Genetic diversity of four protected indigenous chicken breeds in China using microsatellite markers

The genetic diversity of four protected indigenous chicken breeds was evaluated with 25 microsatellite markers. Polymorphism information content (PIC), heterozygosity with the estimator of genetic differentiation FST and Nei’s genetic distance were evaluated. The results showed that these four protected local chicken populations showed high levels of diversity. The proportion of inter-population subdivision among the four protected local chicken populations was 16.0%. The average heterozygosity was 0.514, 0.581, 0.567 and 0.589 in Dongan, Xuefeng black-bone, Xianghuang and Taoyuan chickens, respectively, while the average PIC estimates were 0.455, 0.581, 0.557 and 0.576. A phylogenetic tree was constructed using genetic distance and the neighbour-joining method. Its topology reflects the general pattern of genetic differentiation among the four chicken breeds. The results also showed high genetic diversity and genetic variation among all the breeds. The information about the four local breeds estimated by microsatellite analysis may be useful as an initial guide for the effective conservation of chicken genetic diversity and developing conservation strategies.


Introduction
Chicken is one of the most widely distributed livestock in China.It plays a significant role as a source of income and high-quality protein for humankind.Indigenous chickens appear to possess enormous genetic diversity, especially in adaptive traits, and the ability to survive harsh conditions and under minimum feeding regimens (Qu et al., 2006).Successful preservation and utilization of these local types depend on accurate assessment of genetic diversity and genetic structure.Four indigenous chicken breeds, namely Dongan, Xianghuang, Taoyuan and Xuefeng black-bone chickens were included in the National Poultry Genetic Resource Directory, and play an important role in socio-economic development and ecological values in Hunan Province.In addition, their precious values as genetic resources are used for the genetic improvement of chicken breeds.Dongan are special meat-type chickens, and have good meat characteristics, high nutritional value and other excellent traits, such as yellow feathers, yellow skins and yellow shanks (Qu et al., 2006).The Xuefeng black-bone chicken is a meat and egg-type chicken, whose qualities include black meat, black bone, black beak and black feet, which are rich in nutritive and medicinal value, and were formed through long-term natural selection in the Xuefeng mountainous region in Hunan Province (Wei et al., 2008).Xianghuang and Taoyuan chickens are excellent meat and egg-type local chicken breeds, which come mainly from Changsha and Taoyuan counties in Hunan Province, respectively (Gao et al., 2008).So far, limited genetic diversity research has been conducted on these four chicken breeds, except for the study of Wei et al. (2008) on Xuefeng black-bone chickens.
Many molecular markers have become excellent means for the study of genetic variation (Chang et al., 2005;Chen et al., 2003), such as random amplified polymorphic DNAs (RAPD), amplified fragment length polymorphisms (AFLP), microsatellite DNA, and sequence-related amplified polymorphism (SRAP) (Zietkiewicz et al., 1994;Li et al., 2001).Among these DNA markers, microsatellites are widely used since they are numerous, randomly distributed in the genome, highly polymorphic, and with co-dominant inheritance (Groen et al., 1994;Kavaca et al., 1999).Many microsatellites have been mapped in chickens, and are used to study the genetic relationships among breeds (Tadano et al., 2007;Kaya et al., 2008).The purpose of this study is to estimate the level of genetic differentiation and phylogenetic relationships among the four indigenous chicken breeds in China.This information can contribute to the conservation and utilization of local chicken breeds.

Materials and Methods
Samples were obtained from 179 unrelated individuals, representing four indigenous chicken breeds in Hunan Province.These included 50 Dongan chickens (DA) from Dongan county, 50 Xianghuang chickens (XH) from Liuyang county, 29 Taoyuan chickens (TY) from Taoyuan county and 50 Xuefeng black-bone chickens (XF) from Hongjiang city.Blood samples (3 mL) were collected with syringes from the wing vein into a tube containing DNA preservation solution as an anti-coagulating agent.All samples were stored at −80 ºC for further analysis.
Genomic DNA was isolated from blood using a phenol/chloroform extraction method (Sambrook, 2002).The DNA was quantified with a spectrophotometer, comparing band intensities with known standards of DNA marker on 1.5% agarose gel.The working solution of DNA (approx.25 ng/µL) was dissolved in sterile double-distilled water.In a preliminary experiment, 40 SSR primers were tested on four random individuals from each breed.Based on the amplification result, 25 microsatellite loci were further investigated, which were listed in Table 1.All primers were synthesized by ShenggongBio-Tech.Co., Ltd.Shanghai, China.
Polymerase chain reaction (PCR) amplifications were performed on PTC-200 thermal cyclers.A total reaction volume of 8 μL with 1 μL of 10 × buffer, 0.6 μL of 25 mmol MgCl 2 , 0.2 μL of 10 mmol dNTPs, 0.1 μL of 5 U/μL Taq DNA polymerase, 0.3 μL of 10 pmol/μL each primer, and approximately 50 ng of genomic DNA were used.The reaction was carried out by initial denaturation at 94 ºC for 3 min, and then denaturing at 94 ºC for 30 s, annealing at the temperature optimized for each primer pair for 30 s and extending at 72 ºC for 30 s for 35 cycles, followed by an extra extension step at 72 ºC for 5 min.The optimized annealing temperatures of different primer pairs are listed in Table 1.The amplification products were separated by electrophoresis on 12% non-denaturing polyacrylamide gels and visualized by silver staining (Su et al., 2006).The images data were analysed with Kodak Digital Science ID Image Analysis Software.
Based on microsatellite genotyping and allele frequencies, the number of alleles, effective number of alleles (Ne), observed heterozygosity (Ho), (Nei, 1987) expected heterozygosity (He), and Wright's (1978) fixation index (Fis) were estimated using the computer software package PopGene version 1.31 (Yeh et al., 1997).Allele frequencies obtained from the microsatellite genotypes were used to calculate PIC (polymorphism information content) values (Botstein et al., 1980) using the computer software package Cervus 3.0 (Marshall et al., 1998;Kalinowski et al., 2007) in order to measure the information obtained by a microsatellite.Based on microsatellite genotyping, Nei's (1978) unbiased genetic distance between breeds was estimated.Software FSTAT (version 2.9.3.2) was used to test pairwise linkage equilibriums at all loci over any two groups to calculate the pairwise genetic differentiation F ST (Weir & Cockerham, 1984).These results were used to construct phylogenetic trees by neighbour-joining cluster analysis with the appropriate options of computer software Mega Version 4.0 (Tamura et al., 2007).

Results
The genetic diversity and differentiation among the four local chicken breeds at the 25 microsatellite loci were estimated.The numbers of alleles per locus and the size range of alleles are listed in Table 1.All loci were polymorphic in the four breeds.The observed numbers of alleles varied from 3 (MCW0330) to 9 (MCW0094) and the mean number of alleles across all loci was 5.84.The observed heterozygosity (Ho) ranged from 0.268 (LEI0254) to 0.726 (LEI0098) (Table 3).The expected heterozygosity (He) was quite high, ranging from 0.448 (LEI0254) to 0.861 (MCW0224) (Table 2).The PIC among loci was highest for MCW0224 (0.854) and lowest for ADL0210 (0.447) (Table 2).
There was highly significant genetic divergence across the four breeds for every locus.The F ST values ranged from 0.058 (MCW0330) to 0.243 (MCW0097).Using the multilocus F ST , approximately 16.0% of the total genetic variation can be explained by breed differences, and the remaining 84.0% was owing to the differences among individuals (Table 2).The estimated multilocus heterozygosities varied from H = 0.514 in DA to H = 0.589 in TY.The mean effective allele number was between 3.20 (XF) and 3.72 (TY).Among breeds, the mean PIC value was 0.455, 0.581, 0.557 and 0.576 for DA, XF, XH and TY, respectively.Wright's fixation index (Fis) values ranged from -0.16 (XF) to 0.14 (DA).Deviation from Hardy-Weinberg equilibrium (HWE) between loci and breeds was tested with FSTAT (version 2.9.3, Goudet, 2001).Significance levels were adjusted using Bonferroni correction for multiple testing.Significant deviations (P <0.05) from HWE were observed at the breed level (DA) (Table 3).Using Nei's (1978) unbiased genetic distance (Table 4) and the neighbour-joining method, a phylogenetic tree was constructed for the four chicken breeds.The smallest genetic distance, between Xianghuang and Taoyuan chickens, was 0.242.The largest genetic distance, between Dongan and Xuefeng black-bone chickens, was 0.507.The neighbour-joining dendrogramme in Figure 1 was drawn using the genetic distances given in Table 4.The Xianghuang, Taoyuan chickens and Dongan chickens and Xuefeng black-bone breeds were clustered as two groups, in order to support the reliability of this analysis.when planning conservation and improvement programmes.The effective conservation of genetic diversity in the chicken gene pool relies essentially on the understanding of genetic diversity patterns of chicken breeds in a conservation region, including the levels and distribution of the diversity (Chen et al., 2006).It is particularly important to conserve the chicken's genetic diversity on farm management (or onfarm conservation), because the combination of farmers' diverse needs with the breeds in different ecosystems has created and accumulated wide genetic variation.This study revealed a high genetic diversity within the four protected local chicken breeds.The results from this study indicated that the genetic variation of chicken breeds is still remarkably rich.The considerably rich genetic diversity of chicken breeds in China can be attributed to its complicated local geographical conditions where different farming practices and agro-ecosystems exist.The diversity may also be significantly associated with its rich culture diversity that promotes miscellaneous needs and applications of chicken breeds.Relative isolation of the various areas has probably played a considerable role in reducing the exchange with modern improved chicken breeds.Such a factor has played an essential role in maintaining the genetic diversity of Chinese chicken breeds.In addition, it is important to carry out more studies in future, which can provide us with useful information for the effective conservation of chicken genetic diversity and a roadmap for conservation strategies of chicken genetic resources.

Conclusion
These four protected indigenous chicken breeds throughout Hunan Province in China are resources of considerable socio-economic value.Their genetic diversity was evaluated with 25 microsatellite markers in this study.The result demonstrated that these breeds showed high polymorphism.A phylogenetic tree was constructed using genetic distance and the neighbour-joining method.The four breeds were clustered as two groups, in order to support the reliability of this analysis.The information about the four local breeds that was estimated by microsatellite analysis may be useful as an initial guide to defining objectives for designing future investigations of genetic variation and developing conservation strategies

Figure 1
Figure 1 Neighbour-joining tree based on Nei's genetic distance.

Table 1
Primers used in the present study Note: F: forward primer; R: reverse primer.

Table 2
Nei's estimation of heterozygosity at every locus average over breeds

Table 3
Within breed genetic variation

Table 4
Genetic distance between breeds