9 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 substitu- tions 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 relation- ships in small populations genetically isolated. (Jpn. Poult. Sci., 34 : 9-20, 1997) Key words : native fowl, Red jungle fowl, DNA fingerprinting, genetic relationship, ge- netic 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 hypervari- able 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 hybridiza- tion 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.