DNA Fingerprinting Analysis of Native and Red Jungle Fowls in Fiji and Western Samoa

DNA Fingerprinting Analysis of Native and Red Jungle Fowls in Fiji and western Samoa

Hideji YAMASHITA1), Takao NISHIDA2), Naoki TSUNEKAWA2), Peter MANUEL3), Shin OKAMOTO1), Yoshizane MAEDA1) and Tsutomu HASHIGUCHI1)

Laboratory of Animal Breeding and Genetics, Faculty of Agriculture, Kagoshima University, Kagoshima 890, Japan Laboratory of Anatomy and Physiology, College of Agriculture and Veterinary Medicine, Nihon University, Fujisawa 252, Japan Animal Health and Production Division, Ministry of Primary Industry, Suva, Fiji

In the present study, genomic DNA was prepared from dried chicken blood sample and subsequently used for analyzing DNA fingerprinting (DFP) of native and Red jungle fowl in the South Pacific Islands. These chicken genomic DNAs were sufficient in quantity and purity for multiple DFP analyses, and chicken blood could be stored for up to 3 months without physical degradation under dry condition. The DFP analyses were carried out by individual and population DFP. Similarities of DFP patterns and genetic distances among fowls in Fiji and Western Samoa were estimated using band sharing (BS) value and the mean number of nucleotide substitutions per nucleotide site, respectively. The results of population DFP were under the influence of data in individual DFP. From the BS values and genetic distances, the native fowls in five different islands of Fiji and Western Samoa could be distinguished in each island, and the difference was consistent with geographical distribution. Fijian and Western Samoan native fowls were found to form two separate groups. The Red jungle fowls in Fiji islands were closely related to each other, but were found to be related remotely to Red jungle fowls in Kagoshima University. Therefore, DFP analysis can be used to estimate relative genetic variability and to reconstruct the evolutionary relationships in small populations genetically isolated. (Jpn. Poult. Sci., 34 : 9-20, 1997) Key words : native fowl, Red jungle fowl, DNA fingerprinting, genetic relationship, genetic distance

Introduction

There are two theories as to whether jungle fowl is the ancestor of domestic fowl, namely the monophyletic origin theory which Red jungle fowl (Gallus gallus) is the ancestor, and the polyphyletic origin theory suggesting Grey jungle fowl (Gallus sonneratii), Ceylonese jungle fowl (Gallus lafayettii) and Green jungle fowl (Gallus varius) other than Red jungle fowl as the ancestors (CRAWFORD,1990). One modern technique for genetic monitoring of domestic animal by using DNA fingerprinting (DFP) was developed by JEFFREYSet al. (1985 a), DFP detects hypervariable minisatellite regions which consist of tandem repeats of short sequence and have high degree of polymorphisms due to differences in the number of repeats at each

Received Feb. 24, 1995 10 Jpn. Poult. Sci., 34 (1)

locus. And then, DUNNINGTON et al. (1990) developed 'population DNA fingerprinting'

using pooled DNA from random samples of individuals within a population.

DFP has been used for identification of individuals (GILL et al., 1985 ; JEFFLREYS et

al., 1985 a, b ; BURKE and BRUFORD, 1987), linkage analysis (JEFFREYS et al., 1986 ; GEORGES

et al., 1990), assessment of genetic distance between populations in domestic animal

(KUHNLEIN et al., 1989 ; DUNNINGTON et al., 1991 ; HABERFELD et al., 1992 ; YAMASHITA et al.,

1994) and estimation of relative genetic variability in natural populations (WETTON et

al., 1987 ; GILBERT et al., 1990).

In the present study, the authors analyzed the DFP patterns obtained from native

and Red jungle fowls in the South Pacific Islands in order to clarify their genetic

relationships.

Materials and Methods

Fowls

Eighteen Fijian native fowls, twelve Western Samoan native fowls, four Red

jungle fowls and three hybrid fowls (Fijian native fowl •~ Red jungle fowl) in Fiji, and six Red jungle fowls in Kagoshima University were used in the present study (Table

1). Blood samples were collected at five different islands (Viti Levu, Vanua Levu , Taveuni, Makogai and Koro) in Fiji and two different islands (Upolu and Savai'i) in

Western Samoa and dried onto 3 MM paper (1.5 •~ 15 cm), and then transported to Japan.

DNA extraction

DNA was extracted from dried blood samples of one to nine individuals from each

island. 3 MM paper fixed blood was cut up and suspended in 1% SDS,10 mM Tris-HCl ,

pH 7.5, 0.1 M NaCl and 1 mM EDTA. The solution was then incubated with 100 ƒÊg/ml of proteinase K at 55•Ž for 3h. After phenol extraction, DNA was precipitated by the

addition of 2 volumes of ethanol and dissolved in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.

For electrophotoric determination of genomic DNA, absorbancy reading was taken

at wavelength of 260 and 280 nm (SAMBROOK et al., 1989). Absorbance at 260 nm (A260)

was used to determine the amount of DNA present. An A260 unit of 1.0 indicates 50 ƒÊg/

Table 1. List of fowls used in this study YAMASHITA et al. : DNA Fingerprinting Analysis in Fowl 11

ml of double-stranded DNA. The ratio of absorbances at 260 and 280 nm (A260/A280)

was used as an indicator of nucleic acid purity.

DNA fingerprinting

DFP analyses were performed by using individual DFP and population DFP, which

used DNA prepared from individuals and pooled DNA from individuals of each

population, respectively. Four of six native fowls in Viti Levu and Savai'i, and all of Red jungle and hybrid fowls in Fiji were used for individual DFP. For population

DFP, six of native fowls in each five different islands of Fiji and Western Samoa, and

six Red jungle fowls in Kagoshima University were used. DNA transfer and hybrid-

ization conditions were similar to those used previously (YAMASHITA et al., 1994). In

brief, 10 ƒÊg DNAs were digested with Hinf I, electrophoresed in 1.2% agarose gel and

transferred to a positive charge nylon membrane. Synthetic (TG)n polynucleotide

(Pharmacia LKB Biotechnology) was labeled with Dig-11-dUTP (Boehringer Mannheim) for use as a probe. Chemiluminescent detection was carried out according

to manufacturer's recommendation.

Similarities of DFP patterns were scored by means of band sharing (BS) value

(JEFFREYS and MORTON, 1987), genetic distances were estimated as the mean number of nucleotide substitutions per nucleotide site (NEI 1987, GENTZBITTEL and NICOLAS, 1990),

and dendrograms were constructed by the unweighted pair-group method (SNEATH

and SOKAL, 1973).

Results

Preparation of chicken genomic DNA from dried blood sample

The results of spectrophototic determination of genomic DNA isolated from chick-

en blood dried onto 3 MM paper are shown in Table 2.

The amount of chicken genomic DNA prepared from blood samples preserved for

one to three months in dry condition were 1.52•}0.63 mg, ranging 0.60 to 2.46 mg.

These amounts of genomic DNA were sufficient for multiple DFP analysis in quan-

tities. These DNAs were suggested to be sufficiently pure, because the ratio of

absorbances at 260 and 280 nm (A260/A280) were ranged 1.85 to 1.99.

The agarose gel electrophoresis patterns of genomic DNA prepared from dried chicken blood sample are shown in Fig. 1.

Table 2. Recovery of genomic DNA from dried chicken blood (n = 10)

Mean•}S.D.

Recovery of genomic DNA from fresh chicken blood 12 Jpn. Poult. Sci., 34 (1)

Fig. 1. Comparison of agarose gel electrophoresis patterns of genomic DNA prepared from dried chicken blood samples preserved under dry condition. Fragment sizes (kb) are indicated on the left.

These chicken genomic DNA showed high molecular weight DNA without tailing and physical degradation when preserved under dry condition. DFP analysis of native fowls in Fiji and Western Samoa islands The DFP patterns, which were produced by Hinf I digestion of DNA from native fowls in five different islands of Fiji (Viti Levu, Vanua Levu and Taveuni) and Western Samoa (Upolu and Savai'i), are shown in Fig. 2. The BS values between respective pairs of DFP patterns intra- and inter-island are summarized in Table 3. In individual DFP analysis, the intra-island BS values of Viti Levu and Savai'i were 0.413 and 0.347, respectively. These intra-island BS values were higher than inter-island BS value (0.195). This fact indicated that intra-island individuals were generally more similar to each other than inter-island individuals. In population DFP analysis, all bands that appeared in the population DFP patterns could be traced back to the individual DFP patterns in same population. The BS values within Fijian and Western Samoan native fowls were relatively high, averaging 0.274 and 0.300, respectively. The BS values between Fijian and Western Samoan native fowls (0.186) were lower than these within Fijian and Western Samoan native fowls. The inter-island BS values from population DFP patterns were similar to those obtained in the comparisons of individual DFP patterns and population DFP patterns. In order to illustrate the similarities observed in DFP patterns for native fowls in five different islands, a phenetic classification was carried out. This done on the basis of genetic distances calculated from BS value and presented in Table 3. The dendrogram draw from the genetic distance matrices is shown in Fig. 3. Fijian native fowls in three different islands belonged to the same cluster, and Western Samoan native fowls in two different islands formed another cluster. And then, the YAMASHITA et al. : DNA Fingerprinting Analysis in Fowl 13

Fig. 2. DNA fingerprints of native fowls in Fiji and Western Samoa islands. DNAs were digested with Hinf I, and hybridization was performed with (TG)n. Vi-1-Vi-4 and Sa-1-Sa-4 are individual DNA fingerprints, and the other are population DNA fingerprints. Fragment sizes (kb) are indicated on the left. Abbreviation of breeding places as follows : Vi, Viti Levu ; Va, Vanua Levu ; Ta, Taveuni ; Up, Upolu ; Sa, Savai'i.

dendrogram was drawn from individual DFP patterns of native fowls in Viti Levu and Savai'i. This dendrogram revealed clustering of all native fowl in Viti Levu and Savai'i, respectively, and showed similar shape to the dendrogram drawn from population DFP patterns. DFP analysis of Red jungle and hybrid fowls in Fiji islands Fig. 4 shows individual DFP patterns of four Red jungle fowls and three hybrid fowls in Fiji islands (Viti Levu, Taveuni, Makogai and Koro), and population DFP patterns of Fijian native fowls in three different islands (Viti Levu, Vanua Levu and Taveuni) and Red jungle fowls in Kagoshima University. The BS values between respective pairs of DFP patterns are summarized in Table 4. In a Red jungle fowl in Taveuni, BS value for a Red jungle fowl in Koro was highest (0.475), and followed in order of two hybrid fowls in Taveuni (0.447) and a Red jungle fowls in Makogai (0.400). In two Red jungle fowls in Makogai, high BS values were found between a hybrid fowl in Viti Levu, a Red jungle fowl in Taveuni, two 14 Jpn. Poult. Sci., 34 (1)

Table 3. Band sharing (BS) values and genetic distances among native fowls in five different islands of Fiji and Western Samoa based on DNA fingerprinting analysis using (TG)n

Values of upper and lower rows are BS values and genetic distances, respectively. The mean value of the four individual DNA fingerprints

Fig. 3. Dendrogram showing genetic relationships among native fowls in Fiji and Western Samoa islands based on DNA fingerprinting analysis. Solid line dendrogram is based on population DNA fingerprinting and broken line dendrogram is based on individual DNA fingerpriting. Abbreviation of breeding places are follow ; Vi, Viti Levu ; Va, Vanua Levu ; Ta, Taveuni ; Up, Upolu ; Sa, Savai'i.

hybrid fowls in Taveuni and a Red jungle fowl in Koro, averaging 0.408, 0.400, 0.398 and 0.374, respectively. The highest BS value of a Red jungle fowl in Koro was found between a Red jungle fowl in Taveuni (0.475). In a hybrid fowl in Viti Levu, high BS values were found between two Red jungle fowls in Makogai (0.408) and native fowls in Viti Levu (0.384). In two hybrid fowls in Taveuni, BS values between a Red jungle fowl in Taveuni were the highest (0.447) and YAMASHITA et al. : DNA Fingerprinting Analysis in Fowl 15

Fig. 4. DNA fingerprints of native, hybrid and Red jungle fowls in Fiji islands produced by Hinf I digestion and hybridization with (TG)n. NF(Vi), NF(Va), NF(Ta) and JF(Ka) are population DNA fingerprints, and the other are individual DNA fingerprints. Fragment sizes (kb) are indicated on the left. Abbreviation of fowls and breeding places are as follows : NF, native fowl ; HF, hybrid fowl ; JF, Red jungle fowl ; Vi, Viti, Levu ; Va, Vanua Levu ; Ta, Taveuni ; Ma, Makogai ; Ko, Koro ; Ka, Kagoshima University. between Red jungle fowls in Kagoshima University were the lowest (0.255), and high BS values were found between native fowls in Taveuni (0.396). The matrices of genetic distances between respective pairs of DFP patterns were calculated from BS values and presented in Table 4, and the dendrogram draw from the genetic distance matrices is shown in Fig. 5. Red jungle fowls in Koro and Taveuni, and hybrid and native fowls in Taveuni belonged to the same cluster. And Red jungle fowls in Makogai and hybrid fowl in Viti Levu formed another cluster. On the other hand, Red jungle fowls in Kagoshima University were relatively remote to Red jungle fowls in Fiji.

Discussion

DFP analysis is useful technique for population genetics (KUHNLEINet al., 1989, 1990 ; DUNNINGTONet al., 1990, 1991 ; HABERFELDet al., 1992 ; YAMASHITAet al., 1994). In the present study, chicken genomic DNA was prepared from dried blood sample, and 16 Jpn. Poult. Sci., 34 (1)

Table 4. Band sharing (BS) values and genetic distances among native, hybrid

Values of upper and lower rows are BS values and genetic distances, respectively. NF, native fowl ; HF, hybrid fowl ; JF, Red jungle fowl ; Vi, Viti Levu ; Va, Vanua Levu ;

the genetic relationships among native and Red jungle fowls in the South Pacific Islands were analyzed by using BS value obtained from individual and population DFP. Chicken blood could be stored for up to 3 months in dry condition, and was still available for the isolation of sufficiently pure genomic DNA. Agarose gel electrophoresis patterns of the isolated genomic DNA showed high molecular weight without tailing and physical degradation when preserved under dry condition. These DNAs were obtained in sufficient quantities for multiple DFP analyses. It seems that nuclear of red blood cell, which is usually used for preparation of genomic DNA in chicken, is protected with tough nuclear membrane. These results indicated that blood sample for DNA preparation could be transported from widespread area, and used for molecular biological analyses in chicken and other bird species. The studies on the genetic relationships among the breeds or species of fowl have been done ecologically, morphologically (NISHIDAet al., 1985 a,1985 b) and biochemically (TANABEand MIZUTANI,1980 ; HASHIGUCHIet al., 1981 ; OKADAet al., 1984 ; MAEDAet al., YAMASHITA et al. : DNA Fingerprinting Analysis in Fowl 17 and Red jungle fowls in Fiji based on DNA fingerprinting analysis using (TG)n

Ta, Taveuni ; Ma, Makogai ; Ko, Koro ; Ka, Kagoshima University

1992). The present study revealed the followings, (1) From the BS value and dendrogram, it was obvious that data of population DFP were much the same as collected data from individual DFP. (2) The native fowls in five different islands of Fiji and Western Samoa could be distinguished in each island, and the difference was consistent with geographical distribution. (3) Fijian and Western Samoan native fowls were found to form two separate groups, respectively. (4) Red jungle fowls in Taveuni and Koro belonged to the same cluster, and Red jungle fowls in Makogai formed another cluster. And a Red jungle fowl in Koro was genetically closer to a Red jungle fowl in Taveuni, even though Koro was geographically closer to Makogai. (5) Red jungle fowls in Fiji islands were more closely related to each other, but were found to be related remotely to Red jungle fowls in Kagoshima University. (6) In Taveuni, hybrid fowl were genetically closer to Red jungle fowl than native fowl. It seems that comparing genetic variability of closely related populations and reconstructing their phylogenetic relationships have been difficult in a population- level. Most of the genetic marker, such as protein polymorphism, evolve at such slow 18 Jpn. Poult. Sc., 34 (1)

Fig. 5. Dendrogram showing genetic relationships among native, hybrid and Red jungle fowls in Fiji islands based on DNA fingerprinting analysis. NF(Vi), NF(Va), NF(Ta) and JF(Ka) are based on population DNA fingerprints, and the other are based on individual DNA fingerprints. Abbreviation of fowls and breeding places are as fowllows : NF, native fowl ; HF, hybrid fowl ; JF, Red jungle fowl ; Vi, Viti Levu ; Va, Vanua Levu ; Ta, Taveuni ; Ma, Makogai ; Ko, Koro ; Ka, Kagoshima University.

rates that closely related populations are often too similar to discriminate. Mitochondrial DNA marker can be used effectively to reconstruct the phylogenetic history of population, because mitochondrial genome evolves 5 to 10 times faster than typical nuclear gene in animal. However, mitochondrial DNA polymorphisms do not provide information about the extent of nuclear gene flow or variability. The num- bers of fowls tested in the present study were limited, but these results indicate that DFP analysis can be used as a quick and cheap technique of estimating the relative genomic variability and reconstructing the phylogeny of small closely related, but isolated, populations.

Acknowledgments

The authors wish to heartily thank Dr. J.N. VAKABUA,Mr. S. DEBALEVUand the member of Animal Health and Production Division, Ministry of Primary Industry, Fiji, Dr. K. LAMETA,Deputy Director and Mr. P. TAAVILIand Dr. F.M. FATA, senior Health Officer, Department of Agriculture, Western Samoa for their kind arrangements and cooperations in the field investigations. This work is supported by Grant-in-Aid for Nihon University International Scien- tific Program (Research Title : Anthropological study on food intake and oral morphol- ogy of Polynesians in specialized oceanic environment, Head Investigator : Dr. E. KANAZAWA,Professor of Anatomy, School of dentistry at Matsudo, Nihon University).

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フィジーならびに西サモア諸島における在来鶏および

赤色野鶏のDNAフィンガープリント分析

山下秀次1)・西田隆雄2)・恒川直樹2)・P. MANUELI3) 岡本新1)・前田芳實1)・橋口勉1)

鹿児島大学農学部,鹿児島市890 日本大学農獣医学部,藤沢市252 フィジー第1次産業省,フィジー

DNAフィンガープリント(DFP)を遺伝指標として, ulation DFPのデータはindividual DFPのデータを集フィジーならびに西サモア諸島における在来鶏および赤積したものであることが確認された。2)フィジーならび色野鶏の遺伝的類縁関係について分析を行った。材料にに西サモア諸島の5島嶼の在来鶏においては,各在来鶏

は,フィジー諸島の5島嶼(Viti Levu, Vanua Levu, はそれぞれの島嶼に区分され,フィジー在来鶏と西サモ

Taveuni, Makogai,およびKoro)ならびに西サモア諸ア在来鶏はデンドログラムにおいてそれぞれ独立したク

島の2島嶼(UpoluおよびSavai'i)において採取したラスターを形成し,遺伝的隔たりが存在していることが

合計37羽の鶏の血液を用いた。核DNAは濾紙に吸示唆された。3)フィジー諸島の赤色野鶏においては,

着・乾燥させた血液より抽出・精製し,制限酵素Hinf I Makogai島の赤色野鶏と,Taveuni島およびKoro島で消化した。DFP分析は,プローブに(TG)nを用い, の赤色野鶏はデンドログラムにおいてそれぞれ異なるク individual DFPおよびpopulation DFPを併用してラスターに属した。さらに,これらの赤色野鶏は鹿児島

行った。DFPの遺伝的類似性の評価には,BS値を算出大学の赤色野鶏とは多少離れた遺伝的類縁関係にあるもし,遺伝的距離の推定にはBS値より算出したサイトあのと考えられた。4) Taveuni島の交雑鶏については,遺たりの塩基置換数を用い,非加重結合法によってデンド伝的にわずかではあるが在来鶏に比べて赤色野鶏サイドログラムを作成した。に近いことが示唆された。

本研究において乾燥した血液から核DNAを抽出・精 (家禽会誌,34:9-20,1997)

製したところ,DFP分析に十分な収量と純度の高分子キーワード:在来鶏,赤色野鶏,DNAフィンガープリンのDNAが精製された.また,DFP分析の結果,1)pop- ト,遺伝的類縁関係,遺伝的距離