Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers

332 Science in China Series C: Life Sciences 2006 Vol.49 No.4 332—341

www.scichina.com www.springerlink.com

DOI: 10.1007/s11427-006-2001-6

Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers

QU Lujiang1, LI Xianyao1, XU Guifang2, CHEN Kuanwei3, YANG Hongjie2, ZHANG Longchao1, WU Guiqin1, HOU Zhuocheng1, XU Guiyun1 & YANG Ning1

1. College of Animal Science and Technology, China Agricultural University, Beijing 100094, China; 2. National Service for Animal Husbandry and Veterinarian, Beijing 100026, China; 3. Institute of Poultry Science, Chinese Academy of Agricultural Science, Yangzhou, Jiangsu 200331, China Correspondence should be addressed to Yang Ning (email: [email protected])

Received November 24, 2004; accepted September 25, 2005

Abstract China is rich in chicken genetic resources, and many indigenous breeds can be found throughout the country. Due to poor productive ability, some of them are threatened by the commercial varieties from domestic and foreign breeding companies. In a large-scale investigation into the current status of Chinese poultry genetic resources, 78 indigenous chicken breeds were surveyed and their blood samples collected. The genomes of these chickens were screened using microsatellite analysis. A total of 2740 individuals were genotyped for 27 microsatellite markers on 13 chromosomes. The number of alleles of the 27 markers ranged from 6 to 51 per locus with a mean of 18.74. Heterozy-gosity (H) values of the 78 chicken breeds were all more than 0.5. The average H value (0.622) and polymorphism information content (PIC, 0.573) of these breeds suggested that the Chinese indige-nous chickens possessed more genetic diversity than that reported in many other countries. The fixa-tion coefficients of subpopulations within the total population (FST) for the 27 loci varied from 0.065 (LEI0166) to 0.209 (MCW0078), with a mean of 0.106. For all detected microsatellite loci, only one (LEI0194) deviated from Hardy-Weinberg equilibrium (HWE) across all the populations. As genetic drift or non-random mating can occur in small populations, breeds kept on conservation farms such as Langshan chicken generally had lower H values, while those kept on large populations within con-servation regions possessed higher polymorphisms. The high genetic diversity in Chinese indigenous breeds is in agreement with great phenotypic variation of these breeds. Using Nei’s genetic distance and the Neighbor-Joining method, the indigenous Chinese chickens were classified into six categories that were generally consistent with their geographic distributions. The molecular information of genetic diversity will play an important role in conservation, supervision, and utilization of the chicken re-sources.

Keywords: chicken breeds, China, genetic diversity, heterozygosity, microsatellite marker.

China is regarded as one of the domestication cen-ters for chickens and archaeological studies provided evidence of chicken domestication in northern China

as early as 6000 BC[1]. At present, China has the larg-est chicken population in the world, representing a rich genetic resource. Many indigenous breeds distribute

Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers 333

extensively throughout the country. Because of China’s unique geography, complicated landform and diversified culture, each indigenous breed has devel-oped unique characteristics. These breeds vary in body size, plumage color, and other characteristics. Many of them are often maintained in small populations be-cause of their medium to low performance in egg production and growth rate. The three-yellow (yellow feather, skin and shank, 3Y) chicken is a popular food item in Southern China, while black-bone chickens, such as the Silkie, are generally regarded as highly nutritious and a tonic. Beijing fatty chickens are often referred to as “royal chickens” because of the won-derful meat flavor and beautiful appearance.

There has been much concern in recent years over the loss of biodiversity in poultry[2]. Human activities worldwide are devastating whole ecosystems and re-sulting in the extinction of large numbers of species. Within-species genetic variation is, however, just as important as between-species diversity. As in many other countries, some indigenous Chinese chicken breeds, such as Bashangchangwei chicken in Hebei Province, are on the verge of extinction because of slow growth rate and poor laying performance. Fur-thermore, many breeds have been changed as a result of hybridization with commercial varieties. The ge-netic diversity can be used as a resource for breeding at present and in the future. The genetic erosion of these indigenous breeds threatens the loss of valuable genetic variability in specific characteristics that are currently unimportant in commercial breeding pro-grams. It can be assumed that indigenous breeds con-tain genes and alleles pertinent to their adaptation to particular environments and indigenous breeding goals. Thus, indigenous breeds represent an indispensable part of the world’s chicken resources.

Recently, a large-scale investigation on the current status of Chinese poultry’s genetic resources was initi-ated. In order to develop a rational conservation strat-egy, it is of special interest to assess the genetic diver-sity among indigenous Chinese breeds. Among the modern molecular techniques, microsatellite, as a ge-netic marker, has been shown to be very suitable for the work on genetic diversity. It has been proven espe-cially useful in analysis among populations of the same species and may help resolve phylogenetic rela-

tionships between closely related populations[3―6] in-cluding chickens[6―13]. The present study utilized mi-crosatellite analysis to evaluate the genetic diversity of 78 indigenous Chinese chicken breeds.

1 Materials and methods

1.1 Chicken Breeds

A total of 78 indigenous chicken breeds throughout China were surveyed and blood samples from 30 to 62 individuals for each breed were taken for this study, with Miyi (only 14 individuals available) and Wahui (only 18 individuals) as exceptions. The numbers, names, locations and characteristics of the 78 Chinese indigenous chicken breeds are shown in Table 1.

1.2 Blood and DNA preparation

Blood samples, 3―5 mL per bird, were collected from the wing vein using ACD (0.48% citric acid, 1.32% citrate sodium, 1.47% glucose) as an anti-coagulation agent. High molecular weight DNA was isolated from 30 μL of fresh blood with the fol-lowing steps: haemolysis, proteinase K incubation, extraction with phenol, phenol/chloroform (1:1, v:v) and chloroform, ethanol precipitation and finally re-suspension in 400 μL TE.

1.3 Microsatellite genotyping

DNA samples from 2740 individuals were subjected to microsatellite genotyping. Twenty-seven primer pairs of microsatellite loci on chromosomes 1―11, 13 and 17, including 21 markers used by the AVIANDIV project[14], were used in the study (Table 2). The PCRs were carried out in 15 μL solution containing 20―50 ng genomic DNA, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-Cl, pH=8.3, 1 mmol/L tetrame-thylam-moniumchloride (TMAC), 0.1% Triton X-100, 0.01% gelation, 200 μmol/L dNTP, 0.5 Unit DNA po-lymerase and 295 nmol/L of each primer.

The amplification conditions were: 5 min denatura-tion at 95℃, followed by 35 cycles of denaturation at 94℃ for 1min, annealing at 46―60℃ for 1 min, and extension at 72℃ for 1min. The PCR products were then visualized in 6%―10% denaturating polyacryla-mide gel after being sliver stained[15]. Nine chicken

334 Science in China Series C: Life Sciences

Table 1 The numbers, names, locations and characteristics of the 78 Chinese indigenous chicken breeds Name (Na)) Location Characteristics Name (Na)) Location Characteristics Aijiao (32) Guizhoub) Long-shank Miyi (14) Sichuanb) Partridge Baier (32) Jiangxic) 3 yellow, white ears Muchuan (48) Sichuanc) Black-bone

Beijing fatty (61) Beijingc) Feather shanks Nandan (31) Guangxic) Partridge or yellow Bian (32) Shanxic) Yellow or partridge Ningdu (32) Jiangxic) 3 yellow

Chahua (31) Yunnanc) Partridge Pudong (32) Shanghaic) Yellow, red or black Congren (32) Jiangxic) Partridge Qiandongnan (31) Guizhouc) Partridge, mini-body

Dagu (62) Liaoningc) Yellow or red Qingyuan (32) Guangdongc) Partridge Dongxiang (32) Jiangxic) Black,blue eggshell Shanbei (31) Shaanxib) Various color

Emei (32) Sichuanb) Black Shimian (32) Sichuanc) Black Fujiansilky (31) Fujianc) Silky, polydactyly Shouguang (32) Shandongc) Black

Guangxi (31) Guangxic) 3 yellow Shuanglian (32) Hubeib) Partridge Gushi (47) Henanc) Various color Silky (48) Jiangxic) Silky, black-bone

Guyuan (32) Ningxiab) Yellow or partridge Taibai (32) Shaanxib) Partridge Haidong (32) Qinghaib) Various color Taoyuan (31) Hunanc) Yellow, high body Henan (48) Henanb) Game bird Tengchong (32) Yunnanc) Black-bone Hetian (32) Fujianc) Light-yellow Tibetan (32) Tibetb) Mini-type

Hongshan (32) Hubeic) 3 yellow Tulufan (48) Xinjiangb) Game bird Huaibeima (32) Anhuib) Partridge Wahui (18) Jiangxic) Partridge Huainan (32) Anhuib) 3 yellow Weining (31) Guizhoub) Partridge

Huaixiang (32) Guangdongc) 3 yellow Wenchang (32) Hainanc) Yellow or partridge Huanglang (32) Hunanc) 3 yellow Wenshang (32) Shandongb) Barred Huiyang (32) Guangdongc) Beard, mini-body Wuding (32) Yunnanb) Red and partridge,tall Luding (32) Sichuanb) Partridge Wumeng (47) Guizhoub) Black-bone

Jianghan (32) Hubeib) Multi-color Xianju (31) Zhejiangc) Yellow or black, Jiangshan (47) Zhejiangc) Black-bone Xiaoshan (32) Zhejiangc) Yellow

Jinhu (48) Fujianc) Black-bone Xiayan (32) Guangxic) 3 yellow Jining (32) Shandongb) Yellow or partridge Xinghua (31) Guangdongc) 3 yellow, mini-body

Jingning (31) Gansub) Yellow or partridge Xingwen (47) Sichuanc) Black or white,black-bone Jiuyuan (31) Sichuanc) Black Xishuangbanna (48) Yunnanb) Game bird Kangle (32) Jiangxic) Yellow Xuanzhou (31) Anhuic) Partridge

Langshan (32) Jiangsuc) Black or white Yangshan (32) Guangdongc) 3 yellow Langya (32) Shandongc) Partridge Yanjin (47) Yunnanc) Black-bone

Liangshan (32) Sichuanb) Partridge Yugan (47) Jiangxic) Black-bone Lingkun (32) Zhejiangb) 3 yellow Yunlong (32) Yunnanb) Short-shank Liyang (32) Jiangsuc) Large type Yunyang (32) Hubeic) White, black-bone

Lueyang (31) Shaanxib) Black-bone Yunyangda (48) Hubeib) Yellow, black-bone Lushi (32) Henanc) Partridge Zhangzhou (47) Fujianb) Game bird Luxi (47) Shandongc) Game bird Zhengyang (32) Henanc) 3 yellow

Luyuan (31) Jiangsuc) Yellow, large type Zhuxiang (32) Guizhoub) Black plumage and skin a) N, The number of the detected samples in each breed; b) breeds protected in conservation region close to the origin habitat; c) breeds protected

in conservation farm which were supported by government. samples from AVIANDIV project in Europe were used as references. All genotyping scores were calibrated relative to these control birds.

1.4 Statistical analysis

Allelic information for each microsatellite locus was analyzed and observed, and expected heterozy-gosity and homozygosity values calculated using Cer-

vus software[16]. Polymorphism information content (PIC) and heterozygosity (H) values were calculated using the Cervus program with equations being adopted from Botstein et al.[17].

Multilocus genotypes were tested for deviations from Hardy-Weinberg equilibrium and for linkage disequilibrium using Fisher’s exact test in Cervus software. Genetic diversity was quantified using H


Table 2 Polymorphism measures of the 27 microsatellites Location Number of alleles

Locus chromosome position

across population per population

H PIC Observed H

ADL0112 10 114 7 2.74 0.496 0.396 0.436 ADL0158 10 19 15 5.56 0.7 0.673 0.516 ADL0210 11 54 17 5.97 0.751 0.712 0.504 ADL0268 1 288 14 4.94 0.725 0.678 0.606 LEI0094 4 225 25 9.78 0.835 0.823 0.741 LEI0166 3 300 7 3.85 0.623 0.549 0.58 LEI0192 6 114 51 12.94 0.887 0.883 0.77 LEI0194 1 55 43 8.74 0.832 0.818 0.42 LEI0234 2 72 51 13.05 0.921 0.916 0.791

MCW0014 6 96 15 4.5 0.679 0.619 0.592 MCW0032 5 116 27 9.69 0.819 0.805 0.696 MCW0034 2 230 15 6.63 0.727 0.697 0.615 MCW0037 3 317 9 3.22 0.656 0.586 0.523 MCW0067 10 61 9 3.32 0.638 0.566 0.605 MCW0069 E46C08W18 23 18 7.91 0.814 0.791 0.703 MCW0078 8 87 9 2.49 0.204 0.196 0.114 MCW0081 5 123 12 3.27 0.45 0.386 0.357 MCW0103 3 210 7 2.24 0.474 0.376 0.423 MCW0111 1 118 20 5.65 0.725 0.683 0.606 MCW0120 7 121 14 6.78 0.829 0.806 0.598 MCW0134 9 132 22 7.47 0.815 0.793 0.72 MCW0145 1 455 27 7.15 0.778 0.749 0.616 MCW0206 2 117 17 5.36 0.708 0.664 0.595 MCW0216 13 28 8 3.63 0.456 0.424 0.366 MCW0222 3 28 6 3.35 0.649 0.579 0.515 MCW0295 4 75 16 6.18 0.745 0.708 0.628 MCW0330 17 41 25 5.67 0.763 0.729 0.649

Total 18.74 6 0.693 0.652 0.566

value and the mean number of alleles per locus. The mean genetic identities (MGI) and mean genetic dis-tances (MGD) between a given population and the other 77 populations were of Nei[18,19]. Genepop, Dis-pan and Mega2.1 were used to calculate the unbiased genetic distances and to construct a dendrogram of all populations.

2 Results

2.1 Diversity of populations

The diversity measures of the 78 populations are presented in Table 3. Chinese indigenous chicken breeds showed high polymorphism and extensive ge-netic diversity, with H values for all populations more than 0.5. Average H and PIC values within the 78 populations across all 27 loci were 0.622 and 0.573, respectively, and the average number of alleles was 6.00. The least polymorphic population was Langshan

which is indigenous to eastern China, with H and PIC values being 0.505 and 0.456 across all markers while the most polymorphic population was Shuanglian in central China, with H and PIC value as high as 0.678 and 0.633, respectively.

2.2 Mean genetic identities and mean genetic dis-tances

MGI and MGD between a given population and all other 77 populations are shown in Table 2. The Nei’s MGD values ranged from 0.043 (Huaibeima) to 0.251 (Zhangzhou game) with a mean being 0.097. Zhang-zhou game had the highest MGD. The average Nei’s MGI values were 0.908 with the maximum being 0.958 of Jianghan and minimum being 0.778 of Zhangzhou chicken.

2.3 Polymrophism of markers

All microsatellite loci showed high polymorphisms (Table 2). Some alleles had very low frequencies in a


number of populations (for example, Beijing fatty chicken) and some populations (for example, Zhang-zhou game chicken) had their own specific alleles. All 27 markers were polymorphic across populations with the mean number of alleles being 18.74 with a range of a maximum of 51 to a minimum of 6.

Average H and PIC of all markers were 0.693 and 0.652, respectively. Among the 27 tested markers, the most polymorphic was locus LEI0234 with 51 alleles across populations, 13.05 alleles per population on average and the mean H and of PIC being 0.921 and 0.916 respectively. At the other extreme, marker MCW0078 was the least polymorphic, with 9 alleles across populations, 2.24 alleles per population, with average H and PIC being 0.204 and 0.196, respec-tively.

2.4 Genetic divergence

The fixation coefficients of subpopulations within the total population (FST) for the 27 loci varied from 0.065 (LEI0166) to 0.209 (MCW0078), with a mean of 0.106. The fixation indices of individuals within the total population (FIT) ranged from 0.061 (MCW0067) to 0.492 (LEI0194), with a mean of 0.187. Individual’s fixation indices within the subpopulation (FIS) varied from –0.008 (MCW0134) to 0.400 (LEI0194), with a mean of 0.091.

Most loci identified in this study were in agreement with HWE, although one locus (LEI0194) deviated significantly from HWE among all populations. The LEI0194 loci also had the largest FIS value, reflecting a smaller amount of observed heterozygosity than ex-pected. Other loci deviating from HWE were not con-sistent among the different populations (data not shown).

2.5 Breeds clustering

A phylogenetic consensus tree was constructed us-ing the genetic distance data and NJ methodology based on genotyping results. The tree indicated that the Chinese breeds could be divided into six categories with distributions running generally consistent to their geographical locations (Fig. 1). Six breeds were clus-tered into Northwest group, 15 breeds into South group, 15 breeds into East group, 5 breeds into Southwest I group, 8 breeds into Southwest II group

and 22 breeds into Central group (Table 3).

3 Discussion

3.1 Microsatellite markers for chicken

Choosing proper markers is essential for microsa-tellite analysis of genetic diversity. The 27 microsatel-lite markers with extensive coverage on genome and high polymorphism worked very well in this study for our samples of Chinese indigenous breeds. Among these markers, 21 were identical to those used in European AVIANDIVT project[8,14]. In general, the number of alleles per microsatellite locus varied from 6 to 51. 41 alleles were found at both loci LEI0192 and LEI0234 in our study, while 37 and 23 alleles were found in AVIANDIV project[14], respectively. The variation in the number of alleles mainly depends on the differences among the breeds used and the dif-ferent levels of genetic diversity in the two studies.

Most of the microsatellite loci in this study were in agreement with HWE, with only one locus (LEI0194) deviating from HWE in all populations. Several rea-sons could explain the deviation, including non-random mating, selection, genetic drift, and small population size. In this study, the populations came from different areas and some were kept in conserva-tion regions as large populations without systematic non-random mating. Therefore, population size and non-random mating should not be the major cause for the deviation from HWE for LEI0194. This deviation may be caused by the association between the loci with genes that affect important economic traits. Null alleles could also contribute to the deviation, with an-other possibility for the deviation being incorrect genotyping. The gel conditions may not have been sufficiently accurate to separate 1bp difference be-tween the large fragments and thus some heterozy-gotes may have been ignored. This explanation is supported by the fact that the locus contained several different alleles overrepresented as homozygotes.

3.2 Diversity of Chinese indigenous breeds

The rich heritage of genetic diversity in Chinese in-digenous chicken breeds identified in the present study is consistent with the domesticated history and could reflect extensive diversity in Chinese indigenous


chickens that was also reported by Du et al.[20] in Ti-betan chickens and Zhang et al.[9,10] in 10 Chinese in-digenous chicken breeds. Meanwhile, the H values in Chinese indigenous chicken breeds were significantly

higher than those of commercial lines including layer and broiler lines[6,21―23] and of indigenous chickens in several countries in Europe[8]. The H values of Shuan-gliang (0.678) and Tibetan chicken (0.662) were even

Fig. 1. Phylogenetic tree of 78 chicken breeds by NJ and DS. According to the clustering results, these breeds could be divided into six groups. AJO, Aijiao; BIN, Bian; BIR, Baier; BJF, Beijing fatty; CHA, Chahua; CRM, Congren; DGU, Dagu; DXB, Dongxiang; EMH, Emei; FJS, Fujiansilky; GSI, Gushi; GXS, Guangxi; GYN, Guyuan; HBM, Huaibeima; HDG, Haidong; HLG, Huanglang; HNG, Henan; HNN, Huainan; HSN, Hongshan; HTN, Hetian; HXG, Huaixiang; HYG, Huiyang; JHN, Jianghan; JHW, Jinhu; JNB, Jining; JNG, Jingning; JSN, Jiangshan; JYH, Jiuyuan; KLE, Kan-gle; LDG, Luding; LIY, Liyang; LKN, Lingkun; LSH, Langshan; LSI, Lushi; LSY, Liangshan; LXG, Luxi; LYA, Langya; LYG, Lueyang; LYN, Lu-yuan; MCW, Muchuan; MYI, Miyi; NDS, Ningdu; NDY, Nandan; PDG, Pudong; QDN, Qiandongnan; QYM, Qingyuan; SBI, Shanbei; SGG, Shouguang; SIL, Silky; SLN, Shuanglian; SMC, Shimian; TBI, Taibai; TCX, Tengchong; TIB, Tibetan; TLF, Tulufan; TYN, Taoyuan; WCG, Wen-chang; WDG, Wuding; WHI, Wahui; WMW, Wumeng; WNG, Weining; WSG, Wenshang; XHA, Xinghua; XJU, Xianju; XSB, Xishuangbanna; XSN, Xiaoshan; XWW, Xingwen; XYN, Xiayan; XZU, Xuanzhou; YGW, Yugan; YJW, Yanjin; YLA, Yunlong; YSN, Yangshan; YYB, Yunyang; YYD, Yunyangda; ZXG, Zhuxiang; ZYG, Zhengyang; ZZG, Zhangzhou.


Table 3 Polymorphism measures and clustering results of the 78 Chinese indigenous chicken breedsa)

Name H PIC P Alleles/ locus

MGD Nei (1978)

MGI Nei (1978) Clustering groupb)

Langshan 0.505 0.456 1 4.37 0.137 0.872 5 Henan 0.532 0.486 1 5.04 0.141 0.868 1 Luyuan 0.533 0.488 0.96 4.81 0.182 0.833 5 Congren 0.537 0.489 0.93 4.74 0.122 0.885 7

Bian 0.543 0.497 0.96 4.74 0.147 0.863 6 Dongxiang 0.556 0.511 0.96 5.15 0.107 0.898 7

Beijing fatty 0.576 0.521 0.96 5.44 0.122 0.884 4 Jiangshan 0.581 0.532 1 5.81 0.085 0.918 6

Baier 0.585 0.533 1 5.11 0.101 0.904 7 Liangshan 0.586 0.541 1 5.37 0.199 0.819 4

Jinhu 0.586 0.539 0.96 5.63 0.099 0.905 5 Xiaoshan 0.589 0.541 1 5.44 0.126 0.881 5

Silky 0.592 0.542 1 5.37 0.093 0.911 6 Luxi 0.593 0.544 0.96 6.11 0.109 0.896 1

Zhangzhou 0.594 0.543 1 5.63 0.247 0.780 2 Lingkun 0.595 0.548 1 5.85 0.082 0.921 6 Haidong 0.597 0.521 0.96 4.62 0.117 0.8896 1

Fujiansilky 0.597 0.549 1 5.56 0.091 0.912 2 Wuding 0.602 0.555 1 6.07 0.093 0.911 4 Guyuan 0.603 0.534 0.96 5.62 0.104 0.901 1 Chahua 0.608 0.554 1 5.26 0.164 0.848 2 Langya 0.609 0.561 1 5.41 0.055 0.946 6 Ningdu 0.609 0.556 1 5.48 0.059 0.942 7 Pudong 0.613 0.559 1 5.07 0.121 0.886 5 Tulufan 0.621 0.532 1 6.2 0.116 0.890 1

Yunyangda 0.621 0.567 1 5.7 0.084 0.919 7 Kangle 0.622 0.574 0.96 5.52 0.099 0.906 5

Huaixiang 0.623 0.574 1 5.96 0.063 0.939 2 Luding 0.625 0.578 1 5.67 0.105 0.900 3

Muchuan 0.625 0.584 1 6.59 0.061 0.940 6 Wenshang 0.625 0.571 0.96 5.59 0.109 0.896 5 Wumeng 0.625 0.576 1 6.19 0.139 0.870 4 Jingning 0.626 0.562 0.96 5.96 0.077 0.925 1 Lueyang 0.626 0.577 1 5.85 0.056 0.945 6 Shanbei 0.626 0.583 1 6.04 0.067 0.935 6 Xianju 0.626 0.581 0.96 5.78 0.063 0.938 2 Liyang 0.627 0.582 1 6.11 0.089 0.915 5

Zhengyang 0.629 0.581 1 5.93 0.090 0.914 5 Hongshan 0.629 0.583 1 6.04 0.052 0.949 6

Yugan 0.629 0.582 0.96 6.41 0.093 0.911 6 Gushi 0.630 0.582 1 7.33 0.041 0.959 6

Qiandongnan 0.630 0.585 0.96 5.74 0.073 0.929 4 Wahui 0.630 0.585 0.96 6.37 0.051 0.950 6 Xiayan 0.632 0.588 0.96 6.41 0.060 0.941 2

Huainan 0.633 0.583 0.96 6.52 0.036 0.964 6 Aijiao 0.633 0.583 1 5.93 0.189 0.828 5

Xingwen 0.635 0.589 1 6.67 0.077 0.925 6 Shouguang 0.635 0.577 1 5.22 0.145 0.865 5 Yangshan 0.635 0.584 1 6.00 0.074 0.928 2

(To be continued on the next page)


(Continued)

Name H PIC P Alleles/ locus

MGD Nei (1978)

MGI Nei (1978) Clustering groupb)

Jiuyuan 0.636 0.586 1 5.67 0.116 0.890 3 Huiyang 0.636 0.587 1 6.19 0.056 0.945 2 Taoyuan 0.637 0.593 0.96 6.26 0.091 0.912 5

Huaibeima 0.640 0.596 1 6.56 0.035 0.965 6 Qingyuan 0.640 0.597 1 6.11 0.057 0.944 2 Jianghan 0.641 0.598 1 6.33 0.035 0.965 6 Xinghua 0.642 0.594 1 6.19 0.069 0.933 2 Shimian 0.644 0.600 1 6.74 0.076 0.927 3 Yunyang 0.644 0.600 0.96 7.00 0.064 0.938 6

Xuanzhou 0.644 0.598 1 6.30 0.063 0.938 6 Hetian 0.644 0.601 0.96 6.52 0.041 0.959 2 Taibai 0.645 0.600 1 6.07 0.084 0.919 5 Lushi 0.645 0.601 1 6.56 0.063 0.938 6 Miyi 0.648 0.604 1 5.81 0.078 0.924 3 Dagu 0.648 0.596 1 6.67 0.058 0.943 7

Wenchang 0.650 0.605 1 6.33 0.085 0.918 6 Yunlong 0.651 0.606 1 6.52 0.074 0.929 4 Yanjin 0.657 0.607 0.96 7.00 0.076 0.931 7 Nandan 0.657 0.609 0.96 6.41 0.072 0.930 2

Huanglang 0.660 0.614 0.96 6.44 0.053 0.948 5 Weining 0.661 0.616 1 6.30 0.117 0.889 4

Xishuangbanna 0.662 0.614 1 7.59 0.091 0.913 2 Tibetan 0.662 0.613 1 6.07 0.088 0.915 2 Emei 0.663 0.618 1 6.52 0.102 0.903 3

Guangxi 0.663 0.616 1 7.19 0.060 0.941 6 Jining 0.666 0.621 1 6.78 0.046 0.954 6

Tengchong 0.669 0.626 1 6.96 0.086 0.918 2 Zhuxiang 0.670 0.624 1 6.30 0.065 0.937 4

Shuanglian 0.678 0.633 1 6.81 0.059 0.942 5 Average 0.622 0.573 0.99 5.99 0.0906 0.914

a) H, Heterozygosity across markers; PIC, polymorphism information content; P, frequency of polymorphic markers; MGI, the means of identities; MGD, means of genetic distances. The genetic distance and genetic identity estimates are of Nei (1978). b) 1, Northwest group; 2, South group; 3, Southwest I group; 4, Southwest II group; 5, East group; 6, Central group; 7, Others. higher than those for the Red Jungle Fowl (0.640 for Gallus Gallus spadiceus and 0.600 for Gallus Gallus gallus) reported by Hillel et al.[8].

The production of quality chickens, which are bred mainly from indigenous chickens, is developing rap-idly in China. The meat flavor, fitness, and even the colorful plumage patterns are the main attributes that attract consumers for this special type of meat. The substantial genetic variation present in individual breeds will facilitate continued genetic improvement through selection of relevant quality traits in these chickens. Breeders can also use genetic diversity pre-sent among breeds for crossbreeding in order to com-bine different characteristics, such as meat quality and

reproduction, as well as to take advantage of hybrid vigor. Given the greatly different environments and production systems in China, rich genetic resources will also be helpful and valuable in choosing suitable breeds for particular circumstances.

Many Chinese indigenous chicken breeds, such as Tibetan chicken, are distributed in relatively isolated areas and protected in conservation regions with large population size. They also have not experienced inten-sive directional artificial selection. Thus genetic drift had not been as strong as in small populations. These breeds generally showed higher genetic diversity as shown in Table 3. On the other hand, most of breeds locating in economically developed areas are seldom


found in natural conditions and protected in small population sizes on conservation farms. The non-random mating or small population might cause genetic drift for these breeds, and therefore much lower H values had been revealed for these breeds. For example, Langshan, Dongxiang and Luyuan chicken have been kept in small populations on conservation farms for more than 20 years and the long-term on-farm conservation could reduce their genetic diver-sity. The H values for these populations were 0.505, 0.511 and 0.533, respectively. The results strongly suggest that better conservation strategies must be de-veloped and implemented for these populations to avoid inbreeding and genetic drift.

The high genetic polymorphism in Chinese indige-nous chickens revealed in the results of this study is in agreement with great phenotypic variation in body size, plumage color, and many other qualitative and quanti-tative traits of these breeds. The indigenous breeds have been raised in a widely dispersed manner and are seldom under intensive and consistent artificial selec-tion. The high genetic diversity in these breeds is im-portant for genetic resource conservation and utiliza-tion. The results could also be summarized into a characteristic database of the Chinese chicken genetic diversity that will play an important role in the future conservation and supervision of these chickens. The results indicate that Chinese indigenous chickens have high polymorphism. The indigenous breeds have been raised in a widely dispersed manner and are seldom under intensive and consistent artificial selection. It is interesting to note that Chinese indigenous breeds have more abundant diversity than the breeds in many other countries[8], which is in agreement with great phenotypic variation in body size, plumage color and many other qualitative and quantitative traits. The ge-netic diversity revealed in the present study is impor-tant for genetic resource conversation and utilization. The information from the DNA markers together with phenotypic performance and population history pro-vides reliable guidelines that can be used in develop-ing practical strategies for conservation purposes.

Although the number of birds sampled for some populations was small, the lack of resolution in recon-structing the phylogenies in some closely related populations was probably due to an insufficient num-

ber of loci and the large number of populations studied rather than an insufficient number of samples per population[24,25]. Nevertheless, based on the tree to-pology obtained from the research, the resolution in phylogenetic analysis did reflect genetic differentia-tion between the populations. The Chinese indigenous chicken breeds can be divided into six categories. It is of interest to notice that five game breeds grouped into two clusters: Xishuangbanna game and Zhangzhou game in the south group and Tulufan, Henan, Luxi game breeds in the Northwest group. Zhangzhou game was shown far distant from other Chinese indigenous chicken breeds, and a similar result was got by Qu with mtDNA D-loop analysis (unpublished). Both re-sults represent more evidences for the Philippine or Indonesian origin of Zhangzhou game[26].

The phylogenetic tree is consistent with the geo-graphic distribution by breeds with a few exceptions (Dagu chickens in Northeast China and Beijing Fatty chickens were placed into the east and south-east group respectively). The fact that the grouping of a few breeds was not consistent with geographical dis-tribution is not surprising. Both sampling error and a limited number of possible mistakes of genotyping may make clustering and grouping complicated.

The results of this study demonstrated the useful-ness of microsatellite markers for the evaluation of genetic variation and biodiversity. The information from the DNA markers together with phenotypic per-formance and population history provides reliable guidelines that can be used in developing practical strategies for conservation purposes.

Acknowledgements The authors would like to thank col-leagues and students in China Agricultural University and Poultry Science Institute of Chinese Academy of Agricul-tural Sciences for their assistance in the sampling and labo-ratory work, and Dr. Michele Tixier-Boichard at INRA, France, for providing the reference samples. This work was supported by the National Outstanding Youth Science Foundation of China (Grant No. 30225032), Doctoral Foundation of Ministry of Education (No. 20020019011) and the Ministry of Agriculture of China.

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Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers