Analysis Of Genetic Diversity And Phylogenetic Relationship Among Chinese Domestic Fowls And Red Jungle Fowls

Combining the technique of multiplex-PCR and the fluorescent automated diction, genetic diversity and phylogenetic relationship among 570 individuals of 14 Chinese domestic chicken breeds(Xianju chicken, Chahua chicken, Luyuan chicken, Baier chicken, Tibetan chicken, Gushi chicken, Dagu Chicken, Henan Game, Langshan chicken, Taihe Silkies chicken, Xiaoshan chicken,Beijing Fatty chicken,Huainan Partridge and Wannan Three-yellow chicken) and two red jungle fowl subspecies(Gallus gallus spadiceus in China and Gallus gallus gallus in Thailand)were evaluated with 29 microsatellite loci. The effects of sample size, sex and number of microsatellite loci on various genetic diversity measures were estimated, and similar part of mtDNA D-loop of these 16 populations were sequenced and analyzed. Genetic variability within populations and genetic differentiation among populations were estimated, thereafter genetic diversity and phylogenetic relationship among red jungle fowls and Chinese domestic fowls were analyzed. Our main results were summarized as follows:1. The genetic variability within populations and genetic differentiation among populations were estimated, a total of 286 alleles were detected in 16 populations with 29 microsatellite markers, and the average number of observed alleles was 9.86±6.36. The overall expected heterozygosity of all populations and PIC of all loci were 0.6708±0.0251 and 0.52, respectively. All 29 microsatellite loci in this study showed high levels of polymorphism. The number of populations deviated from Hardy-Weinberg equilibrium per locus ranged from 0 to 7. In the whole population, the average of genetic differentiation among populations, measured as FST value, was 16.7% (P <0.001), and all loci were contributed significantly (P <0.001) to this differentiation. Significant genetic differentiation was observed among two subspecies of red jungle fowl and 14 Chinese domestic chicken breeds, and the deficit of heterozygote was observed very high (0.015) (P <0.01). Reynolds'distance values varied between 0.036 (Xiaoshan chicken-Luyuan chicken pair) and 0.371 (Gallus gallus gallus–Henan Game chicken pair). The Nm value was ranged from 0.583 (between Gallus gallus gallus and Henan Game chicken) to 5.833 (between Xiaoshan chicken and Luyuan chicken).2. The phylogenetic relationship among Chinese domestic fowls and red jungle fowls were analyzed, an un-rooted consensus tree was constructed using the Neighbour-Joining method and the clustering results are generally in accordance with the results obtained from STRUCTURE. The tree topology revealed two main clusters, although the relationship between breeds was not always supported by high bootstrap values. The heavy-body sized chicken breeds, Huainan Partridge, Wannan Three-yellow chicken, Dagu chicken, Henan Game, Langshan chicken, Beijing Fatty chicken, Luyuan chicken and Xiaoshan chicken formed in one cluster; and the light-body sized chicken breeds, including Red Jungle Fowl in China, Red Jungle Fowl in Thailand, Chahua chicken, Tibetan chicken, Xianju chicken, Gushi chicken, Baier chicken and Taihe Silkies chicken formed in the second main cluster. The results suggested that the Tibetan, Huainan Partridge and Wannan Three-yellow chickens have mixed genetic structures, while the genetic bases of Luyuan chicken and Xiaoshan chicken are nearly same. Chahua chicken and Tibetan chicken had closer genetic relationship with Gallus gallus spadiceus but appeared rather farer phylogenetic relationship with Gallus gallus gallus. The evolutional dendrogram was as follows: evolutional breeds ? primitive breeds (Chahua chicken and Tibetan) ? red jungle fowl in China (Gallus gallus spadiceus ) ? red jungle fowl in Thailand (Gallus gallus gallus )3. The geographical elements may own to the close relationship for particular population pairs, however, the equation FST/ (1-FST) =–1.0283–0.0407ln (d) and the result from Mantel's test (P=0.596) did not provide enough support for a significant correlation between the genetic and geographical pair wise distances. It was no significant correlation between the genetic diversity of mtDNA D-loop and the distributing of these populations. The results concluded that the geographical distributing maybe not the determinant influence on the genetic structure of Chinese chicken populations during the course of their developed history.4. Based on the analysis of genetic diversity in 4 Chinese indigenous chicken breeds at 29 microsatellite loci, the effects of sample size and sex on various genetic diversity measures were estimated, the accuracy of pair wise genetic distance among 4 Chinese native chickens were analyzed at 5, 10, 15, 20 and 25 microsatellite loci levels. The results indicated that the expected heterozygosity was fairly stable when sample size was over 20, the mean number of allele over loci was significantly affected by sample size, while there is no significant correlation between sample size and expected heterozygosity, the suitable sample size in microsatellite analysis ranged from 20 to 25. The sample size needed in the study was affected by the polymorphism of microsatellite loci. When the mean number of allele over loci has been chosen to detect the population diversity, the effect of sample size variation should be assessed deliberately, while the expected heterozygosity was not sensitive to sample size variation and can be used as a reliable parameter to estimate the genetic diversity. The genetic diversity measures were not significantly difference between sexes, and the accuracy of genetic distance estimation increased along with the numbers of the loci increased.5. The applicability of microsatellite primers from chicken to peafowl population was analyzed, the results showed that the 14 of the 29 pairs of microsatellite primers from chicken could amplify peafowl DNA and produce special allele patterns, with 1.71 mean alleles per locus. Seven pairs of primer had higher polymorphism, among them MCW0080 and MCW0098 primers were perfect markers for peafowl. Based on the analysis of genetic diversity within and between green peafowl and the blue peafowl population, our results demonstrated the expected heterozygosity of two peafowl populations were 0.2482 and 0.2744, respectively. Inbreeding index (FST), Reynolds' genetic distance and gene flow between two populations were 0.078, 0.0603 and 3.896, respectively. These results indicated that the heterozygosity and the genetic diversity of two peafowl populations were very low, it was a mix-up tendency between two peafowl populations.6. Part of mtDNA D-loop among 256 individuals of 14 Chinese domestic chicken breeds and two red jungle fowl subspecies were sequenced and analyzed. The result showed that the length of D-loop in this study was about 560 bp.Content of nucleotide A, C,G, T were 25.00%, 37.40%, 4.40% and 33.20%, respectively. The percentage of A+T was 58.2% and G+C was 41.8%, showed high A+T of mtDNA D-loop in 16 chicken populations. There were 44 polymorphic sites represent 7.86% of total analyzed sites.Only transition and transversion but no insertion/deletion were found in this region, the ratio of transition and transversion in this study was 0.13.32 haplotypes,among them 14 haplotypes were shared among some chicken populations, 23 haplotypes were unique for one population. The distribution of all haplotypes among the populations was disequilibrium and the diversity of haplotypes was ranged from 0 to 0.964.The average diversity of haplotypes was 0.909±0.014, indicated that there was existed rather abundant mitochondrial genetic diversity in 16 chicken populations.The Gushi chicken showed the lowest diversity of haplotypes while the Huainan Partridge and Wannan Three-yellow chickens showed the highest one. The average number of nucleotide divergence (K) and average nucleotide diversity (Pi) were 7.276 and 1.851%, respectively. Inter-population Nucleotide Divergence (Dxy) in 16 chicken populations was ranged from 0.747%～3.125%, wheras Inter-population Net Nucleotide Divergence (Da) in 16 chicken populations was ranged from -0.015% to 2.633%. The results indicated that the genetic diversity of 16 chicken populations was very abundant.Kimura 2-parameter distance among these populations ranged from 0.007 to 0.031. Analysis of molecular variance showed that 23.83% of genetic variation was present within populations. FST value was 0.38155, which indicated the genetic variation was significant within populations (P < 0.05). There were significant divergence among 14 Chinese domestic chicken breeds and two subspecies of red jungle fowl.7. The NJ, ME and UPGMA phylogenetic dendograms of 32 haplotypes in 16 chicken populations and as Coturnix japonica an outgroup from GenBank (Accession No. D82924) were constructed. As a whole, the results of three kinds of trees are same and these 32 haplotypes were placed into four lineages. Lineage A included the haplotypes of Gallus gallus gallus, Lineage B and C included the haplotypes of Gallus gallus spadiceus and Lineage D included the haplotypes of both Gallus gallus gallus and Gallus gallus spadiceus. The median-joining networks of the 16 chicken populations in the control region also showed the same results with phylogenetic trees and all 32 haplotypes appeared into four clusters. NJ, ME and UPGMA dendograms based on Kimura 2-parameter distance of mtDNA D-loop sequences in 16 chicken populations were also constructed, three kinds of dendograms all showed that Gushi chicken, Xianju chicken and Gallus gallus gallus were always in the same cluster, while other breeds such as Chahua chicken, Tibetan chicken, Taihe Silkies chicken, Henan Game and Baier chicken were confined into the same cluster.8. Genetic diversity of red jungle fowl in China(Gallus gallus spadiceus) and red jungle fowl in Thailand (Gallus gallus gallus)was evaluated with 29 microsatellite loci and mtDNA D-loop, Reynolds' genetic distance and gene flow between two populations were 0.157 and 1.040, respectively, there was no closer relationship and large gene flow between these two subspecies. Genetic differentiation index (FST) of these two populations was 0.194 (P <0.01) and all loci contributed significantly (P <0.01) to this differentiation. There was no shared haplotypes between these two subspecies, the pair wise genetic differentiation index (Fst) of mtDNA D-loop was also significant (P=0.0360), all results indicated that there were different genetic structure and significant genetic differentiation between red jungle fowl in China and red jungle fowl in Thailand, and this provided enough support to identify these two red jungle fowl subspecies as the different subspecies. 9. Analysis of genetic diversity and phylogenetic relationships among Chinese domestic fowls and red jungle fowls estimated with 29 microsatellite loci and mtDNA D-loop sequences indicated that some Chinese domestic fowls such as Chushi chickens and Xianju chickens derived from some subpopulations of Gallus gallus gallus after their domestication, other Chinese domestic fowls such as Chahua chickens and Tibetan chickens derived from some subpopulations of Gallus gallus spadiceus. Tajima's D values were -1.79995 (P< 0.05)for Gallus gallus gallus, which showed significant difference from neutrality. During the course of population expansion after the domestication, Gallus gallus gallus maybe have some effect on some subpopulations of some Chinese domestic breeds originated from Gallus gallus spadiceus in China, so the genetic contribution of these two subspecies of red jungle fowl can be detected in some Chinese chicken populations. The results in this study concluded that the Chinese domestic fowls don't just derive from red jungle fowl in Thailand or just from red jungle fowl in China and gave support the viewpoint that the red jungle fowls were domesticated several times independently in different place and the domestication of red jungle fowls was the outcome of human activities during the long period.