Journal of Heredity Advance Access originally published online on November 5, 2007
Journal of Heredity 2007 98(7):723-726; doi:10.1093/jhered/esm094
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Brief Communications |
Mitochondrial DNA Sequence and Haplotype Variation Analysis in the Chicken (Gallus gallus)
From Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061 (Guan, Geng, Silva, and Smith); and Department of Animal Sciences, University of Peradeniya, Kandy, Sri Lanka (Silva)
Address correspondence to E. J. Smith at the address above, or e-mail: esmith{at}vt.edu.
Although it is known to be useful for certain genotype:phenotype assignments, our knowledge of the nature and extent of variation in the entire chicken (Gallus gallus) mitochondrial genome (mtGenome) is limited. Here, we used experimental and in silico tools to identify nucleotide variants in the mtGenome, including the coding and non-coding (D-loop) regions. The distribution of the experimentally identified mitochondrial DNA variants in meat- (broilers) and egg-type (White Leghorn) chickens was also assessed. A total of 113 single-nucleotide polymorphisms (SNPs) were identified. The in silico analysis revealed a total of 91 SNPs, with 70 in the coding region and 21 in the non-coding region. Of the 41 experimentally identified SNPs, 27 were in the D-loop. Together, the experimentally identified SNPs in the non-coding region formed 11 haplotypes, whereas the 14 SNPs in the coding region formed 6. Though, 9 of the D-loop region haplotypes were observed only in broilers, 3 of the 6 haplotypes from the coding region occurred at a significantly higher frequency in broilers. To our knowledge, this investigation represents the first whole-mtGenome scan for variation and an evaluation, though limited in sample size, of the haplotype distribution in meat- and egg-type populations, using the SNPs and haplotypes identified.
Mitochondria are the primary sites for oxidative phosphorylation in eukaryotes (DiMauro 2004). It has a mitochondrial genome (mtGenome) that is approximately 16–17 kbp in size in most animals including birds. The mtGenome encodes 22 tRNAs, 2 rRNAs, and 13 polypeptides (Chinnery and Schon 2003). Expression of these genes is essential in vertebrates for energy production, metabolism, cellular homeostasis, and apoptosis (DiMauro 2004). The importance of mitochondria in diverse physiological processes makes mutations in the mitochondrial DNA (mtDNA) an important factor in the incidence and severity of diverse diseases and abnormalities in vertebrates (Linnane et al. 1989; Troen 2003). For example, mutations in mtDNA have been shown to be associated with diseases like Alzheimer's and Parkinson's (Troen 2003), diabetes (Maassen et al. 2004), non-hereditary tumors (Zanssen and Schon 2005), and skeletal and cardiac myopathies (Zeviani and Donato 2004).
Although limited, some studies have reported the non-phylogenetic use of variation in the livestock and chicken mtGenomes. For instance, Li et al. (1998) described a single-nucleotide polymorphism (SNP) in the NADH dehydrogenase subunit IV gene that appeared to be associated with resistance to Marek's disease in White Leghorns. Mannen et al. (2003) showed a significant association between SNPs in the mtGenome with carcass traits. Central to these studies involving both phylogenetic and association analysis was the SNP discovery in the mtGenome. Here, we used in silico and polymerase chain reaction (PCR)–based resequencing of mtGenome to screen for DNA polymorphisms in the chicken. Further, the nucleotide variations were used to define novel haplotypes whose relative distributions in commercial egg- and meat-type chickens were also evaluated.
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Materials and Methods |
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Animals and DNA Extraction
We used a total of 53 birds, including 33 broilers and 20 White Leghorn chickens, in the experimental SNP analysis. The broilers, or meat-type chickens, were obtained from two distinct commercial producers in the United States of America. For obvious proprietary reasons, the companies have requested anonymity. The White Leghorn breed is a major egg-type breed that differs from broilers in size and other major phenotypes associated with high–egg-laying ability. Our rationale for using the broilers and White Leghorns is that egg- and meat-type birds have been subjected to different artificial selection emphasis, thus the divergence in phenotypes.
Pulp was obtained from secondary feathers collected from each bird and used to isolate genomic DNA using a minor modification of the recommended protocol for the DNeasy Tissue Kit (Qiagen, Valencia, CA). The modification included the addition of 100 mg/ml dithiothreitol to facilitate lysis of the feather pulp during incubation.
SNP Analysis
A PCR-based resequencing method (Smith et al. 2001) was used to scan the chicken mtGenome for SNPs. The GenBank Gallus gallus mtGenome sequence, accession number NC_001323, was used to design 3 of the 4 primer pairs used in the experimental SNP analysis (Table 1). The fourth primer pair was previously described by Sorenson et al. (1999) as a universal primer. The FailSafe PCR PreMixes (Epicentre, Madison, WI) were used to optimize the reaction conditions at a primer annealing temperature of 56°C. After the optimization, PCR was carried out and the amplicons purified and sequenced, as previously described (Lin et al. 2006). The sequences were analyzed for SNPs using Phred, Phrap, and Consed, according to Gordon et al. (1998).
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The in silico SNP analysis was carried out using completed whole-mtGenome DNA sequences available in GenBank. A total of 11 sequences, each representing a unique chicken, were used in the ClustalW (Higgins et al. 1994) alignment to screen for SNPs. The choice of sequences, from highly divergent strains, was based on the likelihood to detect variation. The accession numbers of the whole-genome sequences used were AY235570 [GenBank] and AY235571 [GenBank] , distinct New Hampshire Red breed birds that exhibit high and low sperm mobility, respectively (Froman and Kirby 2005); AP003317 [GenBank] , AP003318 [GenBank] , AP003319 [GenBank] , AP003321 [GenBank] , AP003322 [GenBank] , and AP003323 [GenBank] from White Leghorn, White Plymouth Rock, a native chicken in Laos, Red Jungle Fowl/Gallus gallus spadiceus, Red Jungle Fowl/Gallus gallus gallus, and Red Jungle Fowl/Gallus gallus bankiva, respectively (Nishibori et al. 2003); NC_001323 [GenBank] from White Leghorn (Desjardins and Morais 1990); AB086102 [GenBank] from Silkie chicken (Wada et al. 2004); and AP003580 [GenBank] another White Leghorn (Nishibori et al. 2003). A mismatch was considered an SNP only if at least 20 flanking nucleotides were of high quality and/or if the mismatch was observed in multiple individuals.
Statistical Analysis
Haplotypes were displayed using Visual Haplotypes (http://pga.gs.washington.edu/VH1.html). Chi-square test was used to test if haplotypes were in Hardy–Weinberg equilibrium. Pairwise linkage disequilibrium between SNPs was done using GENEPOP (http://wbiomed.curtin.edu.au/genepop; Raymond and Rousset 1995).
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Results and Discussion |
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In total, 113 nonredundant SNPs were identified from the sequence analysis including 35 and 78 in the noncoding and coding regions, respectively (Supplementary Figures S1 and S2). Among the 78 SNPs in the coding region, 19 were non-synonymous, 41 synonymous, and 18 in tRNA and rRNA sequences. A total of 91 SNPs were identified from the in silico analysis of whole-mtGenome sequences. Within the 6296 bp, or about 40%, of the mtGenome that were experimentally scanned, 41 SNPs were identified (Supplementary Figure S3). Nineteen SNPs observed in the in silico analysis were also observed in the experimental analysis, representing a validation of about 25% of the virtual SNPs.
Among the 113 non-redundant SNPs observed, 111 were substitutions and two were deletion/insertion polymorphisms (Supplementary Figures S1 and S2). As expected, a disproportionate number of the SNPs were transitions. For example, in the coding region, 70 of the 78 SNPs or 91% were transitions of either C–T (41) or G–A (29) substitutions. Similarly, in the noncoding region, there was a significantly lower number of transversions: only one or 3% of the 35 SNPs observed in the D-loop was a G–C substitution.
From both the in silico and experimental analyses, 39 haplotypes including 17 and 22 in the coding and noncoding regions, respectively, were observed. Six of the 17 coding region haplotypes and 11 of 22 D-loop haplotypes were based on nucleotide variants identified in the experimental SNP analysis. The distributions of the coding and noncoding region haplotypes in the commercial chicken populations are presented in Figures 1 and 2, respectively. Haplotypes 1, 3, and 4 from the coding region occurred at a significantly higher frequency at a combined level of approximately 88% in the broilers evaluated (Figure 1). D-loop haplotypes 1–9 were found only in broilers, whereas 10 and 11 were detected only in the White Leghorn chickens (Figure 2). Among the 9 broiler haplotypes, haplotypes 1 and 2 occurred at a significantly higher frequency (Figure 2).
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About 42% of the pairwise linkage disequilibrium between the SNPs identified in the analysis in both broilers and White Leghorn chickens in the D-loop were significant (P < 0.05), and approximately 20% of the associations among SNPs were highly significant (P < 0.001, Supplementary Table S1). In the coding region, however, only 8 out of 22 SNP associations evaluated were significant (P < 0.05, Supplementary Table S2).
The SNPs described in the present work provide a foundation for which the role of the mitochondria in metabolic disorders could be further investigated. Some of the SNPs cause a change in amino acids, which may affect important traits in the chicken. The SNPs in the non-coding region could also influence economic traits because the D-loop contains the origin of replication of the H-strand and the promoters for L- and H-strand transcriptions. In humans and model species such as mice, common mtDNA polymorphisms have been associated with important disease traits as well as other characteristics including climatic adaptation and longevity (Wallace 2005). Further association studies will thus be needed to show the value of our resources in mtDNA genotype:phenotype correlations in the chicken.
The data presented here represent, to our knowledge, the first investigation of variations across the entire chicken mtGenome. The results described could be useful for defining, as has been done in other animals, the molecular basis of many metabolic disorders, diseases, and abnormalities that affect chickens. For example, Moreno-Loshuertos et al. (2006) reported that the differences in reactive oxygen species production were correlated with mtGenome haplotypes in mouse. Using SNPs and haplotypes described by Johnson et al. (2001) and Jenuth et al. (1997), it was shown that variations in the dihydrouridine loop of tRNA arginine were associated with the production of hydrogen peroxide, a free radical, which influences respiratory performance. Therefore, the novel SNPs and haplotypes may be useful in biochemical and molecular studies of economically important phenotypes in chicken. Additionally, although limited in scope, the differences between White Leghorn in haplotypes and frequency of haplotypes may provide a baseline for further studies of genotype x environment interactions in chickens.
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Supplementary Material |
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Supplementary Figures S1–S3 and Tables S1 and S2 can be found at http://www.jhered.oxfordjournals.org/.
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Funding |
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Funding for our work was provided in part by the National Human Genome Research Institute and Virginia Agricultural Council.
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Acknowledgments |
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We are grateful to Drs Audrey McElroy and Ronald Okimoto for providing feather pulp from commercial meat-type chickens.
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Footnotes |
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Corresponding Editor: Rob Fleischer
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References |
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