Biochem Genet DOI 10.1007/s10528-015-9668-y ORIGINAL ARTICLE

Mitochondrial DNA-Based Analyses of Relatedness Among Turkeys, Meleagris gallopavo Xiaojing Guan1 • Pradeepa Silva1,2 • Kwaku Gyenai1 • Jun Xu1 • Tuoyu Geng1,2 Edward Smith1

•

Received: 28 June 2013 / Accepted: 27 February 2015 Ó Springer Science+Business Media New York 2015

Abstract The domesticated turkey, Meleagris gallopavo, is believed to be a single breed with several varieties whose relatedness and origins remain poorly understood. Using the mitochondrial genome sequence (GenBank accession no. EF153719) that our group first reported, we investigated the relationships among 15 of the most widely occurring turkey varieties using D-loop and 16S RNA sequences. We included, as a non-traditional outgroup, mtDNA sequence information from wild turkey varieties. A total of 24 SNPs, including 18 in the D-loop and 6 in the 16S rRNA, was identified, validated and used. Of the 15 haplotypes detected based on these SNPs, 7 were unique to wild turkeys. Nucleotide diversity estimates were relatively low when compared to those reported for chickens and other livestock. Network and phylogenetic analyses showed a closer relationship among heritage Electronic supplementary material The online version of this article (doi:10.1007/s10528-015-9668-y) contains supplementary material, which is available to authorized users. & Edward Smith [email protected] Xiaojing Guan [email protected] Pradeepa Silva [email protected] Kwaku Gyenai [email protected] Jun Xu [email protected] Tuoyu Geng [email protected] 1

Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA

2

Department of Animal Sciences, University of Peradeniya, Kandy 20400, Sri Lanka

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varieties than between heritage and wild turkeys. The mtDNA data provide additional evidence that suggest a recent divergence of turkey varieties. Keywords

Turkeys  mtDNA  SNPs  Heritage varieties  Relatedness

Introduction The turkey, Meleagris gallopavo, is native to North America and exists widely as both an important wild bird and the second most widely consumed poultry meat species in developed countries. The domesticated turkey, derived by cross- and linebreeding, is believed to be a single breed with eight distinct varieties and numerous sub-varieties (Kennamer et al. 1992). The major varieties include those described as heritage by the American Livestock Breed Conservancy (http://www. livestockconservancy.org/) and the American Standard of Perfection (American Poultry Association 2001), including the Black, Bronze, Narragansett, Slate, Beltsville Small White, Bourbon Red, Royal Palm and White Holland. It should be noted, however, that most contemporary commercial turkey varieties raised for meat are believed to be crosses involving the White Holland and Bronze followed by selection for breast size and body weight (Austic and Nesheim 1990). The genetic relationships among the strains that comprise the crosses remain without a consensus. In Europe, for example, the European Association of Poultry recognizes 12 breeds of turkey based primarily on the type of turkey found in Germany (http://en. wikipedia.org/wiki/List_of_turkey_breeds and references therein). In the Americas, the turkey ‘‘variety,’’ as defined here, is basically how the American Poultry Association (APA) decided to define the different colors of turkeys. The logic behind this is that the colors do not differ in comb type or other morphologic details, as is typical of chicken breeds. Therefore, the APA was unwilling to make this level of variation into ‘‘breeds’’ in the case of the turkey, but opted for ‘‘varieties’’ instead. This is totally different from ‘‘bloodlines,’’ which are just groups of birds from a specific breeder (Dr. Phillip Sponenberg, Virginia Tech, personal email communication). Basically, ‘‘variety’’ is not as differentiated as ‘‘breed’’ in the mind of the APA. Relationships among wild turkeys, primarily for conservation purposes, have been extensively evaluated using both morphological and mitochondrial molecular tools. For example, using amplified fragment length polymorphic DNA markers and the mitochondrial control region derived using heterologous primers from the chicken, Mock et al. (2002) evaluated genetic variation among wild turkeys distributed widely in the USA. The molecular analyses revealed relationships among turkeys from distinct geographic regions that were also consistent with earlier morphological designations of the varieties (Mock et al. 2002). Szalanski et al. (2000) also used heterologous primers from the chicken and quail to evaluate the extent of genetic variation and differentiation among some subspecies of the wild turkey. The PCR-RFLP analyses of variants identified in the screening of 721 bp of the turkey mitochondrial genome (mtGenome) sequence of 116 birds from 18 subspecies did not, unlike that of Mock et al. (2002), support sub-species

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status in the turkey. Speller et al. (2010) used a 438-bp fragment of the mtDNA control region to study the North American turkey domestication. There were 12 haplotypes identified in that study of prehistoric turkey remains. These analyses revealed at least two occurrences of turkey domestication in pre-contact America, one involving the South Mexican wild turkey and a second involving Rio Grande/ Eastern wild turkey populations. Differences among the turkey varieties have also been investigated at the molecular level using diverse nuclear DNA marker systems (Kamara et al. 2007 and Smith et al. 2005) including microsatellites and SNPs. For example, by using microsatellites, Kamara et al. (2007) showed that the Blue Slate, Bourbon Red and Narragansett were genetically closely related to the commercial varieties. Smith et al. (2005) used randomly amplified polymorphic DNA (RAPD), microsatellites and SNPs to evaluate genetic relatedness among different turkey varieties. Although the investigations of Kamara et al. (2007) and Smith et al. (2005) showed a lack of congruence about some relationships, both suggested that Royal Palm is distinct from the other heritage breeds, although more closely related to Narragansett. A mtDNA-based evaluation of relatedness may provide further support of earlier preliminary analyses about the distinctness of the Royal Palm from other heritage and commercial turkeys (Vawter and Brown 1986). Here we used the 16S rRNA and D-loop regions of the previously described turkey mtGenome sequence [GenBank accession no. EF153719, (Guan et al. 2008)] to evaluate the phylogenetic relationships based on haplotype diversity, network analysis and pairwise genetic distance estimates. We reasoned that using two mitochondrial genomic regions with differing rates of evolution strengthen the relationships defined here. Since the D-loop is prone to homoplasy, the phylogenetic noise arising from this can be controlled for using variation in the 16S rRNA. We do realize that the whole nuclear genome sequence, like those recently reported for a large number of birds (Jarvis et al. 2014), can offer a significantly higher level of resolution than mtDNA and a few nuclear DNA sequences.

Methods DNA Samples A total of 126 birds from 15 turkey varieties including heritage (www.albc.org), non-heritage, wild and commercial (British United Turkeys, BUT) were used (Table 1). The heritage varieties, obtained from breeders, were Narragansett (N), Royal Palm (RP), Blue Slate (BS), Spanish Black (SB), Bourbon Red (BR), Midget White (MW), White Holland (WH) and Standard Bronze (StB). The non-heritage turkey varieties were Broad Breasted Bronze (BBB) and Broad Breasted White (BBW). The wild subspecies were Rio Grande (R) or Meleagris gallopavo intermedia, Eastern (E) or Meleagris gallopavo silvestris, Merriam’s (M) or Meleagris gallopavo merriami, and Osceola (O) or Meleagris gallopavo osceola. The samples were from birds randomly selected from each domesticated variety, and the numbers are consistent with other phylogenetic investigations in which two individuals

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Biochem Genet Table 1 Number and source of birds from each turkey variety used in the study Turkey variety

Source Privett Hatchery

Welp Hatchery

Purdue University

Total

Blue slate

4

6

0

10

Spanish black

2

7

0

9

Narragansett

4

4

0

8

Royal palm

4

6

0

10

Bourbon red

0

6

0

6

Midget white

0

10

0

10

Broad breasted bronze

0

9

0

9

Broad breasted white

0

10

0

10

White holland

0

8

0

8

Standard bronze

0

6

0

6

British United Turkeys

0

0

0

6

Eastern wild

0

10

1

11

Rio Grande wild

0

12

2

14

Merriami’s wild

0

0

5

5

Osceola

0

0

4

4

from each population are included in the analyses (Nei and Kumar 2000). Mitochondrial DNA was extracted from diverse sources including whole blood collected in EDTA, pulp obtained from secondary feathers and blood sampled on FTA cards (Whatman, Piscataway, NJ) using previously described protocols (Guan et al. 2007). Phylogenetic and Network Analysis In addition to the sequences generated from the varieties in Table 1, publicly available wild turkey DNA sequences in GenBank were also used for the phylogenetic analyses. The wild turkey sequences provide a reference consistent with the use of an ‘‘outgroup.’’ The names, accession numbers and references for the GenBank sequences were: commercial [AJ297180 (Lucchini et al. 2001)], M. g. merriami [Merriam’s, AY037889 (Mock et al. 2001)], M. g. mexicana [Gould’s, G, AY037888, (Mock et al. 2001)], M. g. intermedia [Rio Grande, AF487119 (Mock et al. 2001)], M. g. silvestris [Eastern, AF486929 (Mock et al. 2001)], and AF532414 (Drovetski 2002), and M. g. osceola [Osceola/Florida, AF486959, (Mock et al. 2001)]. For each wild turkey, the multiple in silico sequences were used, along with the experimental sequence generated in our laboratory, to obtain a consensus sequence that was used in the phylogenetic and network analyses (DNASTAR Lasergene MegAlign: DNASTAR, Inc., Madison, WI). Primer pairs developed from the turkey mtDNA sequence (GenBank accession no. EF153719) for the resequencing were 50 -CCAAGGATTACGGCTTGAAA-30 (forward), 50 -TTAAGCTATGGGGGCTGTTG-30 (reverse) for the D-loop and 50 ACAACCAAGCAAAGCGAACT-30 (forward) and 50 -ATGGGCTCTTGGAGGAG ATT-30 (reverse) for the 16S rRNA-based analyses.

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The primers were optimized using the FailSafe PCR PreMixes from Epicenter Technologies (Madison, WI) as previously described (Guan et al. 2007). Sequences generated from the amplicons were analyzed for nucleotide variants using Phred, Phrap and Consed (Lin et al. 2006). DnaSP was used to estimate haplotype and nucleotide diversities (Rozas et al. 2003). Parsimony network analysis was carried out using 933 bp (1,451–2,383) and 578 bp (15,666–16,243) of 16S rRNA and D-loop sequences, respectively, using the TCS computer program, version 1.21 (Clement et al. 2000). Following alignment of sequences using Clustal-X (Thompson et al. 1997), PAUP* version 4.0 (Swofford 2002) was used in the analysis of genetic relatedness and to generate trees using minimum evolution, neighbor-joining, maximum parsimony and maximum likelihood methods. One thousand bootstrap replicates were used to assess the confidence in the grouping (Felsenstein and Kishino 1993). The Modeltest 3.8 (Posada 2006) was used to select an appropriate model for maximum likelihood analysis. Using the Akaike information criterion, the Hasegawa-Kishino-Yano (Hasegawa et al. 1985) and F81uf model (Felsenstein 1981) with equal rates for all sites were selected for D-loop and 16S rRNA, respectively. Pairwise genetic distance estimates of aligned sequences were also calculated using PAUP* version 4.0 (Swofford 2002). In our use of network analyses, we hope to provide some data that may help us understand the evolutionary relationships of heritage and commercial turkeys to other turkeys. Like other birds, turkeys remain very little studied (Jarvis et al. 2014; Zhang et al. 2014).

Results and Discussion A total of 24 SNPs, 6 in the 16S rRNA and 18 in the D-loop, were detected (Table 2). As expected, 75 % of the SNPs were either C/T or A/G transitions. Two SNPs, at positions 15,741 and 15,762, were deletions. Also as expected, the number of SNPs in the sequences of the D-loop was higher than that in the 16S rRNA sequences. The prevalence of purine–purine or pyrimidine–pyrimidine substitutions among SNPs is consistent with the characteristics of SNPs observed in the mtDNA described in other organisms (Zhang and Gerstein 2003; Wakeley 1996). Using the 18 SNPs detected from our experimental analysis, 15 haplotypes were identified (Table 3). The haplotypes ranged in overall frequency in the turkey varieties with multiple birds from 0.009 to 0.333. Haplotype diversity ranged from 0.00 to 0.83 and from 0.00 to 0.38 based on the D-loop and 16S rRNA, respectively. Nucleotide diversities ranged from 0.00 to 0.08 for variants in the D-loop and from 0.00 to 0.001 in the 16S rRNA (Table 4). The parsimony network analyses in the variants detected are presented in Figs. 1 and 2 for 16R rRNA and D-loop, respectively. Four haplogroups based on the six 16S rRNA variants were entitled. The root haplogroup included the MW, WH and BBW varieties. The largest of haplogroup included all the heritage birds, the commercial and one wild turkey (Fig. 1). The other two haplogroups consisted only of wild varieties. The D-loop analysis also revealed, including the root, four haplogroups (Fig. 2). The root consisted of sequences from one wild and three

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Biochem Genet Table 2 Context and position in the turkey mtGenome sequence of the SNPs detected in the control (D-loop) region and 16S rRNA of the turkey mitochondrial genome

Region D-loop

a

Position within the reference sequence (GenBank accession number EF153719) in Genbank

Context

15,575

CTAAC(C/T)CCCCT

15,673

GTAC(T/Cb)ATATAC

15,674

GTACT(A/G)TATAC

15,677

CTGTA(T/Cc)ACTAT

15,679

GTATA(C/Tc)TATAT

15,686

ATATA(C/T)GTACT

15,708

ATGTA(G/Ac)ACGGA

15,740

TTCTCC(C/Ab)AATGA

15,741

TTCTCCC(T/-b)AATGA

15,762

AAACAT(-/Gb)CCAATGA

15,782

TCCTT(C/T)CTACC

15,793

CCCAA(C/Tc)ATCCA

15,800

ATCCAT(A/Gb)CCAACCC

15,808

AACCC(T/Cc)CAAGA

15,845

CATAC(C/T)TCTAA

15,893

CGTACCA(G/Ab)ATGGAT

15,953

TACTC(C/T)ATGAC

15,999

TCGCCCT(C/Tb)CTTGC

1,623

CCTCC(C/A)CCAGC

1,951

CGGCA(T/A)ATTAA

Alleles were found only in the in silico sequences

2,128

TCTTA(A/G)CTGTC

2,197

GACGA(G/A)AAGAC

c

2,279

TGGTC(G/A)ACATT

2,340

CACAA(T/C)TCTTC

b

Alleles were found both in the experimental and in silico sequences

16S rRNA

Positiona

heritage varieties. As expected, the breeds that comprised each of the haplogroups were different from those based on the 16S rRNA variants. Sequence divergence estimates among the turkey varieties and selected Galliformes based on 16S rRNA and D-loop sequences are presented in supplementary Tables S1 and S2, respectively. Divergence estimates between heritage and commercial turkey varieties were similar between the domestic and wild turkeys. For example, both the D-loop and 16 S rRNA-based divergence estimates showed that the wild turkeys including O, R, M, E subspecies had similar relatedness to each as to commercial and heritage birds. Phylogenetic relationships among the varieties based on both the 16S rRNA (Fig. 3) and D-loop (Fig. 4) were generally consistent with those from the network analysis (Figs. 1, 2). The 16S rRNA-based phylogenetic analysis showed that heritage and commercial varieties were separated by apparently equal distances from the common node, but the wild turkeys formed two divergent clusters (Fig. 3). The D-loop analyses revealed closer relationships among the wild-type turkeys including M, O and G with heritage varieties BR, MW and WH, while the wild E turkey variety was more closely related to BS and SB, and R was on the same

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Biochem Genet Table 3 Mitochondrial D-loop and 16S rRNA-based haplotype structure and frequency Haplotype

Haplotype sequencea

Frequency

VTMgD16H1

-C-A-T-C-C-G-C-C-T-C-T-C-A-A-G-G-T-

0.239

VTMgD16H2

-C-A-T-C-C-G-C-C-T-C-C-C-A-A-G-G-T-

0.333

VTMgD16H3

-C-A-C-T-C-G-C-T-C-C-C-C-A-A-G-G-T-

0.009

VTMgD16H4

-C-A-T-C-C-G-C-C-C-C-C-C-A-A-G-G-T-

0.017

VTMgD16H5

-T-A-C-T-C-G-T-T-C-T-C-C-A-A-G-G-T-

0.026

VTMgD16H6

-C-A-C-C-C-G-C-C-C-C-C-C-A-A-G-G-T-

0.009

VTMgD16H7

-C-G-T-C-C-G-C-C-T-C-C-C-A-A-G-G-T-

0.085

VTMgD16H8

-C-G-T-C-C-A-C-C-T-C-C-C-T-A-G-G-T-

0.009

VTMgD16H9

-C-A-T-C-T-G-C-C-T-C-C-C-A-A-G-G-T-

0.034

VTMgD16H10

-C-A-T-C-C-A-C-C-T-C-C-C-A-A-G-G-T-

0.034

VTMgD16H11

-C-A-T-C-C-G-C-C-C-T-C-C-A-A-G-G-T-

0.171

VTMgD16H12

-C-A-T-C-C-G-C-C-T-C-T-A-A-A-G-G-T-

0.009

VTMgD16H13

-C-A-C-T-C-G-C-T-C-C-C-C-A-A-A-G-C-

0.009

VTMgD16H14

-C-A-T-C-C-G-C-C-C-C-C-C-A-G-G-A-T-

0.009

VTMgD16H15

-C-A-C-T-C-G-C-T-C-T-C-C-A-A-G-G-C-

0.009

a

Haplotypes based on experimentally verified SNPs in both D-loop and 16S rRNA regions shown in Table 2

Table 4 Haplotype (Hd) and nucleotide diversity (p) in different turkey varieties

Turkey variety

D-loop

16S rRNA

Hd

p

Hd

p

Blue Slate

0.000

0.000

0.000

0.000

Spanish Black

0.417

0.001

0.000

0.000

Narragansett

0.000

0.000

0.000

0.000

Royal Palm

0.524

0.002

0.378

0.001

Bourbon Red

0.800

0.004

0.000

0.000

Midget White

0.378

0.002

0.000

0.000

Broad Breasted Bronze

0.694

0.004

0.000

0.000

Broad Breasted White

0.733

0.002

0.000

0.000

White Holland

0.000

0.000

0.000

0.000

Standard Bronze

0.600

0.002

0.000

0.000

British United Turkeys

0.833

0.027

0.000

0.000

Eastern Wild

0.644

0.003

0.200

0.001

Rio Grande Wild

0.725

0.018

0.143

0.001

Merriami’s Wild

0.700

0.006

0.000

0.000

Osceola Wild

1.000

0.084

0.833

0.001

branch with commercial, N and RP. The lack of congruence between the 16S- and D-loop-based phylogenetic analyses may be due to the different rates of evolution between the two mtDNA segments. It could also be due to the relatively small

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Fig. 1 Parsimony network (minimum spanning tree) of 933 bp of 16S rRNA-based haplotypes of sequences from turkey varieties. Each circle represents a unique haplotype. The line connecting two circles, independent of length, indicates a single base pair difference between the two haplotypes. The rectangle indicates the root haplotype based on 95 % probability. Filled dots on the line (between any two haplotypes) represent intermediate haplotypes (theoretical) not found in the present analysis. The size of each circle, although not to scale, is proportional to the frequency of the haplotype. The names of the varieties associated with each haplotype are listed in the circle. The superscripts C, W, H and NH represent commercial, wild, heritage and non-heritage varieties, respectively. The acronyms for the varieties are defined in Table 1

number of birds sampled from each variety. Depth and nature of sampling, however, are only important considerations if the separation of the populations is short. For example, Niu et al. (2002) only used five specimens per breed to study the origin and genetic diversity of five Chinese native chicken breeds based on D-loop sequences. Also, Drovetski (2002) only used 38 samples representing all 9 species of Locustella and 21 individuals from 10 species representing other warbler genera to study the mitochondrial phylogeny of Locustella and related genera. Among the species they evaluated, most of them only had one sample to represent. Here, two segments of the mtGenome sequence normally reported to be under different rates of divergence in the mitochondria genome were used to evaluate the genetic relatedness among turkey varieties. The relatively low level of variation in the D-loop and 16S rRNA suggest that, unlike other animals, existing varieties of the turkey are more closely related. In chickens, an earlier report by Guan et al. (2007) identified a total of 27 SNPs in the D-loop and, like the number in the present

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Biochem Genet Fig. 2 Parsimony network (minimum spanning tree) of 578-bp of D-loop sequences from different turkey varieties. Each circle represents a unique haplotype. The line connecting two circles, independent of length, indicates a single base pair difference between the two haplotypes. The rectangle indicates the root haplotype based on 95 % probability. The size of each circle, although not to scale, is proportional to the frequency of the haplotype. The names of the varieties associated with each haplotype are listed in the circle. The superscripts C, W, H and NH represent commercial, wild, heritage and non-heritage varieties, respectively

work, found only 5 in the 16S rRNA when diverse chicken varieties were screened. From comparisons of native Chinese chicken breeds, Niu et al. (2002) detected 24 SNPs in 539 bp of the D-loop region, about half the sequence evaluated here for the turkey. Mitochondrial nucleotide variants in the mtGenome of humans and other animals have been found to be associated with disease (Herrnstadt and Howell, 2004; Wallace 2005) and fitness (Castro et al. 2007). For example, a report by Arbustini et al. (1998) suggested that the mtDNA mutation may be associated with dilated cardiomyopathy. Such reports are however very few in birds and other livestock species. In the chicken, only one suggestive association between variation in the mtGenome and a phenotype has been reported (Li et al. 1998). In addition to providing insight into genetic relatedness among diverse turkey varieties, the SNPs identified here may provide a foundation to begin to more widely evaluate the role of the mitochondrial genome in differences in performance traits. Further, these SNPS and haplotypes may be useful in avian forensics. Recently, Rojas et al. (2010) described a PCR-based procedure that could distinguish among meats from different birds, both commercial and wild. Since it is a popular game bird, and sometimes a concern in illegal hunting, the variants described here could form a foundation for identifying turkey and resolving disputes.

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Fig. 3 The 16S rRNA sequences-based maximum likelihood tree of commercial, non-heritage, heritage and wild turkeys. The maximum likelihood tree was congruent with those from neighbor joining, minimum evolution and maximum parsimony methods. Confidence of the groupings was estimated using 1,000 bootstrap replications. The Arabic numerals at the base of a node are the bootstrap values derived from the maximum parsimony, neighbor joining and minimum evolution analysis, respectively. Bootstrap values lower than 50 % are not presented. The superscripts C, W, H and NH represent commercial, wild, heritage and nonheritage varieties, respectively

One can argue that our efforts here to answer the important question of the origin of the commercial turkey relative to the wild and heritage varieties, although important, have not been fully addressed because of the limited numbers of birds and varieties. Obviously, the resources we have described can be used by others in the community to extend the phylogenetic analyses conducted in the present work. We are aware, of course, that there have been other investigations of the history of the domestic turkey. Using 438 bp of the DNA sequence from the control region, for example, Speller et al. (2010) reported that domestication of the turkey was a complicated process that may have involved intense breeding and selection. The very close relationships described here, which involved the whole D-loop and 16S rRNA, appear to support this.

Conclusions By using mtDNA sequences from both experimental and in silico sources, this study represents the most comprehensive evaluation to date of relatedness among both wild and domesticated turkeys. Although the sample sizes of the varieties were

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Fig. 4 D-loop DNA sequence-based maximum likelihood tree of the relatedness of commercial, nonheritage, heritage and wild turkey varieties. The tree, constructed using the maximum likelihood algorithm, was congruent with those from neighbor joining, minimum evolution and maximum parsimony methods. Confidence of the groupings was estimated using 1,000 bootstrap replications. The Arabic numerals at the base of a node are bootstrap values derived from the maximum parsimony, neighbor joining and minimum evolution analyses, respectively. Bootstrap values lower than 50 % are not shown. Superscripts are defined as follows: C, commercial; W, wild; H, heritage; NH, nonheritage. *Polymorphism detected in the multiple sequences included in the analyses. Therefore, a consensus sequence from both experimental and in silico sequences was used for the analysis

relatively small for intra-varietal diversity, because of the limited number of birds raised by fanciers and enthusiasts and cost considerations, some inferences regarding inter-varietal relationships could be made from our SNP- and haplotypebased data. The wild and commercial domestic turkeys appear to be highly variable with the highest number of haplotypes, and the heritage turkeys and Broad Breasted varieties were the least variable. Based on the low genetic distance estimates, our analysis appears to provide mtDNA sequence-based support for the view [American Standard of Perfection (APA 2001)], based primarily on morphological data, that turkeys may be a single variety. The relationships defined from the D-loop sequences appear to be consistent with previous reports by Smith et al. (2005) from nuclear DNA markers, which showed RP to be more closely related to N but distant from BS and SB heritage varieties. Acknowledgments We are grateful to Dr. Olin Rhodes, Purdue University, for DNA from some wild turkeys and to Dr. Jake Tu, Biochemistry Department, Virginia Tech, for help with the phylogenetic

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Biochem Genet analyses. Funding for this work was provided in part by Virginia Tech, the National Institutes of Aging, National Institutes of Health, and National Institute of General Medical Sciences.

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