关键词: 线粒体COI基因, DNA条形码, 地方鸡种, 品种鉴定, 遗传多样性

Abstract: 【Objective】 The aim of this study is to determine the feasibility of utility of mitochondrial cytochrome C oxidase subunit I (COI) gene as DNA barcoding to identify indigenous chicken breeds with nearer appearances.【Method】 COI gene of 648 bp in length was obtained from nine indigenous chicken breeds of South China (Huaixiang, Qingyuan spotted, Huiyang bearded, Zhongshan shalan, Yangshan, Xinghua, Wuhua three-yellow, Wenchang and Guangxi yellow) and one commercial breed, Recessive White, with the method of direct sequencing of PCR products; while other COI sequences were downloaded from GenBank, including Chinese indigenous chickens and wild jungles and mallard (Anas platyrhynchos). These sequences were then used to analyze genetic diversity and genetic distance, construct median-joining network and phylogenetic tree based on haplotypes, as well as define breed specific haplotypes. 【Result】 COI gene of 695 bp in length was obtained after deletion of the primers sequences. And then 648 bp of standard barcoding was used for analysis. A total of 110 mutation sites were detected from 203 individuals of 10 breeds with 16.98% in all sites, of which 90 were singleton variable sites and the remaining 20 were parsimony informative sites. The average nucleotide diversity and haplotype diversity were 0.00394 (0.00349-0.00560) and 0.832 (0.763-0.905), respectively. Wuhua three-yellow chicken had the highest levels of genetic diversity, Zhongshan Shalan chicken had the second higher one, but Wenchang chicken had the lowest ones. A total of 84 haplotypes were defined, haplotype 1 had the highest frequency in nine indigenous chicken breeds. Haplotyes 9 and 5 were sharing both in indigenous and Recessive White chicken breeds, with the frequencies of 29 and 19, respectively. Each breed had its own haplotypes. Guangxi yellow, Wuhua three-yellow and Zhongshan shalan chickens had most 13 haplotypes, while Qingyuan spotted and Recessive White had least 8 haplotypes. The distribution of haplotypes of different breeds had a little of difference. For example, the haplotypes of Xinghua chicken was mostly distributed at 1, where Qingyuan spotted, Huiyang bearded and Recessive White was mostly distributed at 1 and 9, 1, 5 and 9, and 9 and 79, respectively. The genetic distance and net genetic distance between 10 breeds were ranged from 0.003 to 0.006 and from 0 to 0.003, respectively. The genetic distance among breeds was higher than those of within a breed; those of between Anas platyrhynchos and chickens were higher than 0.2. The 84 haplotyes of median-joining networks of were classified into three clusters with the characteristic of breed specific. For example, the cluster originated from in haplotype 9 had no Guangxi yellow and Wenchang chicken breeds. Other haplotypes were the descendent of 1. The Neighbor-joining tree showed that indigenous chickens in China and Gallus gallus gallus were clustered into one branch, separating from Gallus lafayetii, Gallus sonneratii and Gallus varius. No branch with breed specificity was found. 【Conclusion】The results presented herein indicated that COI gene can be used as a candidate molecular marker for elucidate genetic diversity of indigenous chickens. It’s less effectivity of utility of standard COI gene as DNA barcoding to identify indigenous chicken with nearer appearance, there is highly need to incorporate multiple molecular markers such as COI gene, Cytochrome b gene, AFLP (Amplified Fragment Length Polymorphism), SNP (Single Nucleotide Polymorphism) and breed specific appearances.

Key words: mitochondrial cytochrome C oxidase subunit I gene, DNA barcode, indigenous chicken, breed identification, genetic diversity