The Genetic Variability and Relationships of Japanese and Foreign Chickens Assessed by Microsatellite DNA Profiling

This is the first study in which genetic variability and relationships of a large number of Japanese chicken breeds were revealed along with those of several foreign breeds by using microsatellite DNA polymorphisms. Twenty-eight breeds (34 populations) of native Japanese chickens and seven foreign breeds or varieties were analyzed. The mean number of alleles per locus, the proportion of the polymorphic loci, and the expected average heterozygosity ranged from 1.75 to 4.70, from 0.55 to 1.00, and from 0.21 to 0.67, respectively. Microsatellite alleles being unique to a particular population were detected in some populations. The DA genetic distance between populations was obtained from allele frequency for every pair of the populations to construct a neighbor-joining tree. According to the phylogenetic tree, excluding a few exceptions, native Japanese chicken breeds and foreign breeds were clearly separated from each other. Furthermore, the tree topology divided native Japanese chickens into four main classes, which was almost in accordance with the classification based on body morphology; that is, (1) Cochin type, (2) Malay type, (3) layer type, and (4) intermediate type between Malay and layer types. This is the first finding for native Japanese chickens. (


INTRODUCTION
In Japan, there are approximately 50 breeds of native chickens (Tsudzuki, 2003).Most of today's Japanese chicken breeds are thought to have been mainly established from three original breeds, Jidori, Shoukoku, and Oh-Shamo during Japan's period of isolation (1635-1854) (Oana, 1951).Exactly speaking, Jidori is not a word to specify a breed, but a generic name used for various breeds whose ancestors inhabited Japan more than 2,000 years ago.It is thought that in ancient Japan there were many kinds of Jidoris throughout the country, although there are only three or a bit more breeds of Jidoris in recent Japan.The Shoukoku is thought to have been introduced into Japan from China between the 8th and 12th centuries.The Oh-Shamo is thought to have been derived from a Malay-type chicken introduced into Japan from Thailand in the early 17th century.Most Japanese chicken breeds were developed for special plumage, body shape, crowing, and cockfighting.In addition to these ornamental breeds established during Japan's isolation period, some breeds were established to produce eggs and/or meat during the late stages of the 19th century to the early stages of the 20th century.Oana (1951) first presumed the origin and lineage of native Japanese chickens based on external appearance and old literature.Thereafter, his hypotheses have become a kind of "Bible" for fanciers and researchers of native Japanese chickens.Later, to confirm the hypotheses of Oana (1951), osteometrical and somatometrical studies were carried out (Nishida et al., 1985a, b).In addition, on the basis of blood groups and/or blood protein polymorphisms, several phylogenetic and variability studies were performed (Hashiguchi et al., 1981;Okabayashi et al., 1998;Okada et al., 1980Okada et al., , 1984Okada et al., , 1989;;Tanabe and Mizutani, 1980;Tanabe et al., 1991).However, the genetic relationships of native Japanese chicken breeds resulted from these studies were not always in concordance with those from the morphological and literature studies.So, the genetic relationships among native Japanese chicken breeds are still unclear.
Microsatellites show simple repeat sequence polymorphisms (Tautz, 1989).Microsatellites alleles differ in the number of repetitive di-, tri-, or tetra-nucleotide units, and this length variation is detected with polymerase chain reaction (PCR) by using pairs of primers.Recently, microsatellites have become the preferred type of genetic markers because of their abundance, ease of identification, random distribution, codominant inheritance, high variability, and possibility of automated detection (Milligan et al., 1994;Goldstein and Pollock, 1997;Petit et al., 1997;Paszek et al., 1998).Microsatellite markers have been proven to be useful for assessing genetic variation and diversity in livestock (Buchanan et al., 1994;MacHugh et al., 1994;Martinez et al., 2000;Pandey et al., 2002;Dorji et al., 2003).A large number of chicken microsatellite markers are currently being developed (Groenen et al., 2000;Schmid et al., 2000) and have been employed to evaluate genetic diversity of chicken populations (Crooijmans et al., 1996;Ponsuksili et al., 1996;Vanhala et al., 1998;van Marle-Köster and Nel, 2000;Emara et al., 2002;Hilell et al., 2003).Recently, we studied genetic relationships of native Japanese chickens based on microsatellite DNA polymorphisms (Osman et al., 2004(Osman et al., , 2005(Osman et al., , 2006)).However, these studies were conducted on a small number of breeds in each work.
The aim of this study is to synthetically investigate genetic relationships between a large number of native Japanese breeds of chickens, along with other imported commercial breeds present in Japan.
Based on chicken morphology and old literature, Oana (1951) discussed the origin and lineage of native Japanese chicken breeds in his book.Among the Japanese breeds used in this study, GJI, MJI, and TJI belong to the Jidori group.As described in the introductory section, the ancestors of SHO and OSM are thought to have been imported from China and Thailand, respectively.In addition to these, the ancestors of CHA and UKO are thought to have been imported from Vietnam and China (or India), respectively, around the early stages of the 17th century (Oana, 1951).KUM, MYA, NAG, and TKU are utility breeds for eggs and/or meat production that were bred, through the late stages of the 19th century to the early stages of the 20th century, by mating Japanese indigenous chickens and newly imported foreign breeds (e.g.Cochin, Black Minorca) (Mitsui, 1979).Although the historical events of ADU and EHM are unclear, other Japanese breeds (HIN, JTK, KIN, KOE, KRK, KSM, KWA, MIN, OHK, ONA, SAT, TMA, TOT, UZU, and YKD) are thought to have been established by the middle stage of the 19th century based on various kinds of crossbreeding with SHO, OSM, and various types of Jidoris, and subsequent selective propagation (Oana, 1951).

Statistical analysis
The genetic variability of each population was assessed by calculating the mean number of alleles per locus (MNA), proportion of polymorphic loci (P poly : Lewontin and Hubby, 1966), and unbiased expected heterozygosity (H e : Nei, 1978).
Genetic divergence between the populations was calculated according to D A genetic distance (Nei et al., 1983) using DISPAN computer program (Ota, internet source), based on the allele frequency of each locus and population directly calculated from the observed genotypes using the program Excel Microsatellite Toolkit (Park, internet source).Takezaki and Nei (1996) studied the efficiencies of many methods for calculating genetic distances from microsatellite DNA data, for example, D A (Nei et al., 1983), D c (Cavalli-Sforza and Edwards, 1967), D sw (Shriver et al., 1995), and (δµ)2 (Goldstein et al., 1995) distances.Their results indicated that the D A and D c distances are most efficient in obtaining the correct tree topology.Since the D A distance is the modified D c distance, we selected the D A distance in our study.
The Phylogenetic tree was constructed using neighbourjoining (NJ) clustering method (Saitou and Nei, 1987) based on the D A distance.Bootstrap resampling (n = 1,000) was performed to test the robustness of the dendrogram topologies.The tree was visualized using the TREEVIEW program (Page, internet source).

Genetic variability
The allele and genotype frequencies of 20 microsatellite loci were determined in 34 populations of 28 breeds of native Japanese chickens and seven foreign chicken populations.Genetic variability estimated for each population is summarized in Table 2.The allele size range Twenty-three populations had one or more private alleles, that is, alleles unique to a single population (Table 3).There were four private alleles in the SHO-B; three in the JTK and KWA, two in the CHA, EHM, GJI-G, KRK, MIN, MJI, NAG, and WL, and one in the KSM-K, MYA, NHR, ONA OSM-H, RC, SAT, SHO-A, UKO-B, UKO-W, TMA, and YKD-B.There were no private alleles in 18 populations (ADU, BPR, GJI-T, HNI, KIN, KOE, KSM-H, KUM, OHK, OSM-K, RIR, TJI, TKU, TOT, UZU, WC, WPR, and YKD-A).

Genetic distance
The D A genetic distance matrix estimated between every pair of populations is presented in Table 4.There were 820 possible population pairs, of which the D A values ranged from 0.103 to 0.716 with the mean value (±SD) of 0.463±0.095.The lowest (0.103) and highest (0.716) distances were estimated between OSM-H and OSM-K and between KOE and NAG populations, respectively.The D A values estimated between Japanese breed populations and each of the BPR, NHR, RC, RIR, WC, WL, and WPR ranged from 0.373 to 0.621, from 0.236 to 0.550, from 0.309 to 0.576, from 0.271 to 0.570, from 0.378 to 0.674, from 0.427 to 0.648, and from 0.271 to 0.577, respectively, with the mean values (±SD) of 0.508±0.056,0.432±0.071,0.447±0.062,0.431±0.071,0.548±0.078,0.552±0.057,and 0.453±0.071,respectively.

Phylogenetic relationships
Figure 1 visualizes the genetic relationships among breeds/populations as the NJ tree reconstructed based on the D A distance matrix (Table 4).According to the NJ tree, excluding a few exceptions, native Japanese chicken breeds and foreign breeds were clearly separated from each other (Clusters A and B).As for exceptional cases, four Japanese breeds (KRK, TMA, KUM, and TKU) were combined with six foreign breeds (WL, BRB, WPR, RIR, NHR, and RC).In addition, an American breed WC was clustered with three Japanese breed populations (HIN, UKO-B, and UKO-W).
Within native Japanese breeds, there were two major clusters: one was composed of HIN, UKO-B, and UKO-W (Cluster C), and a second consisted of 22 breeds (27 populations) (Cluster D).In Cluster C, both HIN and UKO breeds have a Cochin-type (meat-type) body, although their body size is not so large.Cluster D was divided into two subclusters: one included KOE, MJI, OSM-H, OSM-K, YKD-A, YKD-B, KIN, KSM-H, and KSM-M (Cluster E),  I).These four breeds have a larger number of tail feathers and saddle hackles, and these feathers are quite long and tend to drag on the ground, even though they have the usual layer-type body shape.

Genetic variability
Genotyping at various microsatellite loci across the genome indicated that the chicken populations examined were genetically different.The microsatellite allelic composition and frequencies differed among the 41 chicken populations, and some alleles were observed as breed/population-private alleles.Nowadays, native Japanese chickens are mated to imported breeds to produce specialized delicious meat (Japan Chicken Association, 2003), because native Japanese chicken meat is high in quality.Breed/population-private alleles may be used as a diagnostic marker to trace the origin of meat when blandchicken meat has been fraudulently transacted.
In the present study, the number of alleles per locus across all populations studied were three to 18 (Table 2).This result is generally similar to those of Crooijmans et al. (1993Crooijmans et al. ( , 1996)), Cheng et al. (1995), Ponsuksili et al. (1996), andHilell et al. (2003).While, the MNA in each breed varied from 1.75 to 4.70.This result also resembles that of van Marle-Köster and Nel (2000), in which the MNA varied from 2.30 to 4.30 in five chicken lines from South Africa (Koekoek, New Hampshire Red, Naked-Neck, Lebowa-Venda, and Ovambo) and two chicken populations each from Mozambique and Botswana.Furthermore, Emara et al. (2002) andHilell et al. (2003) also observed similar values of MNA in their studies.
In our study, the H e value ranged from 0.21 to 0.67.This result generally resembles that of van Marle-Köster and Nel (2000) mentioned above, in which the H e values ranged from 0.31 to 0.61.Also, using microsatellite markers, Vanhala et al. (1998) reported similar H e values ranging

Genetic relationships among breeds/populations
The phylogenetic dendrogram (Figure 1) well reflected origins and body morphology of chicken breeds.Japanese breeds and foreign commercial breeds were clearly separated (Clusters A and B), although some exceptions were recognized.In cluster A, Japanese breeds KUM and TKU were combined with foreign commercial breeds.However, this seems to be a reasonable result, because the KUM and TKU are utility breeds that were created from the Cochin breed.The Cochin breed also has contributed to the establishment of most of foreign commercial breeds (Roberts, 1997).In cluster A, Japanese breeds KRK and TMA were also combined with foreign commercial breeds far from almost all Japanese breeds.This result is well consistent with the report of Okabayashi et al. (1998), in which they analyzed genetic relationships of chicken breeds based on blood protein polymorphisms.In the report, they suggested the possibility that the TMA would receive the gene flow from some Chinese breed.Oana (1951) also assumed that TMA was derived from Oh-Toumaru, which was a large breed imported from China in the early stage of the 17th century.Although Oh-Toumaru has been extinct and details of this breed are unknown, there is a possibility that Oh-Toumaru was a breed genetically similar to Cochin.In this case, the phenomenon might occur that TMA is genetically close to foreign commercial breeds, which were also genetically influenced by the Cochin.However, both KRK and TMA do not have cochin-type body, but have rather layer-type body.Oana (1951) presumed that KRK and TMA were genetically close to SHO.However, from the present study, we could not obtain the evidence that SHO is genetically close to KRK and TMA.Further studies will be necessary to confirm the origin of KRK and TMA and genetic relationship between these two breeds and other Japanese breeds.
In Japanese chickens (Cluster B), UKO and HIN comprised of one cluster (Cluster C).So far, there is no report or hypothesis that UKO and HIN are genetically close.However, both of these breeds show Cochin-type body shape, which is rarely seen in Japanese fancy breeds.Thus, there might be some close breeding history between them.Additional studies will be necessary to confirm it.The ancestor of UKO is thought to have been introduced into Japan at the early stages of the 17th century (Oana, 1951).There is no literature or assumption that, from the introduction time to the present day, crossbreeding has been frequently done between the UKO and other breeds.The tree topology of Figure 1 seems to well reflect this history, because Cluster C, to which UKO belonged, was clearly distinguished from Cluster D in which many Japanese breeds were included.
Cluster E of Figure 1 was composed of so-called Shamo-group and Shamo-related breeds.Based on the external morphology, OSM, YKD, KIN, and KSM are classified as Shamo-group breeds having Malay-type body shape (Oana, 1951).KOE has a similar body shape to OSM, and is thought to be a Shamo-related breed (Oana, 1951).Oana (1951) presumed that one more ancestral breed of KOE is TMA.However, it is difficult from this study to think that TMA is an ancestral breed of KOE, because TMA showed a far distance from KOE in Figure 1.As an exceptional case, MJI was combined in Cluster E. MJI is one of Jidoris and has a layer-type body shape, greatly differing from the morphology of Shamo-group and Shamorelated breeds.It is likely that random gene drift and/or bottleneck effect may lead to this result.According to anonymous fanciers, the number of MJI is very small in recent Japan.
MIN, NAG, JTK, and SAT were combined together in Cluster G.This result supported Oana's hypothesis based on morphological observation that MIN, JTK, and SAT are genetically close (Oana, 1951).Having a pea comb, thick tail feathers, and a somewhat erect body shape are common in these breeds.On the other hand, NAG has a single comb and a Cochin-type body, greatly differing from these three breeds in external appearance.NAG was established in Aich Prefecture by crossbreeding of Cochin and some native Japanese breed (Oana, 1951).However, the name of the Japanese breed is unknown.The origin of MIN is also in Aich Prefecture.Thus, there is a possibility that the MIN breed was used to create the NAG.
In Cluster H, TJI, KWA, and MYA showed a close relationship.According to the assumption of Oana (1951), TJI contributed to the establishment of KWA, which was supported by the present study.MYA was a breed established in Kochi Prefecture by crossbreeding Black Minorca and Kamochi-dori (Sawada, 1978).The Kamochidori has been extinct and details are unknown about this breed.However, it is clear that the origin of the Kamochidori is in Kochi Prefecture.TJI has its origin also in Kochi Prefecture.Accordingly, there is a possibility that the Kamochi-dori has close genetic relationship to TJI, which might result in the somewhat close relationship between TJI and MYA.  1 for the abbreviations of breed/population names.
In Cluster H, CHA was combined with EHM.According to anonymous fanciers, EHM is not a true Jidori, but a descendant of crossbreds between CHA and some other breeds, although EHM has the word Jidori in its breed name, Ehime-Jidori.Our result supported the fanciers' view.
OHK, ONA, TOT, ADU, and SHO were combined in Cluster I as seen in Figure 1.OHK, ONA, TOT, and SHO are grace breeds whose males have a large amount of flowing tail feathers and saddle hackles.These feathers are long and frequently drag on the ground.Oana (1951) assumed based on the external appearance of these breeds that OHK, ONA, TOT, and SHO are genetically close.This has been supported by the present study at a DNA level.
According to anonymous fanciers, ADU is not a true Jidori, but a descendant of hybrids between SHO and some other breed, although ADU has the word Jidori in its breed name, Aidu-Jidori.Our result supported the fanciers ' opinion because ADU showed a close relationship to SHO.
GJI showed a somewhat close relationship to the breeds, OHK, ONA, TOT, and SHO, having thick and long tail feathers.The GJI shows usual morphology in tail feathers and saddle hackles as typically seen in the Leghorn breeds.So far, there is no report or assumption that GJI is genetically close to these long tailed breeds.Further studies will be necessary to confirm the genetic relationship between the GJI and long tailed breeds.

Figure 1 .
Figure1.Neighbour-joining tree showing the genetic relationships among 28 breeds (34 populations) of native Japanese chickens and seven foreign breeds or varieties, using D A genetic distance calculated from 20 microsatellite loci.Bootstrap values less than 25% are not shown.See Table1for the abbreviations of breed/population names.

Table 1 .
List of chicken breeds/populations used in this study Experimental Station on Animal Husbandry, Aomori Prefectural Agriculture and Forestry Research Center. 4Ibaraki Prefectural Livestock Research Center and Shizuoka Prefectural Livestock Experiment Station. 5 Livestock Research Division, Mie Prefectural Science and Technology Promotion Center.
2 Hyogo Station, National Livestock Breeding Center.

Table 2 -
i). Statistics of microsatellite variability in terms of the number of different alleles at each locus, the mean number of alleles per locus (MNA), proportion of polymorphic loci (P poly ), and mean expected heterozygosity (H e ), estimated for 41 chicken breeds
1 See Table 1 for the abbreviation. 2Number of different alleles per locus across breeds/populations.(ADL0262) to 18 (LEI0209 and MCW0301) with the MNA of 10.85.The MNA in each population ranged from 1.75 (KOE) to 4.70 (SAT).The lowest value of the P poly (0.55) was estimated in the KOE population, while 17 populations (BPR,

Table 3 .
Microsatellite alleles specific to each breed/population

Table 4 . i)
Pairwise genetic distance (D A ) estimated between 41 chicken breeds/populations

Table 4 .
ii) Pairwise genetic distance (D A ) estimated between 41 chicken breeds/populations 1 See Table1for the abbreviation.