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GENETICS |

* Laboratory of Animal Breeding and Genetics, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan; and
Tohoku National Fisheries Research Institute, Shiogama, Miyagi 985-0001, Japan
1 Corresponding author: tsudzuki{at}hiroshima-u.ac.jp
| ABSTRACT |
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Key Words: genetic diversity genetic relationship Japanese long-tailed chicken breed microsatellite marker Shoukoku
| INTRODUCTION |
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Native Japanese chicken breeds were mainly improved as ornamental breeds before the opening of Meiji era (A.D. 1868). About 30 unique and artistic breeds were established up to the time (Mitsui, 1979; Tsudzuki, 2003). Seventeen types of them have been designated as "natural monuments" by the Japanese Government (Kuroda et al., 1987). Today, most of these breeds are conserved by fanciers on a small scale, and some breeds face the brink of extinction.
Oana (1951) studied relationships among native Japanese chicken breeds based on ancient documents, pictures, and morphological characters such as body shape, comb, and tail structure. In his report, native Japanese breeds, except for some breeds, were largely classified into 3 types according to their characters; that is, Jidori (similar to Leghorn body shape), Shoukoku (having a larger number of tail feathers than usual), and Shamo (having an erect body shape). Also, he believed that the Shoukoku was one of the oldest breeds, and its ancestor had been introduced into Japan from China in the Heian era (A.D. 794 to 1192) and that several breeds were thereafter generated based on the Shoukoku. They are the Koeyoshi, Kurokashiwa, Minohiki, Ohiki, Onagadori, Satsumadori, Toumaru, and Toutenkou (Figure 1
). Among them, Minohiki, Ohiki, Onagadori, and Toutenkou have long and thick tail feathers and saddle hackles as in Shoukoku. Although Kurokashiwa has long and thick tail feathers, its saddle hackles are not as long. Koeyoshi, Satsumadori, and Toumaru have thick tail feathers; however, these tail feathers are not as long as that of Shoukoku. Saddle hackles are also not long in these breeds.
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The microsatellite marker is a valuable tool to evaluate genetic diversity and relationships of domestic livestock including chickens, because it shows a higher degree of polymorphisms and ease of identification than other markers, such as allozyme assay or random amplified polymorphic DNA analysis (Zhang et al., 2002a). Up to now, several studies using microsatellite markers have been conducted to evaluate genetic diversity and relationships among populations of various domestic chicken breeds and jungle fowls (Vanhala et al., 1998; Wimmers et al., 2000; Romanov and Weigend, 2001; Zhang et al., 2002b; Hillel et al., 2003). Some studies have been conducted (Takahashi et al., 1998; Osman et al., 2004, 2005, 2006) for native Japanese chicken breeds as well. However, these studies were generally performed from a relatively small number of microsatellite loci and individuals per breed or population. Takezaki and Nei (1996) suggested that at least 30 microsatellite markers should be used to obtain exact genetic information about phylogenetic relationships in a case of a lower level of diversity among populations or species. Furthermore, FAO has recommended that 25 or more individuals should be used in this kind of study (FAO, 1998). In the present study, genetic diversity was measured and relationships evaluated for 9 native Japanese chicken breeds, which have Shoukokutype tail morphology, and 2 commercial breeds from a larger number of microsatellite loci and individuals per breed than earlier studies.
| MATERIALS AND METHODS |
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PCR Amplification and Microsatellite Genotyping
Forty microsatellite markers (Table 2
) were chosen from the US Poultry Genome Project Web site (http://poultry.-mph.msu.edu/) and the ArkDB database Web site by the Roslin Bioinformatics Group (http://www.thearkdb.org/), among which 14 markers were the recommended markers by a joint International Society of Animal Genetics-FAO working group for biodiversity study (http://dad.fao.org/en/Home.htm). The 40 microsatellite markers covered 23 linkage groups.
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The DNA fragments produced by the PCR amplification were electrophoresed with a size standard Genescan-350 TAMRA (Applied Biosystems), using an ABI 310 automated DNA sequencer (Applied Biosystems). Fragment length was determined with GeneScan software version 3.1 (Applied Biosystems), and microsatellite genotypes were assigned to the birds using Genotyper software version 2.5 (Applied Biosystems).
Statistical Analysis
The genetic diversity of each breed was assessed by calculating the number of alleles per locus and its mean (MNA), observed heterozygosity (HO), unbiased expected heterozygosity (HE; Nei, 1987), and polymorphic information content (PIC; Botstein et al., 1980), using the CERVUS version 2.0 software package (Marshall et al., 1998). Additionally, F-statistics [fixation coefficient of an individual within a subpopulation (FIS), fixation coefficient of an individual within the total population (FIT), and fixation coefficient of a subpopulation within the total population (FST)] per locus across 9 native Japanese breeds (Weir and Cockerham, 1984) were calculated using the GENEPOP version 3.4 program (Raymond and Rousset, 1995).
Genetic distances among 11 breeds were evaluated by modified Cavalli-Sforza chord distance (DA; Nei et al., 1983). A phylogenetic tree was constructed based on the DA genetic distance by using the neighbor-joining (NJ) method (Saitou and Nei, 1987). The robustness of tree topologies was evaluated with a bootstrap test of 1,000 resampling across loci. These processes were conducted using the DISPAN computer program (Ota, 1993). The phylogenetic tree was edited using TreeView program (Page, 1996).
| RESULTS |
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Table 3
shows measures of genetic diversity in 11 chicken breeds for the 40 microsatellite markers, such as MNA, HO, HE, and PIC. The MNA ranged from 2.60 (Minohiki) to 4.07 (Shoukoku). The HO and HE ranged from 0.273 (Koeyoshi) to 0.523 (Shoukoku) and from 0.293 (Koeyoshi) to 0.545 (Satsumadori), respectively. The PIC ranged from 0.250 (Koeyoshi) to 0.478 (Satsumadori). In particular, Koeyoshi exhibited a lower degree of genetic diversity than all the other breeds in all measures of genetic diversity (MNA = 2.75, HO = 0.273, HE = 0.293, and PIC = 0.250). Conversely, a high degree of diversity was observed in Satsumadori and Shoukoku. (Satsumadori: MNA = 3.90, HO = 0.496, HE = 0.545, PIC = 0.478; Shoukoku: MNA = 4.07, HO = 0.523, HE = 0.527, and PIC = 0.464).
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| DISCUSSION |
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In native Japanese long-tailed chicken breeds, the lowest diversity was observed in Koeyoshi (MNA = 2.75, HO = 0.273, HE = 0.293, PIC = 0.250). Among the measures, heterozygosity was extremely lower than in the other breeds. This result seems to reflect the present breeding state of Koeyoshi, in which inbreeding is generally carried out among closely related birds in narrow geographical areas in north Japan to improve the long crowing trait. Low genetic diversity of Koeyoshi has also been reported by Osman et al. (2004) using a set of 20 microsatellite loci and 24 birds, in which MNA and HE were 1.65 and 0.206, respectively.
In contrast, high diversity was observed in Satsumadori (MNA = 3.90, HO = 0.496, HE = 0.545, PIC = 0.478) and Shoukoku (MNA = 4.07, HO = 0.523, HE = 0.527, PIC = 0.464). Today, Satsumadori and Shoukoku are kept by fanciers as ornamental breeds in large areas of Japan, which leads to breeding from many individuals. This condition seems to result in a higher degree of diversity than other breeds. Osman et al. (2004) also reported that Satsumadori showed a high degree of diversity (MNA = 4.70 and HE = 0.670).
The MNA in White Leghorn and White Plymouth Rock was not as high when compared with native Japanese long-tailed breeds that are generally reared in a small-size population. The HO:HE in these commercial breeds was apparently larger than that of the Japanese breeds. These results suggested that the population size was relatively small at the starting point of these 2 commercial breed populations; thereafter, sufficient random matings were performed for the breeds with a large population size.
The HO and HE observed in native Japanese chicken breeds in the present study were similar to or slightly lower than those reported for European or American breeds or lines (Vanhala et al., 1998). On the other hand, native Chinese breeds possess higher HO. According to Zhang et al. (2002b), HO ranged from 0.707 to 0.861. It was believed that the population size was large in each Chinese breed, and no intensive control has been performed for these Chinese breeds.
Genetic Relationships
In a NJ tree, Shoukoku, Ohiki, Onagadori, and Toutenkou showed a close relationship. This result supports the supposition of Oana (1951) based on literature and morphological studies. Osman et al. (2005) also found the same result from 20 microsatellite loci analyses.
Kurokashiwa and Toumaru showed no close relationship to each other in the present study. On the contrary, Osman et al. (2006) reported a relatively close genetic relationship between the 2 breeds. Although Kurokashiwa samples used in the present study were partly the same as those used by Osman et al. (2006), the Toumaru samples were collected from a different population. There is a possibility that some genetic differentiation occurred among different Toumaru populations. Furthermore, studies will be necessary to reveal a genetic relationship between Kurokashiwa and Toumaru.
The NJ tree showed that Toumaru is genetically distant from other Japanese breeds. Also, 9 breed-specific alleles, the largest number of breed-specific alleles, were detected in Toumaru. Although breed-specific alleles were generally detected with very low frequency, 5 of the 9 breed-specific alleles detected in Toumaru showed relatively high frequency (>0.200). Takahashi et al. (1998) also reported that DA between Toumaru and other native Japanese breeds are relatively far. Oana (1951) supposed that Toumaru originated from Oh-Toumaru that had been imported from China in the 16th or 17th century and became extinct by the opening of Meiji era (A.D. 1868). There is a possibility that the breeding history of Toumaru is different from other Japanese breeds. In the present study, Koeyoshi, Toumaru, and Toutenkou did not exhibit close DA between each pair. Conversely, Komiyama et al. (2004) reported that these 3 breeds are genetically close to each other, based on the polymorphism analysis for the mitochondrial D-loop region. Further studies will be necessary in the future to reveal genetic relationships among these 3 breeds.
Oana (1951) mentioned that Kurokashiwa was a direct descendant of Shoukoku or a variety of Shoukoku. In the present study, however, Kurokashiwa showed no close relationship to Shoukoku. So far, Osman et al. (2006) also obtained the same result. Thus, the hypothesis of Oana (1951) should be rejected. It was concluded that Shoukoku and Kurokashiwa are not genetically close.
Minohiki and Satsumadori showed a close relationship. Osman et al. (2006) also reported that these 2 breeds are genetically close. Moreover, Komiyama et al. (2004) also reported a close relationship between the 2 breeds based on mitochondrial DNA polymorphisms. Thus, the hypothesis of Oana (1951), in which he supposed that these 2 breeds have a common ancestor, is thought to be supported by 2 kinds of DNA analyses (i.e., nuclear and mitochondrial DNA analyses).
In the present study, a precise study on genetic diversity and relationships was performed in and among chicken breeds from microsatellite markers and approximately 48 birds per breed. Previously, Osman et al. (2004, 2005, 2006) performed similar studies based on 20 microsatellite markers and approximately 24 birds per breed, the results of which are the same in almost all points as those of the present study. Thus, it is thought that the use of 20 microsatellite markers and approximately 24 birds might be adequate to obtain accurate results.
Evaluating genetic diversity might be useful for conservation of native Japanese chicken breeds as a genetic resource and natural monument. For instance, judging from the result in the present study, conservation of the Koeyoshi breed should be an emergency. It was confirmed through the present study that the microsatellite is a valuable tool to evaluate genetic diversity of chicken breeds.
| ACKNOWLEDGMENTS |
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Received for publication July 21, 2006. Accepted for publication September 16, 2006.
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