The Origin and Genetic Diversity of Chinese Native Chicken Breeds

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Jun 8, 2002 - The Origin and Genetic Diversity of Chinese. Native Chicken Breeds. Dong Niu,1 Yan Fu,1 Jing Luo,2 Hui Ruan,3 Xu-Ping Yu,1 Gong Chen,1.
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C 2002) Biochemical Genetics, Vol. 40, Nos. 5/6, June 2002 (°

The Origin and Genetic Diversity of Chinese Native Chicken Breeds Dong Niu,1 Yan Fu,1 Jing Luo,2 Hui Ruan,3 Xu-Ping Yu,1 Gong Chen,1 and Ya-Ping Zhang2,4 Received 13 March 2001—Final 19 September 2001

The first 539 bases of mitochondrial DNA D-loop region of six Chinese native chicken breeds (Gallus gallus domesticus) were sequenced and compared to those of the red junglefowl (Gallus gallus), the gray junglefowl (Gallus sonneratii), the green junglefowl (Gallus varius) and Lafayette’s junglefowl (Gallus lafayettei) reported in GenBank, and the phylogenetic trees for the chickens were constructed based on the D-loop sequences. The results showed that the four species of the genus Gallus had great differences among each other, the G. g. domesticus was closest to the red junglefowl in Thailand and its adjacent regions, suggesting the Chinese domestic fowl probably originated from the red junglefowl in these regions. The two subspecies of Thailand, G. g. gallus and G. g. spadiceus, should belong to one subspecies because of their resemblance. In the case of native breeds, there existed a great difference between the egg breeds and general purpose breeds, which suggested different maternal origins of the two types. KEY WORDS: Chinese native chicken breeds; junglefowl; mitochondrial D-loop sequence; genetic diversity.

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Animal Science College, Zhejiang University, Hangzhou 310029, People’s Republic of China. Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, People’s Republic of China. 3 Agricultural Engineering and Food Science College, Zhejiang University, Hangzhou 310029, People’s Republic of China. 4 To whom correspondence should be addressed; e-mail: [email protected]. 163 C 2002 Plenum Publishing Corporation 0006-2928/02/0600-0163/0 °

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INTRODUCTION It is well known that the Chinese native chicken breeds are the results of human efforts. Compared with commercial breeds, these chickens have desirable properties such as suitability to surroundings, high disease resistance, good reproductive capacity, and fine meat quality, etc. But now the native chicken breeds are becoming endangered or even extinct because of their poorer commercial performances, e.g. slow growth and low efficiency of feed utilization, compared with commerical breeds. Thus there has been a great reduction of chicken genetic diversity. These breeds form a gene pool which can be utilized to culture new, better breeds by various means. It is urgent now to investigate and estimate the genetic diversity of native breeds, which would not only be beneficial to the livestock industry but also contribute to world animal protection. Classical approaches to genetic analysis of the structure and origin of native chicken breeds are based on cytogenetics and morphological studies (Cheng et al., 1996; Li et al., 1996; Liu and Cheng, 1997; Ma and Lu, 1993). More recently studies on DNA divergence are becoming more attractive in population genetic analysis. Mitochondrial DNA (mtDNA) is an available molecular tool for investigating evolutionary relationships and genetic variations within and between species because of its maternal inheritance and more rapid variability than nuclear DNA (Avise et al., 1987; Zhang and Shi, 1992, 1993a,b). It has been demonstrated that the mtDNA RFLPs (restriction fragment length polymorphism) of junglefowl have more extensive polymorphism than those of domestic fowl, which suggests the domestic fowl has a single and recent ancestor ( Glaus et al., 1980; Wakana et al., 1986; Wang et al., 1994). However, sequencing a specific fragment of mtDNA gives more accurate information on evolution and genetic diversity (Akishinonomiya et al., 1994, 1996). The D-loop region does not encode protein and evolves much faster than other regions of the mtDNA genome, so it is the most valuable and sensitive region in population genetic analysis, especially suited for genetic variation studies within species. In this study, we sequenced the first 539 bases of the mtDNA D-loop region of five Chinese native chicken breeds and the White Leghorn chicken breed, compared the sequences to those of the red junglefowl, the gray junglefowl, the green junglefowl, and Lafayette’s junglefowl reported in GenBank, and constructed a phylogenetic tree for these birds. The results demonstrate the origin, classification, and genetic differentiation of the Chinese domestic fowl; this may be beneficial for the protection and development of native chicken breeds.

MATERIALS AND METHODS Twenty-five samples of five native chicken breeds (five specimens per breed) were collected from five districts in China. The five breeds are Xiaoshan, Xianju,

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Lingkun, Baiyiner, and Wugu, taken from the Xiaoshan district, Xianju district, Lingkun island, Jiangshan district of Zhejiang province, and the Shangrao district of Jiangxi province, respectively. Five specimens of the foreign breed, White Leghorn, served as the control. Total genomic DNA was isolated from the heart tissue of 30 specimens of six breeds according to the procedure adapted by Zhang and Ryder (1993). The conserved primer pair, L16750 (50 -AGGACTACGGCTTGAAAAGC-30 ) and H547 (50 -ATGTGCCTGACCGAGGAACC-AG-30 ), was used to amplify the first 539base segment of the D-loop region of the 30 birds. L and H refer to the light and heavy chains and the number designates the position of the 3’ end of the primer in the reference sequence (Desjardins and Morais, 1990). PCR amplification was performed in a 50 µL reaction volume using 1.5 units of Pfu DNA Polymerase, a high fidelity DNA polymerase (Sangon Co.). The PCR cycle profile was 94◦ C for 2 min before the first cycle, then 94◦ C for 1 min, 63◦ C for 1 min, and 72◦ C for 1 min for 35 cycles. After the last cycle, the PCR mixture was incubated for a further 5 min at 72◦ C. The D-loop fragment of the same bird was amplified twice to determine the error in the PCR. The PCR product was purified by a DNA gel extraction kit (Watson Biotech. Co.) and directly sequenced by the dideoxy method (Sanger, 1977), with sequenase 2.0 (United States Biochemical), using heat denaturation. The original PCR primers could be used as sequencing primers. Our procedure was according to the protocol provided by the manufacturer as follows: 2–2.5 µL of gel-purified DNA was mixed with 1 µL of sequencing primer (10 µmol/L) of L or H chain of the D-loop region, then heated it at 92◦ C for 2 min, followed by rapid cooling on ice; 2 µL of solution of the sequencing kit (containing the high fidelity DNA polymerase, buffer, dNTP and ddNTP labeled with fluorescein isothiocyanate) was added to the mixture, followed by deionized water up to the total volume of 10 µL; then the sequencing reaction was carried out. Products of these reactions were purified and electrophoresed, and the sequence was read by a DNA sequencer 377 (PE Co.). Both the L and H chains of the two D-loop PCR products of the same bird were sequenced respectively and checked by eye to ensure the correctness of the sequence. We analyzed the sequences using the PC/GENE 6.6 program software and constructed the phylogenetic trees for these birds on the basis of the D-loop sequences. RESULTS AND DISCUSSION Origin of Chinese Domestic Fowl The genus Gallus is composed of four wild species, G. gallus (red junglefowl), G. varius (green junglefowl), G. sonneratii (gray junglefowl), and G. lafayettei (Lafayette’s junglefowl). The G. gallus species includes five subspecies: G. g. gallus, G. g. spadiceus, G. g. bankiva, G. g. marghi, and G. g. jabouillei. The

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former two inhabit Thailand and its immediate surroundings, and are named as the continental population; G. g. bankiva inhabits the Indonesian islands of Java, and is considered an island population (Akishinonomiya et al., 1996). In this study, we chose the first three subspecies (G. g. gallus, G. g. spadiceus, and G. g. bankiva) of G. gallus to do the sequence analysis; for the last two the sequence reports are not in the GenBank, and their classification status remain uncertain. The 31 nucleotide sequences of four wild species (G. gallus, G. varius, G. sonneratii, and G. lafayettei), the Japanese quail (Coturnix coturnix japonica), and the Chinese quail (Coturnix coturnix chinensis) from the GenBank and the six nucleotide sequences of domestic fowl sequenced in this study were aligned (shown in Fig. 1), and 115 variation sites, i.e., 28% sequence divergence, were detected. There are 32 haplotypes in the four species of genus Gallus studied, which indicates there exist great differences among the species and their separation from each other occurred much earlier than supposed. The results also showed considerable variation within the species of green junglefowl and red junglefowl (G. g. gallus and G. g. bankiva), suggesting there is great genetic diversity within these species. Among the species of the genus Gallus, the tandem duplication of one 60-base unit containing a nearly invariant tetradecamer, AACTATGAATGGTT, is found in this study (shown in Fig. 1), and the copy number of the unit varies in different species. For G. varius, two copies were found in two individuals, whereas three or four copies were found in the remaining three. All specimens of G. lafayettei and G. sonneratii have three copies. All subspecies of G. gallus have two copies, of particular interest is the fact that all the domestic fowl have the same copy number as G. gallus, implying that the domestic fowl likely originated from G. gallus alone, whereas this 60-bp unit is present as a single unit in Japanese and Chinese quail. These observations on copy number of the 60-bp unit are in close agreement with the reports of Akishinonomiya et al. (1994, 1996), who also found there existed only one 60-bp unit in various pheasants of the genus Phasianus, suggesting tandem duplication of the 60-base unit within the control region as a genus-specific trait of Gallus. It is most interesting that for the species with two copies of the 60-bp unit, there exist large differences between the first and second copy in the same individual; for the species with three or four copies, only the last copy is very different from the others. G. sonneratii is a notable exception, in which sequence differences among the three copies appear to be great. The above data indicate that the initial duplication producing the rather different copy likely occurred a long time ago, probably antedating the separation of the four species from a common stem. It is likely that there existed two copies before the formation of four species of genus Gallus. A great variation among the three copies of G. sonneratii implies that the split of G. sonneratii from the common stem probably took place earlier than the other three species, G. gallus, G. lafayettei and G. varius, which likely branched out from the stem almost simultaneously and differentiated in different directions.

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Fig. 1. The tandem duplication of 60-base unit within the D-loop region of the domestic fowl and four wild species of genus Gallus. The invariant 14-base sequence in the center of each 60-base unit is underlined.

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We grouped the sequences of the same breed or species together, constructing an unrooted neighbor joining (NJ) population tree shown in Fig. 2. Among the domestic fowl, the egg breeds (Xianju and Leghorn) belonged to the same cluster as the continental populations of G. g. gallus and G. g. spadiceus in Thailand and its adjacent areas, while the general purpose breeds (Xiaoshan, Lingkun, Baiyiner, and Wugu) formed a separate cluster. The two subspecies of Thailand, G. g. gallus and G. g. spadiceus, should belong to one subspecies because of their similarity; the subspecies status of G. g. spadiceus might be questioned, but the other subspecies, G. g. bankiva, an island population, forming a separate cluster, is very different from the continental populations; thus the subspecies status given to G. g. bankiva is reasonable. From the phylogenetic tree (Fig. 2), we can draw the conclusion that the domestic fowl probably originated from a single domestication event in Thailand and its neighbor regions, which is consistent with the notions of Akishinonomiya et al. (1994, 1996). In their study, the domestic fowl from the Indonesian islands have large genetic differences compared with G. g. bankiva from the same place; in sharp contrast, all Thailand junglefowls are very close to these Indonesian domestic fowls, which clearly excludes the involvement of G. g. bankiva in the domestication event, further indicating that the red junglefowl of Thailand may suffice as the matriarchic ancestor of all domestic fowl.

Fig. 2. Neighbor joining population tree of the fowl species (on the basis of the D-loop sequence, quail was chosen as the outgroup).

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It can also be inferred from Fig. 2 that there exists a closer relationship between the egg breeds (Xianju and Leghorn) and Thailand red junglefowl (G. g. gallus and G. g. spadiceus), and that these egg breeds still retain a genetic similarity to red junglefowl, while the general purpose breeds (Xiaoshan, Lingkun, Baiyiner, and Wugu) chosen as meat sources are considerably different from their ancestor the same red junglefowl. In the study of Cheng et al. (1996), it was found that Chinese domestic fowl had a closer relationship to Chinese red junglefowl than to Thailand red junglefowl and Nepal red junglefowl, which suggests that the Chinese domestic fowl probably originated from Chinese red junglefowl. That the Chinese domestic fowl has, according to their study, the closest relationship with Indonesian red junglefowl, G. g. bankiva, is different from our and Akishinonomiya’s conclusions. Because the sequence of the Chinese red junglefowl has not been reported in GenBank and the samples were not collected in this study, whether the Chinese red junglefowl belongs to the same subspecies as Thailand red junglefowl still remains an open question. It is also indicated from Fig. 2 that the Chinese native chicken breeds are the next of kin to red junglefowl in Thailand and its adjacent regions, and close to the red junglefowl in Indonesia, Lafayette’s junglefowl, gray junglefowl, green junglefowl, and Chinese and Japanese quails. Our finding places the original site of domestication of the chicken in Thailand and its neighboring areas inhabited by the subspecies of G. g. gallus and G. g. spadiceus. The reason for the domestication of the chicken is commonly thought to be its use as a food source in the forms of egg and meat, the cock as a harbinger of the rising sun, cockfighting as a divine offering, as well as the religious significance attached to chicken. For these reasons, domestic fowl are likely to have been distributed from their domestication site, and thus the diverse native chicken breeds were formed. Genetic Diversity of Chinese Native Chicken Breeds The first 539 bases of the mtDNA D-loop region of twenty-five Chinese native chicken and five White Leghorn chicken samples were sequenced and aligned with each other (shown in Fig. 3). The results indicate that there are 23 and 1 sites, respectively, characterized by transitions and transversions, i.e. 4.45% sequence divergence, is detected among the 30 DNA sequences, indicating a relatively low variability in the native chicken breeds. It might be interpreted that chicken mtDNA has gone through an evolutionary bottleneck during the course of domestication. There are 13 haplotypes in the native breeds studied, of which four are found in Xianju and Xiaoshan chicken breeds, respectively, which suggests the two breeds have a great variation within breeds. That most of Baiyiner and Lingkun chickens belong to the same haplotype indicates a very close relationship between the two

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Fig. 3. The alignment of mitochondrial D-loop sequences of the Chinese native chicken breeds. Dots indicate identity with the sequence in the first row. Small letters indicate the variation sites in the sequence. Chicken breeds: A, Xianju; B, Baiyiner; H, Leghorn; L, Lingkun; W, Wugu; and X, Xiaoshan. The 14-base core sequence of each 60-base unit is underlined.

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Fig. 3. (Continued )

breeds. The fact that the five Leghorn individuals belong to the same haplotype suggests there exists a genetic similarity within the Leghorn breed. The red junglefowl is chosen as the outgroup for rooting the phylogenetic tree of the native breeds based on the D-loop sequences (shown in Fig. 4). The phylogenetic tree demonstrates that the six breeds belong to two different maternal lineages, one including Xianju and White Leghorn chicken, and the

Fig. 4. Phylogenetic tree of the native chicken breeds (on the basis of the D-loop sequence, the red junglefowl was chosen as the outgroup). Chicken breeds: A, Xianju; B, Baiyiner; H, Leghorn; L, Lingkun; W, Wugu; X, Xiaoshan; and GL, red junglefowl.

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other including Lingkun, Baiyiner, Wugu, and Xiaoshan chicken. Among the latter lineage, Lingkun chicken has a closer relationship to Baiyiner than to the Wugu and Xiaoshan chicken breeds. Considering appearances, breed-forming histories (Baiyiner chicken has a long history, and Lingkun chicken is a new breed formed in 1950s), and DNA sequence similarity comprehensively, Lingkun chicken most likely originated maternally from Baiyiner chicken. In the native chicken breeds studied, there are some exceptional individuals that are different from others of the same breed (e.g., A3, L2, X5, B2, B3, W1, X3). It can be interpreted that another maternal ancestor was involved during the course of breed formation. The NJ population tree for the native breeds is shown in Fig. 5, the relationship of the chicken breeds is in close agreement with that of the above individual tree (Fig. 4). In summary, our results indicate that (1) Chinese domestic fowl most likely originated maternally from the red junglefowl in Thailand and its adjacent areas;

Fig. 5. Neighbor joining population tree of the native chicken breeds (on the basis of the D-loop sequence).

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(2) the two subspecies from Thailand, G. g. gallus and G. g. spadiceus, should belong to one subspecies; (3) the genetic diversity of Chinese native chicken breeds is relatively low; (4) the egg breeds and general purpose breeds in this study have different matriarchic origins; (5) the egg breeds in this study are genetically the same as the red junglefowl in Thailand; (6) the Lingkun chicken most likely originated maternally from the Baiyiner chicken.

ACKNOWLEDGMENT This work was supported by Zhejiang Provincial Natural Science Foundation of China (NO. 300249).

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