Journal of Poultry Science, .+ : 10ῌ2,, ,**.

Yasuhiko Wada+), ,ῌ*, Yoshiyuki Yamada+ῌ, Masahide Nishibori-ῌ and Hiroshi Yasue.ῌ

+ῌ Faculty of Agriculture, Saga University, Saga, 2.*ῌ**,1 ,ῌ , Japan Science and Technology Corporation, Tokyo, +*,ῌ2000 -ῌ Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, 1-3ῌ2/,2 .ῌ Genome Research Department, National Institute of Agrobiological Sciences, Tsukuba, -*/ῌ*3*+

Whole mitochondrial DNA of the Silkie fowl (Gallus gallus var. domesticus)was sequenced in order to elucidate the genetic position of the Silkie fowl in its related species based on mitochondrial DNA sequence. The length of Silkie mitochondrial DNA was found to be +0,12. base pairs. The similarity between Silkie and White Leghorn was found to be 33.11ῌ (-3 nucleotide di#erences in +0,12. base pairs). Since the sequences of NADH dehydrogenase subunit , and cytochrome b genes have been reported in six species [Japanese quail : japonica, Ring-neck pheasant : Phasianus colchicus, Grey : perdix, Chinese partridge : Bambusicola thoracia, Chicken (White Leghorn) : Gallus gallus var. domesticus, and Redhead : Aythya ameri- cana], nucleotide sequences of these two genes were used for a phylogenetic analysis of Silkie against these six species using neighbor joining and maximum likelihood methods. The Silkie and White Leghorn are in the same phylogenetic cluster with a bootstrap value of +***/+***.

Key words : whole mitochondrial DNA, Silkie fowl, phylogenetic analysis

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Introduction

Silkie fowl is distinct from the other chicken breeds in that the Silkie fowl has silky plumage, purple-colored skin and the bluish meat, purple to dark blue colored wattles, beak and comb, the fifth hind toe pointing slightly upwards, beards and feathering in the feet. In addition, as compared to the other breeds, the Silkie fowl is placid and tamable ; and its behavioral characteristic is persistent broodiness, which are often used for “surrogate broody” The Silkie is one of twenty-seven Japanese native breeds, which are defined to have habituated in Japan before the beginning of Edo era (Muramatsu, +313), and was appointed as national natural treasure of Japan in +3., (Nirasawa et al., +331). Concerning the origin of the Silkie fowl, although it has been considered that the fowl

Received : July ,/, ,**-, Accepted : November 1, ,**- * Correspondence to : Dr. Y. Wada, Faculty of Agriculture, Saga University, Saga, 2.*ῌ**,1, Japan. Tel/Fax (2+) 3/,ῌ,2ῌ2121, e-mail : [email protected] Wada et al. : Sequence of Mitochondrial Genome in Silkie Fowl 77 has originated in India and that the breed of Silkie fowl has been established in China and Japan (Roberts, +331), its original habitat in Asia has not been clarified. When these facts are taken into account with the fact that, as described above, the Silkie fowl has many genetic features distinct from those observed in the other chicken breeds, it is interesting to determine the genetic position of the Silkie fowl in fowls of genus Gallus including chicken breeds. Therefore, in the present study, in order to provide information for elucidation of the genetic position, we determined mitochondrial DNA (mtDNA) sequences of the Silkie fowl, and performed the phylogenetic analysis using a part of mitochondiral sequences of species in genus Gallus so far available.

Materials and Methods

DNA was prepared from peripheral blood of a male Silkie kept at a Hiroshima University, as described by Sambrook and Russell (,***). The blood sampling was performed in accordance with the guideline for experiment, Hiroshima Univer- sity. MtDNA fragments were amplified from mitochondorial genome using the LA-PCR Kit (Takara Shuzo, Ohtsu, Japan) with Silkie DNA as the template and the LA-PCR primer sets described below, following the procedure described previously (Nishibori et al., ,**+, ,**,, ,**-). Two primer sets were used for the LA-PCR :The first primer set was LA+0SF, /ῌ-CCTACGTGATCTGAGTTCAGACCGGAGCAA- TCCAG--ῌ ;andLA+0SR, /ῌ-TGCACCATTAGGTTGTCCTGATCCAACATCGA- GGT--ῌ. The second primer set was LACytbF, /ῌTACACGAATCAGGCTCAAAC- AACCCCCTAGGCATC--ῌ ;and LACytbR, /ῌAGATACAGATGAAGAAGAATG- AGGCGCCGTTTGCG--ῌ.TheLA-PCR was conducted at 3.ῌ for + min, followed by -* cycles consisting of -* s denaturation at 3.ῌ, and +0 min annealing and extension at 02ῌ, using a Techgene (Nalge Nunc International K.K., NY). Amplified fragments with +0 kb were then isolated through agarose gel electrophoresis, and used for segmental amplification using the -1 primer sets (Nishibori et al., ,**+). DNA fragments obtained through the segmental amplification were sequenced using the Exo SAP-IT (Amersham Biosciences, Buckinghamshire, England) and an ABI PRISMTM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster, CA). Se- quences were assembled using AutoAssember ver. ,.+ software (Applied Biosystems, Forester, CA) and analyzed with the GENETYX program package (Software Develop- ment Inc, Tokyo, Japan), with reference to the structure of White Leghorn (AP **--+1) (Nishibori et al., ,**-). Since the reliability of the phylogenetic analysis is generally heightened by increase of sequence length for the analysis (Cao et al., +33. ; Hasegawa and Adachi, +330), sequence information as much as possible was used for phylogenetic analysis together with six , Japanese quail (Coturnix japonica), Ring-neck pheasant (Phasianus colchicus), Grey partridge (Perdix perdix), Chinese (Bambusicola thoracia), Chicken (White Leghorn) (Gallus gallus var. domesticus), Redhead (Aythya americana)(Table +). The phylogenetic analysis was done by neighbor-joining (NJ) method (Saitou and Nei +321) and maximum likelihood (ML) method (Adachi and 78 J. Poult. Sci., .+ (+)

Table +. Species examined and accession number of sequence data for ND, and Cytb availabled from DDBJ/EMBL/GenBank

Accession number Group/species Common name References ND, Cytb Duck Aythya americana Readhead AF*3*--1 AF*3*--1 Mindel et al. (+333) Pheasants Phasianus colchicus Ring-neck pheasant AF,,,/0+ AF*,2132 Kimball et al. (+333)and Dimche# et al. (,***) Gallus gallus var. domes White Leghorn AP**--+1 AP**--+1 Nishibori et al. (,**-) Gallus gallus var. domes Silkie fowl AB*20+*, AB*20+*, In this study Perdix perdix Grey partridge AF,,,/0* AF*,213+ Kimball et al. (+333)and Dimche# et al. (,***) Bambusicola thoracica Chinese bamboo AF,,,/-2 AF*,213* Kimball et al. (+333)and partridge Dimche# et al. (,***) Coturmix japonica Japanese quail AP**-+3/ AP**-+3/ Nishibori et al. (,**+)

Hasegawa +330). In the NJ and ML analyses, bootstrap values were obtained by +,*** replications.

Results and Discussion

The nucleotide sequence of the Silkie whole mtDNA (AB*20+*,) was determined according to the procedure described above. The Silkie mtDNA consists of +0,12. bp, which is . bp shorter than the White Leghorn mtDNA (AP**--+1) (Nishibori et al., ,**-). The Silkie mtDNA sequence was annotated and presented in Table ,. Briefly, the +0SrRNA of the Silkie was . bp shorter than that of White Leghorn. A single extra base, which had been reported in NADH dehydrogenase subunit - (ND-) of chicken (breed not specified), Redhead, and Peregrine falcon (Mindell et al., +332), was observed in ND- of the Silkie. The cytochrome oxidase subunit I (COI) gene of the Silkie had an additional amino acid, as compared to that of White Leghorn. Five amino-acids substitutions between them were found in / genes, COI, COII, ND,,ND- and ND/. The initiation and stop codons of +- genes were as same as those of White Leghorn and Japanese quail (Table ,). In order to examine phylogenetic position of the Silkie, the NJ analysis was performed for the Silkie, Japanese quail, Ring-neck pheasant, Grey partridge, Chinese bamboo partridge, White Leghorn and Redhead using the concatenated sequence of ND, and Cytb. (The ND, and Cytb sequences of White Leghorn were the same as that of White Plymouth Rock (AP**--+2), so that, only the White Leghorn sequences were used for the phylogenetic analysis.) The NJ phylogenetic tree thus obtained is shown in Fig. + with the bootstrap values. In order to examine the tree, ML method was applied for the sequences, generating the tree, which is exactly the same as that of NJ analysis, (data not shown). As shown in Fig. Wada et al. : Sequence of Mitochondrial Genome in Silkie Fowl 79

Table ,. Localization of genes, features and length of each gene (base pair) in the mitochondrial genome of Silkie fowl

Size Codon/ Codon/ Genes and features Location (bp) Initiation Stop Control region (D-loop) +ῌ +,-+ +,-+ tRNA-Phe +,-,ῌ +-** 03 +,S ribosomal RNA (+,SrRNA) +-*+ῌ ,,10 310 tRNA-Val ,,11ῌ ,-.3 1- +0S ribosomal RNA (+0SrRNA) ,-/*ῌ -31+ +0,, tRNA-Leu -31,ῌ .*./ 1. NADH-dehydrogenase subunit + (ND+) .*//ῌ /*,3 31/ ATG TAA tRNA-Ile /*-*ῌ /+*+ 1, tRNA-Gln* /+*1ῌ /+11 1+ tRNA-Met /+11ῌ /,./ 03 NADH-dehydrogenase subunit , (ND,) /,.0ῌ 0,20 +*.+ ATG TAG tRNA-Trp 0,2/ῌ 0-0* 10 tRNA-Ala* 0-01ῌ 0.-/ 03 tRNA-Asn* 0.-3ῌ 0/++ 1- tRNA-Cys* 0/+-ῌ 0/12 00 tRNA-Tyr* 0/12ῌ 00.2 1+ Cytochrome oxidase subunit + (CO+) 00/*ῌ 2,** +//+ GTG AGG tRNA-Ser* 2+3,ῌ 2,00 1/ tRNA-Asp 2,03ῌ 2--1 03 Cytochrome oxidase subunit , (CO,) 2--3ῌ 3*,, 02. ATG TAA tRNA-Lys 3*,.ῌ 3*3+ 02 ATPase subunit 2 (ATPase2) 3*3-ῌ 3,/1 +0/ ATG TAA ATPase subunit 0 (ATPase0) 3,.2ῌ 33-+ 02. ATG TAA Cytochrome oxidase subunit - (CO-) 33-+ῌ+*1+0 120 ATG T tRNA-Gly +*1+0ῌ+*12- 02 NADH-dehydrogenase subunit - (ND-)# +*12.ῌ+++-/ -/, ATG TAA tRNA-Arg +++-1ῌ++,*. 02 NADH-dehydrogenase subunit . light chain (ND.L) ++,*/ῌ++/*+ ,31 ATG TAA NADH-dehydrogenase subunit . (ND.) ++.3/ῌ+,21. +-2* ATG T tRNA-His +,21-ῌ+,3.+ 03 tRNA-Ser +,3.-ῌ+-**1 0/ tRNA-Leu +-**3ῌ+-*13 1+ NADH-dehydrogenase subunit / (ND/) +-*2*ῌ+.231 +2+2 ATG TAA Cytochrome b (Cytb) +.3*,ῌ+0*.. ++.- ATG TAA tRNA-Thr +0*.2ῌ+0++0 03 tRNA-Pro* +0++1ῌ+0+20 1* NADH-dehydrogenase subunit 0 (ND0)* +0+3-ῌ+01+. /,, ATG TAA tRNA-Glu* +01+1ῌ+012. 02 mitochondrial genome +012. * : coded on the complementary (L) strand, # : insertion of one extra base in Silkie fowl 80 J. Poult. Sci., .+ (+)

Fig. +. Phylogenetic tree of Silkie, Japanese quail, Ring-neck pheasant, Grey partridge, Chinese bamboo partridge, Chicken (White Leghorn), and Redhead. The tree was calculated for the concatenated sequence of ND, and Ctyb genes by NJ method (Saitou and Nei, +321) based on the Kimura’s two-parameter (Kimura, +32*). Numeral on each branch represents the bootstrap value obtained with +,*** repetitions. The horizontal scale bar indicates the number of substitutions/site.

+, the Silkie and White Leghorn are in the same phylogenetic cluster and the Silkie/ White Leghorn cluster formed a cluster with Chinese bamboo partridge. This clustering is consistent with our previous observation that chicken was clustered with Chinese bamboo partridge (Nishibori et al. ,**,). Thirty-nine nucleotide substitutions and .* nucleotide substitutions were observed between the Silkie in our study and the White Leghorn (AP**--+1) and between the Silkie and the White Plymouth Rock (AP**--+2), respectively. These substitutions are much greater that those (0 substitutions) between the White Leghorn (AP**--+1)and the White Plymouth Rock (AP**--+2) (Nishibori et al., ,**-). These observations taken together indicate that the genetic relationship between the Silkie and the current representative commercial breeds, i.e., the White Leghorn and White Plymouth Rock, is remoter than that between the commercial breeds. However, since no whole mitochondrial DNA sequences are available for other chicken breeds such as Japanese native chicken breeds (Nirasawa et al., +331), Chinese native chicken breeds (Niu et al., ,**,), and Indian native chicken breeds (Wimmers et al., ,***), it is, at present, impossible to elucidate the origin of the Silkie fowl. In the future, if those sequences become available, we will examine the genetic status of the Silkie fowl in the chicken breeds using those sequences. Wada et al. : Sequence of Mitochondrial Genome in Silkie Fowl 81

Acknowledgments

We are grateful to Dr. M. Tsuduki at Hiroshima University for providing the whole blood of the Silkie. The part of this study was carried out under the ISM Cooperative Research Program (,**,-ISMῌCRP-,*-2).

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