown in the Fig 1. compared the published sequences for chickens1), quails2), Data analysis. The homologies of Cyt b gene sequences and Black silky fowls3) after which we designed, between the Chinese painted quail and Japanese quail, synthesized, and tested many primers and primer pair sets. Black silky fowl, and White Leghorn chicken were Finally the following 6 primers were chosen and used in estimated using GENETYX-MAC 9.0 computer program. Corresponding: Takao NAKAMURA (fax: +81(0)58293-2830, e-mail: [email protected]) Anim. Sci. J. 70 (4): 240-242, 1999 240 SHEN, TSUDZUKI and NAKAMURA Fig. 1. Strategies for amplification and sequencing of cytochrome b gene of Chinese painted quail. Bars depict the cytochrome b gene and the flaking genes for ND5 (NADH dehydrogenase 5), threonine tRNA, and ND6 (NADH dehydrogenase 6). Arrows indicate the six oligonucleolide primers described in detail in the text. Fig. 2. Complete cytochrome b gene sequence of mtDNA in the Chinese painted quail (Excalfactoria chinensis). Fig. 3. Complete amino acid sequence of Chinese painted quail cytochrome b gene, and alignment with those of the Japanese quail, Black silky fowl, and White Leghorn chicken (* showed stop codon, TAA). Anim. Sci. 3. 70 (4): 240-242, 1999 241 Complete Cytochrome b Gene of Chinese Painted Quail Results and Discussion respectively (Fig. 3). The results of nucleotide sequence The three pairs of PCR primers were designed for the homology and amino acid homology showed that the annealing temperature at 54℃, the CG content at 45 to Chinese painted quail is genetically nearer to the Japanese 50%, the oligo length at the 20 to 22. All the primers quail rather than to the Black silky fowl and the White possessed comparatively high specificity for cytochrome Leghorn chicken. The Chinese painted quail can be used b gene region and the amplified nucleotide fragments as an outgroup of the family Phasianidae while covered the complete gene region. The theoretical comparing the genetic diversity among the different temperature of annealing for primers and the practical genuses within the family Phasianidae. The results of the annealing temperature in PCR just matched each other. homology analysis among these four poultry populations The PCR and sequencing were performed following the were in accordance with the general biological strategies as shown in the Fig. 1. classification methods. The Cyt b genes in the Chinese painted quail were beginning with a conserved initiating methionine (ATG) Acknowledgement codon and terminating with a TAA stop codon Fig. 2, We would like to thank Dr. S. ITO of Gifu University which were the same as the codons described in the White Leghorn, Japanese quail and Black silky fowl chicken3). for his critical comment and reading of the manuscript. The entire Cyt b gene sequences for Chinese painted quail consisted of 1,143bp nucleotides, with no apparent References deletions, or insertions. The nucleotide sequence base 1) Desjardins P, Morais R. Sequence and gene composition of Chinese painted quail was 326 A organization of the chicken mitochondrial genome. Journal of Molecular Biology, 212:599-634. 1990. (28.52%); 399 C (34.91%); 281 T (24.58%); 137 G (11.98%). Comparison of complete nucleotide sequences 2) Kornegay JR, Kocher TD, Williams LA, Wilson AC, of Cyt b genes among Chinese painted quail, Japanese Pathway of lysozyme evolution inferred from the quail2),Black silky fowl, and White Leghorn1)revealed sequence of cytochrome b in birds. Journal of that the Chinese painted quail showed 89.0% with MolecularEvolution, 37: 367-379. 1993. Japanese quail, 86.4% with Black silky fowl, and 86.2% 3) Shen X-J, Baba T, Iwasawa A, Nakamura T. Complete with White Leghorn. cytochrome b gene information in the silky fowl The entire Cyt b gene of Chinese painted quail encoded (Gallus gallus) (Black feather line). Animal Science for a 380 amino acid sequence, and it consisted of 237 Journal, 70: 98-100. 1999. (62.37%) hydrophobic, 85 (22.37%) neutral, and 58 4) Tsudzuki M. Excalfactoria quail as a new laboratory (15.26%) hydrophilic amino acid. The amino acid research animal. Poultry Science, 73: 763-768. 1994. sequence homologies of Chinese painted quail showed 5) Yamashina Y. A World List of Birds With Japanese 98.2%, 95.3%, and 95.3% with those of the Japanese Names. Daigaku-ShorinC. Tokyo. 1986. quail, Black silky fowl, and White Leghorn chicken, Anim. Sci. J. 70 (4): 240-242, 1999 242.