Research Note Microsatellite Marker-Based Genetic Analysis of Relatedness Between Commercial and Heritage Turkeys (Meleagris gallopavo) D. Kamara, K. B. Gyenai, T. Geng, H. Hammade,1 and E. J. Smith2 Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg 24061 gansett, Bourbon Red, Blue Slate, Spanish Black, and Royal Palm. Primer pairs specific for 10 previously described turkey microsatellite markers were used. The phylogenetic analysis showed that the Blue Slate, Bourbon Red, and Narragansett were genetically closely related to the CO strain, with a Nei distance of 0.30, and the Royal Palm and Spanish Black were the least related to the CO strain, with Nei distances of 0.41 and 0.40, respectively. The present work provides a foundation for the basis of using heritage turkeys to genetically improve CO populations by introgression.
ABSTRACT The turkey is second only to the chicken in importance as an agriculturally important poultry species. Unlike the chicken, however, genetic studies of the turkey continue to be limited. For example, to date, many genomic investigations have been conducted to characterize genetic relationships between commercial (CO) and non-CO chicken breeds, whereas the nature of the genetic relatedness between CO and heritage turkeys remains unknown. The objective of the current research was to use microsatellites to analyze the genetic relatedness between CO and heritage domestic turkeys including Narra-
Key words: heritage turkey, commercial turkey, microsatellite, genetic relatedness, genetic analysis 2007 Poultry Science 86:46–49
Genetic variation within and among CO turkey populations was previously evaluated by Smith et al. (1996), Zhu et al. (1996), and Ye et al. (1998). Though Smith et al. (2005) also analyzed the genetic relatedness of 5 heritage turkey varieties, the relatedness between the CO and heritage turkeys has never been investigated. Information regarding the genetic relatedness between CO and heritage turkey varieties can be used for genetic improvement of the different turkey strains such as the introgression of novel genes important for economic traits including disease resistance. Commercial turkeys have been highly selected for increased BW and growth rate. In turn, they have a relatively higher rate of susceptibility to diseases (Li et al., 2001; Huff et al., 2005) that may be due to a relatively narrow genetic background. There are only a few highly selected strains of the Large White variety, and these may lack the genetic diversity they need to be able to resist or tolerate diverse disease conditions. Indications are that emphasis in the turkey breeding programs on meat quantity and quality, but not on disease resistance, could have led to the increased vulnerability of the CO turkey to diseases (Christman and Hawes, 1999). The relatively high vulnerability of CO birds to various disease conditions is of serious concern to the turkey industry. Because there is no comprehensive linkage map available for the turkey, exploring the turkey genome and thus identifying QTL for economic traits such as disease resistance in CO birds is impossible. The need to evaluate the status of turkey genetic diversity is attracting attention from poultry scientists, because it is now realized that
INTRODUCTION Heritage turkeys have become of interest to the United States consumer (Shriver, 2003). As their number increases and more farmers grow them for the US market, the need to increase biological information about them increases. Though the turkey varieties are considered a single breed (American Poultry Association, 2001), evidence is emerging about significant strain differences among the populations (Hartman et al., 2006) and between the heritage turkeys and commercial (CO) birds (Gyenai, 2005). In his unpublished thesis work, Gyenai (2005) evaluated phenotypic differences in CO and heritage turkey populations for their response to toxic levels of furazolidone to make inferences about the genetic basis of the incidence and severity of dilated cardiomyopathy in turkeys. Variety differences in the turkey’s response to diets containing furazolidone were observed, suggesting that an animal’s response to furazolidone-induced dilated cardiomyopathy is genetically based. More recently, among heritage varieties, Hartman et al. (2006) observed significant strain differences for plasma uric acid concentration, a biomarker for diverse phenotypes including oxidative stress.
2007 Poultry Science Association Inc. Received June 27, 2005. Accepted August 10, 2006. 1 Visiting Fulbright scientist from Aleppo University, Syria. 2 Corresponding author:
[email protected]
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RESEARCH NOTE Table 1. Pairwise genetic distance estimates based on microsatellite allelic frequencies in 6 turkey populations Genetic group1 NA BR BS SB RP CO
Genetic group NA
BR
BS
SB
RP
CO
0.000000 0.089772 0.068903 0.131444 0.108381 0.306311
— 0.000000 0.139911 0.153629 0.180905 0.299395
— — 0.000000 0.152738 0.069508 0.297193
— — — 0.000000 0.211093 0.401194
— — — — 0.000000 0.411111
— — — — — 0.000000
1 NA = Narragansett; BR = Bourbon Red; BS = Blue State; SB = Spanish Black; RP = Royal Palm; CO = commercial strain.
the diversity of the non-CO turkey varieties are essential genetic resources that will enable breeders to improve their birds’ health and vigor or to respond to changing environmental conditions, production systems, or consumer needs (Christman and Hawes, 1999). The objective of the current research was to conduct molecular genetic analysis of relatedness between CO and heritage turkey varieties including Bourbon Red (BR), Blue Slate (BS), Narragansett (NA), Royal Palm (RP), and Spanish Black (SB). Understanding this genetic relationship between CO and heritage turkeys may be useful in breeding programs that could involve the introgression of novel genes important for economic traits including disease resistance.
at 95°C, followed by 38 cycles of denaturation for 45 s at 95°C; annealing for 45 s at optimized temperature (Table 1) and extension for 45 s at 72°C; with final extension for 7 min at 72°C. Electrophoresis of the amplified products was performed on a 4% metaphor agarose gel with 1% ethidium bromide at 40 V for 7 h). To test the efficiency of metaphor agarose in distinguishing among microsatellite alleles, PCR products of 2 SB samples amplified by the TUM20 primer and 1 amplified by the RHT0011 primer were randomly selected and genotyped using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA), and allele sizes were observed using GeneScan 3.7 (Applied Biosystems).
Statistical and Phylogenetic Analysis MATERIALS AND METHODS Samples Twenty-five birds were used from each of CO, NA, BR, BS, SB, and RP varieties. Birds from the heritage varieties were obtained from Privett Hatcheries (Portales, NM) as day-old birds and raised at the Virginia Tech Turkey Farm using standard protocols. Though the genetic background of the heritage turkeys is unknown, the varieties are true breeding (Smith et al., 2005). Blood was collected by brachial venipuncture in tubes containing 0.5 M EDTA. Aliquots of 50 L of blood were used for genomic DNA isolation according to Smith et al. (1996). Isolated genomic DNA from each sample was air-dried and dissolved in sterile water to a concentration of 50 ng/L before use in PCR.
Microsatellite DNA Analysis The sequences for the 10 primer pairs used in the present work are presented in Table 1. These primers were previously described by Burt et al. (2003). The PCR amplifications were carried out in a final volume of 10 L. Each reaction contained 50 ng of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM Mg2+, 200 M each deoxynucleotide triphosphate, 160 ng of each primer, and 1 U of AmpliTaq DNA Polymerase (Eppendorf, Westbury, NY). Amplifications were carried out in a Mastercycler Gradient Thermocycler (Eppendorf) under the following conditions: an initial denaturation step of 5 min
The microsatellite alleles were analyzed by direct count within each population, and allele frequencies were computed according to Hartl and Clark (1997). Genetic distance was estimated according to Nei (1972), and these estimates were used to construct a consensus tree with bootstrap values using the neighbor-joining method in PHYLIP (Felsenstein, 1989) and visualized using TREEVIEW (Page, 1996).
RESULTS AND DISCUSSION Alleles observed using the metaphor agarose gel were consistent with those observed by Burt et al. (2003) on the ABI PRISM 3100 Genetic Analyzer for TUM20 and RHT0011. An example of the metaphor-resolved alleles is presented in Figure 1 for primer TUM20 with SB birds as template for the PCR. Two alleles were observed for ADL0023; 3 for the RHT0024, RHT0294, RHT0095, RHT0009, RHT0131, RHT0011, and TUM16 loci; and 4 for TUM20 and RHT0216. The genetic distance estimates among the 6 populations are presented in Table 1. The lowest genetic distance was observed between the BS and the NA (0.07) and RP (0.07). The highest genetic distance was observed between the CO and the SB (0.40) and RP (0.41). The extended majority rule consensus tree with bootstrap values (Figure 2) depicts the genetic relationship among the 6 different populations, clustering the RP with the BS and the BR with the CO population. The clustering of the turkey populations was unstable, as indicated by the low bootstrap values, suggesting that these
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Figure 1. Metaphor agarose gel patterns of TUM20 amplicons produced using template from Spanish Black (SB) heritage turkeys. Alleles ranged from ∼110 to 170 bp. The M1 and M2 represent DNA ladder I and II, respectively (Gene Choice Inc., Frederick, MD). Each numbered lane contains the amplicon from an individual SB bird.
turkey varieties were very closely related and therefore no group clustering was observed at a significantly high frequency. Phylogenetic trees showing genetic relationships among taxa are accepted by many biologists in diverse
Figure 2. A neighbor-joining tree using Nei’s (1972) distance for all population samples at 10 microsatellite loci. Bootstrap values indicated at the nodes were calculated from 100 resamplings. NA = Narragansett; BR = Bourbon Red; BS = Blue State; SB = Spanish Black; RP = Royal Palm; and CO = commercial strain.
areas of research, because they can provide meaningful insights into different biological phenomena (Soltis and Soltis, 2003). Examples of their use in diverse areas of research include evaluating population history and genetic variation across historical ranges of animals for implications of animal conservation (Eizirik et al., 2001; Mock et al., 2002). Here, the genetic relatedness between CO and heritage turkeys was investigated using microsatellites. These markers are valuable for assessing pedigree, family, and population relationships due to their abundance in the genome and their ability to reveal polymorphisms (O’Brien et al., 1999). The genetic relatedness among the heritage varieties appears to be consistent with that observed by Smith et al. (2005), but with a slight difference. When a tree was constructed using only the heritage varieties (data not presented), the SB clustered with the BR, which was the same observed by Smith et al. (2005). However, the tree showed a closer relatedness between RP and BS, unlike that observed by Smith et al. (2005), which showed closer relatedness between RP and NA. Considering that the NA was the second closest strain to RP, results of the current research and that of Smith et al. (2005) were actually showing that the BS, NA, and RP heritage varieties were genetically closely related. The current studies provide a foundation on which CO turkeys could be improved using heritage turkeys. Though the bootstrap values were generally low, the use of randomly distributed microsatellite markers provides a level of support for the relationships estimated in the present work. Explanations that have been advanced for low bootstrap values, including a short internal branch (Hirt et al., 1999), could be responsible for the lack of statistical significance in the estimates observed here. However, the lack of statistical significance appears to support the long-held view that all turkey varieties are a single breed, because the use of 10 markers distributed on different chromosomes may represent the most unbiased estimate of the relationships to date. Using agarose in the genotyping will facilitate the use of these markers
RESEARCH NOTE
by a larger number of laboratories. For example, agarose gel-based genotyping of these microsatellite markers could be used to provide additional support for estimates of genetic relatedness among CO strains previously reported by Smith et al. (1996), Zhu et al. (1996), and Ye et al. (1998).
ACKNOWLEDGMENTS We thank the staff of the Virginia Tech turkey facility for their help. Financial support was provided by the Virginia Agricultural Council (Midlothian) and the National Human Genome Research Institute (Bethesda, MD). This work is part of the thesis submitted to the Virginia Tech Graduate School in partial fulfillment of the MS degree for Davida Kamara. We are also grateful to the editor and 2 anonymous reviewers for suggestions that were used to extensively revise the manuscript.
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