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Poult Sci 2006. 85:2124-2129
© 2006 Poultry Science Association
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GENETICS

A Method for Discriminating a Japanese Chicken, the Nagoya Breed, Using Microsatellite Markers1

A. Nakamura*, K. Kino*, M. Minezawa{dagger}, K. Noda* and H. Takahashi{dagger},2,3

* Poultry Laboratory, Animal Husbandry Research Division, Aichi-ken Agricultural Research Center, Sagamine, Yazako, Nagakute, Aichi 480-1193, Japan; and {dagger} Laboratory of Animal Genetic Resources, Genebank, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan

2 Corresponding author: naoe{at}affrc.go.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Nagoya breed native to Japan is popular as a dual-purpose breed for eggs and meat. The current study describes a method to discriminate between the Nagoya breed and other breeds and commercial stocks of chicken. Four strains of the Nagoya breed established at the Aichi-ken Agricultural Research Center were analyzed using 25 microsatellite markers. In these strains, 5 of the markers (ABR0015, ABR0257, ABR0417, ABR0495, and ADL0262) had a single allele. Other chicken samples (448) of various breeds and hybrids were analyzed using the same 5 markers. None of these chicken samples had the same allele combination as the Nagoya breed strains. These 5 microsatellite markers provide a practical method to accurately discriminate the Nagoya breed from other chicken breeds.

Key Words: chicken • breed discrimination • microsatellite marker • Nagoya breed • genetic uniformity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Food traceability is defined as the ability to follow particular foods through all stages of the food chain, from production to sale (Committee on the Guidelines for Introduction of Food Traceability Systems, 2003). The establishment of traceability systems for livestock products in Japan is necessary, especially since 2001, when a cow of bovine spongiform encephalopathy disease was found for the first time in Japan. Beef produced in Japan has been managed by an individual identification number since the Beef Traceability Law was enforced in 2003 (Ministry of Agriculture, Forestry and Fisheries of Japan, 2003). Consumers can find information on beef by inputting the identification number on the meat package into a Web site (National Livestock Breeding Center of Japan, 2003). The Japanese Agricultural Standard for Disclosed Production Information of the Pork was enforced in 2004 (Ministry of Agriculture, Forestry and Fisheries of Japan, 2004). Based on this standard, pigs must be managed by both individual and lot or herd identification number. There is no law and public standard system for chicken meat traceability. However, guidelines for the introduction of egg traceability systems have been proposed (Food Marketing Research and Information Center, 2004).

To insure food traceability systems, scientific inspection is indispensable. Microsatellite markers, amplified fragment length polymorphism (AFLP) markers, and single nucleotide polymorphism markers have been used for individual identification and parentage testing in cattle (Glowatzki-Mullis et al., 1995; Usha et al., 1995; Ajmone-Marsan et al., 1997; Heyen et al., 1997; Heaton et al., 2002). It is possible to discriminate chicken individuals using these markers, however, individual management is not realistic for chicken. So, breed identification with conclusive proof is more desirable than individual identification in chicken.

The Nagoya breed, a dual-purpose breed for eggs and meat, is a popular native chicken in the Aichi Prefecture of Japan. In the early 1880s, local chicken around the city of Nagoya was crossed with the Chinese Buff Cochin. After that, offspring that had a buff color was selected. In 1905, the chicken was recognized as the first practical breed for poultry farming in Japan. After removal of the shank feathers and fixation of the gray-colored legs, the Nagoya breed was formally established in 1919. The pure-bred (Nagoya breed x Nagoya breed) has been commercialized, so the commercial chickens are produced by intrastrain mating. The Nagoya breed has 4 strains (NG1, NG2, NG3, and NG4) that were established at the Aichi-ken Agricultural Research Center (Nakamura and Noda, 2001). They are maintained at the Aichi Livestock and Poultry Breeding Center Okazaki, Aichi, Japan, which supplies parent stocks to the hatcheries using the 4 strains. Therefore, all commercial Nagoya breed chicken are derived from 4 strains. Because the taste, palatability, and texture of Nagoya breed meat are well recognized in Japan, the market price of Nagoya breed meat is much higher than that of contemporary broiler meat. However, cut-up meat of the Nagoya breed and commercial broilers cannot be easily distinguished by appearance. Therefore, a technology to discriminate between the Nagoya breed and the broiler is vital for preventing false sales and guaranteeing the quality of meat. The objective of the present study was to develop a method to discriminate between the Nagoya breed and all other chicken on the market using microsatellite DNA markers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Individuals (382) belonging to 4 strains of the Nagoya breed at the Aichi Livestock and Poultry Breeding Center, 82 NG1, 100 NG2, 100 NG3, and 100 NG4, were studied. Chicken genomic DNA for PCR amplification was extracted from blood or myocardium by the conventional phenol-chloroform extraction method or using a DNA extraction kit (SepaGene, Sanko-Junyaku, Tokyo, Japan).

Twenty-five chicken microsatellite markers including twenty ABR (ABR0015, ABR0028, ABR0046, ABR0075, ABR0223, ABR0228, ABR0257, ABR0258, ABR0297, ABR0343, ABR0378, ABR0417, ABR0419, ABR0495, ABR0506, ABR0526, ABR0617, ABR0624, ABR0634, and ABR0645; Takahashi et al., 2005), 3 MCW (MCW0080, MCW0217, and MCW0304; Crooijmans et al., 1996, 1997), 1 ADL (ADL0262; Cheng et al., 1995), and 1 LEI (LEI0066; Gibbs et al., 1997) marker were used in the current study. The PCR amplifications were performed in a 6-µL reaction volume, which included 2.5 pmol of each primer, 100 µM each deoxynucleotide triphosphate, 1.2 mM MgSO4, 0.0125 units of KOD plus DNA polymerase (KOD-201, Toyobo, Tokyo, Japan) that was isolated from Thermococcus kodakaraensis, 1x reaction buffer provided by the supplier, and 30 ng of genomic DNA in a 384-well plate on an iCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). The PCR was performed as follows: hot start 75 s at 94°C, followed by 10 cycles of 15 s at 94°C, 30 s at 60°C, and 60 s at 68°C, followed by 10 cycles with the same conditions except that the annealing temperature was 55°C; 30 cycles with an annealing temperature of 50°C; and, finally, an elongation time of 9 min at 68°C. The PCR products were run with the internal size standard GeneScan 400HD [ROX] Size Standard (Perkin-Elmer, Foster City, CA) on an ABI PRISM 3100 DNA Sequencer (Perkin-Elmer). The size of fragments was analyzed using the GeneScan (Version 3.7) and GeneMapper (Version 2.0) programs (Perkin-Elmer). Alleles were designated according to PCR product size, and allelic frequencies were calculated directly from observed genotypes.

From the allele frequencies observed, the expected homozygositity of Nagoya-type (NT) in each autosomal locus was calculated as


Formula

where pi = the allele frequency of the ith locus, which shows 1 fixed allele in the Nagoya breed. In a locus on the Z chromosome, the expected homozygositity of NT was calculated as


Formula

In crossbreds between Nagoya and a purebred, the expected homozygosity of NT in each locus was essentially equal to allele frequencies of NT homozygous alleles in each purebred. However, in a female offspring crossing between a Nagoya male and a purebred female, the expected homozygosity of NT in a locus on the Z chromosome was 1.000.

The exclusion probabilities (PE) of the Nagoya breed were calculated as


Formula

where n = the number of loci that show one fixed allele in the Nagoya breed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In 4 strains of the Nagoya breed at Aichi Livestock and Poultry Breeding Center, 5 markers (ABR0495, ABR0257, ADL0262, ABR0015, and ABR0417) showed 1 fixed allele each (Table 1Go). The commercial chickens derived from these strains must have fixed alleles at these 5 marker loci. These 5 markers were used to determine whether they could discriminate between the Nagoya breed and other chickens. Samples (448), including 7 purebreds, broiler chickens, and crossbreds between the Nagoya breed and another purebred, were analyzed using the 5 markers. Allele frequencies for all populations by locus are shown in Table 2Go. These fixed alleles in the Nagoya breed were not specific to the Nagoya breed. From the data of allele frequencies in Table 2Go, genotype frequencies showing NT homozygosity in various chicken populations and the PE of Nagoya breed were calculated (Table 3Go). The expected PE in which 7 purebreds are totaled and broiler chicken were 99.997 and 99.92%, respectively (Table 3Go). The expected PE of female individuals crossing Nagoya males and White Leghorn, Rhode Island Red, and White Plymouth Rock A females were 96.61, 90.58, and 94.10%, respectively. The observed homozygositity of the NT at each locus and the PE in each population are shown in Table 4Go. Because alleles not expected in the Nagoya breed were detected in all samples, they are not the Nagoya breed.


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  		Table 1. Microsatellite markers that showed 1 fixed allele in 4 strains of Nagoya breed
 

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  		Table 2. Allele frequencies for all populations by locus1
 

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  		Table 3. Expected genotype frequencies showing Nagoya-type homozygosity and the exclusion probabilities (PE) for all populations1 by locus
 

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  		Table 4. Observed genotype frequencies showing Nagoya- (NG) type homozygosity, the exclusion probabilities (PE) for all populations by locus, and accuracy of discriminating NG breed from other chicken breeds using 5 microsatellite markers1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The PCR-based markers random amplified polymorphic DNA, AFLP, and microsatellites are widely used for cultivar identification in autogamous plants (Garland et al., 1999; Ohtsubo et al., 2002; Shirasawa et al., 2004). For example, kits to identify the Japanese rice cultivar, Koshihikari, are commercially available (RR211A and RR213, PCR Kit I & II for Rice Identification, Takara Bio Inc., Otsu, Shiga, Japan), which provides sequence-tagged site primers derived from discriminating DNA bands of random amplified polymorphic DNA.

Breed identification in livestock is much more difficult than cultivar identification in autogamous plants, because each individual is heterozygous and each breed has genetic diversity. In swine, Okumura et al. (2000) reported that polymorphic information in MC1R and KIT genes were useful for distinguishing pig breeds that are commercially produced in Japan. Alves et al. (2002) reported the usefulness of AFLP markers to discriminate between purebred and crossbred in Iberian pigs. In cattle, markers to discriminate between the Japanese Black breed and F1 (Japanese Black x Holstein) crosses were reported (Sasazaki et al., 2004). The AFLP and 6 single nucleotide polymorphism markers absent in the Japanese Black but present in the Holstein were identified. From allelic frequencies of the 6 markers in both genetic groups, the combined probability of identifying F1 and misjudgment was estimated at 88.2 and 2%, respectively.

Genetic diversity and relationships among various populations of chicken breeds have been studied using microsatellite markers (Takahashi et al., 1998; Vanhala et al., 1998; Romanov and Weigend, 2001). These reports indicated alleles peculiar to several populations, although there were a limited number of samples per population, ranging from 12 to 24 (Takahashi et al., 1998), 12 to 31 (Vanhala et al., 1998), and 6 to 24 (Romanov and Weigend, 2001) were analyzed. Hence, a sufficient number of samples (382) belonging to 4 strains of the Nagoya breed were analyzed in the current study to discriminate the Nagoya breed from other chicken breeds. Because the commercial chickens of the Nagoya breed are originally from the 4 strains at the Aichi Livestock and Poultry Breeding Center, they must have fixed alleles for the 5 microsatellite markers (ABR0015, ABR0257, ABR0417, ABR0495, and ADL0262). The probability of mistakenly identifying the Nagoya breed using the 5 markers was 0%. The expected and observed PE of the Nagoya breed in purebreds and broiler chicken were almost 100%. In the market, it is mostly thought that hybrid chickens (i.e., broiler chickens) are falsely named the Nagoya breed. Therefore, it is a method to identify the Nagoya breed from commercial broiler stocks. However, the expected PE of female chickens developed from a cross between Nagoya males and females from the other breeds were relatively low (90.58 to 96.61%). In this case, many more markers on autosomes that have fixed alleles in the Nagoya breed may be required to improve the discrimination ability.

In conclusion, a method for discriminating the Nagoya breed from other chicken breeds using only 5 microsatellite markers has been described. Because this method is useful for discrimination between the Nagoya breed and broiler chickens on the market and can contribute to checking the validity of labeling the Nagoya breed, this is being applied to the Japanese market, and a kit will be commercially available soon.


   ACKNOWLEDGMENTS
 
We thank K. Noguchi-Takahashi for technical assistance and D. A. Vaughan for his help in preparing the manuscript in English.


   FOOTNOTES
 
1 This study was supported in part by grants from the Integrated Research Program for Functionality and Safety of Food Toward the Establishment of a Healthy Diet, a program by the Ministry of Agriculture, Forestry and Fisheries of Japan and the Ito Foundation, Tokyo, Japan. Back

3 Present address: Animal Breeding and Reproduction Research Team, National Institute of Livestock and Grassland Science, Ikenodai 2, Tsukuba 305-0901, Ibaraki, Japan. Back

Received for publication March 15, 2006. Accepted for publication July 4, 2006.


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Alves, E., C. Castellanos, C. Ovilo, L. Silio, and C. Rodriguez. 2002. Differentiation of the raw material of the Iberian pig meat industry based on the use of amplified fragment length polymorphism. Meat Sci. 61:157–162.

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