Progress 07/01/02 to 06/30/07
Outputs
OUTPUTS: Among the turkey genome projects conducted in our lab in the past year, the following accomplishments and progress were madefrom SNP analysis of functionally important turkey genes: Determining variation in genes is fundamental to understanding their function in the disease state. Cardiac troponin T (cTnT) and phospholamban (PLN) genes have been implicated in the incidence of dilated cardiomyopathy (DCM) in human and model species. In order to investigate the role of these two candidate genes for DCM in the turkey, Meleagris gallopavo, knowledge of sequence variants and map position distribution is necessary. To this end, a total of 1,854 and 1,771 bp of cTnT and PLN gene sequence, respectively, was scanned for single nucleotide polymorphisms (SNPs) in a random bred population. A total of 15 SNPs was identified in the cTnT and PLN genomic sequences. Nine haplotypes, five in cTnT and 4 in PLN were identified. Observed heterozygosities (0.02-0.39) in the turkey population were
low for both genes. Within each gene, one SNP corresponding to a restriction enzyme site was identified and used to develop a PCR-RFLP genotyping assay. The PLN gene was genetically mapped to turkey chromosome 2, equivalent to Gallus gallus chromosome (GGA) 3, and cTnT mapped to a turkey microchromosome. Though limited because of the relatively small sample size of 55 birds, the data from SNP analysis of PLN and cTnT provide a foundation for evaluating the function of cTnT and PLN in the turkey. Knowledge of the distribution of the SNPs and haplotypes will facilitate future association and linkage studies.
PARTICIPANTS: Kwaku Gyenai Jun Xu Xiaojing Guan Davida Kamara Kuan-chin Lin Roger Coulombe, Uta State University Kent Reed, University of Minnesota Bill Pierson, Virginia-Maryland Regional College of Veterinary Medicine
TARGET AUDIENCES: Academic and industry
Impacts
We have described resources that will be useful in future studies of genetic contributions to round heart disease and related abnormalities (which a rereported to account for about 5% in annual losses to farmers) in the turkey. The emphasis here was on identifying and mapping SNPs and developing haplotypes to provide resources for assigning function to troponin T and phospholamban.
Publications
- Jena Highkin, Kwaku Gyenai, Olivia Ho-Shing, James Martin Jr., Craig Thatcher, and Ed Smith, 2007. Molecular and Biochemical Analysis of Oxidative Stress in Toxin-Induced Dilated Cardiomyopathy Turkey (Meleagris gallopavo). Abstract. Paper presented at 2007 IGERT-MILES/MIIS Research Symposium.
- Lin, K.C., Xu J., Kamara, D., Geng, T., Gyenai, K., Reed, K.M., and Smith, E.J.. DNA sequence and haplotype variation in two candidate genes for dilated cardiomyopathy in the turkey Meleagris gallopavo. Genome. 2007 May;50(5):463-9.
- Kamara, D., Gyenai, K.B., Geng, T., Hammade, H., and Smith, E.J. 2007. Microsatellite marker-based genetic analysis of relatedness between commercial and heritage turkeys (Meleagris gallopavo). Poult Sci. 2007 Jan;86(1):46-9.
- Gyenai, K., J. Xu, T. Geng, L. Pyle, and C. Larson, 2007. Oxidative stress and toxin-induced dilated cardiomyopathy in the turkey (Meleagris gallopavo). Poster. Annual meeting of Poultry Science Association, San Antonio, TX. Poult. Sci. 86: Suppl. I. Abstract.
- Guan, X., K. Gyenai, C. Larson, and E. Smith. 2007. Age-specific species variation in oxidative stress in birds. Poster. Annual meeting of Poultry Science Association, San Antonio, TX. Poult. Sci. 86: Suppl. I. Page 87. Abstract M265.
- Guan, X., Kwaku B. Gyenai, Jun Xu, Tuoyu Geng and Edward J. Smith, 2007. The complete sequence of the turkey (meleagris gallopavo) mitochondrial genome. Chicken Genomics Workshop, 2007. Barcelona, Spain. Abstrac. Poster.
- Smith, E., Xiaojing Guan, Kwaku Gyenai, Kuan-chin Lin, and Jun Xu, 2007. Dissecting the turkey genome. Abstract. Poster presented at the Second Annual Deans Forum. Virginia Tech. November 5-7, 2007.
- Guan, X., K. B. Gyenai, C. T. Larson and E. J. Smith. Biochemical analysis of oxidative stress in diverse avian species. Deans' Forum on Health, Food, and Nutrition, Blacksburg, VA. November 5, 2007.
- Guan, X. 2007. Genomic and biochemical analysis of oxidative stress in birds with diverse longevities. Virginia Tech. 170 pp. PhD Dissertation.
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Progress 10/01/05 to 09/30/06
Outputs
Heritage turkeys have become an attractive food item for a significant fraction of the US consumer. Among the turkey genome projects conducted in our lab in the past year, the following accomplishments and progress were made: 1. The objective of the current research was to use microsatellites to analyze the genetic relatedness among commercial and heritage domestic turkeys including Narragansett, 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 commercial strain with Nei distance (D) of 0.30, and the Royal Palm and Spanish Black were the least related to the commercial strain with D=0.41 and D=0.40, respectively. The present work provides a foundation for the basis of using heritage turkeys to genetically improve commercial populations by
introgression. 2. Determining variation in genes is fundamental to understanding their function in the disease state. Cardiac troponin T (cTnT) and phospholamban (PLN) genes have been implicated in the incidence of dilated cardiomyopathy (DCM) in human and model species. In order to investigate the role of these two candidate genes for DCM in the turkey, Meleagris gallopavo, knowledge of sequence variants and map position distribution is necessary. To this end, a total of 1,854 and 1,771 bp of cTnT and PLN gene sequence, respectively, was scanned for single nucleotide polymorphisms (SNPs) in a random bred population. A total of 15 SNPs was identified in the cTnT and PLN genomic sequences. Nine haplotypes, five in cTnT and 4 in PLN were identified. Within both genes, tag SNPs were identified and used to develop PCR-RFLP genotyping assays. The PLN gene was mapped to turkey chromosome 2, and cTnT mapped to a turkey microchromosome. The data provide a foundation for evaluating the
function of cTnT and PLN in the turkey. 3. The mitochondrial genome (mtGenome) is an extra-chromosomal and maternally inherited DNA molecule whose expression is essential in vertebrate biology for processes such as energy production, metabolism, cellular homeostasis, and apoptosis. Despite this importance, the mtGenome has been very little studied in the turkey, for which there is no publicly available whole genome mitochondrial sequence. In this study, we used PCR-based methods with 37 primers developed from mitochondrial DNA of the chicken and other species to develop a complete turkey mtGenome sequence (GenBank accession number: EF153719). We then used two pairs of primers to screen for single nucleotide polymorphisms (SNPs) in the D-loop and 16S rRNA of the new sequence. The SNPs and the whole genome sequence were further used to establish and validate phylogenetic relationships among heritage turkeys and between the turkey and 47 different avian species for which mitochondria
sequences have previously been reported. The SNPs will be useful in subsequently defining a role for the turkey mitochondria in metabolic disorders.
Impacts
Round heart disease or dilated cardiomyopathy is reported to cause more than $200 million in losses annually, primarily due to mortality of birds between two and four weeks of age. Our research work has described two genes that appear to be responsible for some of the incidence and severity of the disease. Since the disease also affects humans and is the primary cause of heart disease, our research also provides additional information useful for reducing or treating the incidence of heart disease.
Publications
- Lin KC, Gyenai K, Pyle RL, Geng T, Xu J, Smith EJ. 2006. Candidate gene expression analysis of toxin-induced dilated cardiomyopathy in the turkey (Meleagris gallopavo). Poultry Sci. 85(12):2216-21.
- Hartman S, Taleb SA, Geng T, Gyenai K, Guan X, Smith E. 2006. Comparison of plasma uric acid levels in five varieties of the domestic turkey, Meleagris gallopavo. Poultry Sci. 85(10):1791-4.
- Gyenai, K.B. and Smith, E. J. 2006. Differences among turkey (Meleagris gallopavo) varieties for the difference and severity of toxin-induced dilated cardiomyopathy. Poultry Sci. 85. W52, Abstract. Page 121. Poster presented at the 2006 Poultry Science Association meeting, Edmonton, Canada.
- Guan, X., K. B. Gyenai, F. A. McNabb, and E. J. Smith. 2006. An assessment of antioxidant and oxidant status in diverse poultry species. Poultry Sci. 85. M34, Abstract. Page 15. Poster presented at the 2006 Poultry Science Association meeting, Edmonton, Canada.
- Lin, KC. Candidate gene expression and SNP analysis of toxin-induced dilated cardiomyopathy in the turkey. 2006. MS. Thesis. Virginia Tech, Department of Animal and Poultry Sciences.
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Progress 10/01/04 to 09/30/05
Outputs
Heritage turkeys have become an attractive food item for a significant fraction of the US consumer. Previously, and as reported last year, we evaluated the genetic relatedness of five heritage turkey strains using genomic reagents developed in our laboratory. To complement the molecular genetic analyses of differences among these strains, our lab recently started evaluating differences among the strains for phenotypes that include feed efficiency, growth rate and round heart disease or dilated cardiomyopathy (DCM), an abnormality that affects approximately 5 percent of commercial turkeys. Based on these differences, we have initiated association studies between candidate genes and turkey phenotypes: i. Expression variation of two candidate genes: Dilated cardiomyopathy is a disease that affects different animals including humans and poultry. Pathologically, DCM can cause enlarged ventricles and cavity dilatation in hearts. Recently, our lab showed that the response of
turkeys to a toxin that induces DCM may be genetic. Here, our objective was to compare the level of mRNA expression of two candidate genes using reverse transcription polymerase chain reaction (RT-PCR) including troponin T (TNT) and phospholamban (PLN) in DCM-affected and -unaffected hybrid turkeys. Relative to control birds, the expression levels of both TNT and PLN genes in cardiomyoathic hearts were significantly lower (0.18-fold and 0.27-fold). In the liver, though both genes were also differentially expressed in DCM-affected birds, only TNT differential expression was significant. The data suggest that TNT and PLN, as has been reported in spontaneously occurring DCM, maybe candidate genes for toxin-induced dilated cardiomyopathy in vertebrates. ii. SNP and haplotype analysis of candidate genes for dilated cardiomyopthy in the turkey: Since TNT and PLN have both been implicated in the incidence of DCM, complementary investigations were initiated by scanning the TNT and PLN genes
for single nucleotide polymorphisms (SNPs) which could be useful in evaluating their role in the incidence of stress-induced dilated cardiomyopathy. While a TNT1 sequence is available, we used the recently released chicken genomic DNA sequence to develop a turkey phospholamban (tPLN) sequence. A total of 1059 and 666 bp of TNT and tPLN, respectively, were scanned for SNPs using unrelated turkeys from five strains that differ in the incidence and severity of stress-induced cardiomyopathy. The turkey sequence of the chicken PLN-derived amplicon had a 90 percent identity with the GenBank chicken PLN reference sequence. Within these sequences, a total of 5 SNPs were identified in each of the tPLN and TNT genes. The 5 SNPs in TNT were all in introns and were used to identify six haplotypes. Both the SNPs and haplotypes of the TNT gene were shown to be present in heritage and commercial turkeys. These genomic reagents may be useful to us and others interested in assigning phenotypes to
genotypes in the turkey. Additionally, they will help us validate observations in other model species of the effect of TNT and PLN on DCM, a major cause of heart failure in humans and other animals.
Impacts
Heritage turkeys have become an attractive food item for a significant fraction of the US consumer. Our current reseach provides information that will increase the utility of these strains in improving commercial turkeys as well as to develop breeding programs to improve the performance of the heritage birds. The current work also describes DNA sequence variants and other genomic reagents that will facilitate selection of breeders for economic traits such as round heart disease. The genome and SNP analysis now provide us an ability to assign these genes to phenotypes that influence the productivity of turkeys.
Publications
- Lin, K.C. and Smith E.J. 2005. Genome analysis of candidate genes for dilated cardiomyopathy in the turkey. Abstract. Poster presentation. Abstract. Poster. May 8-11. Cold Spring Harbor. Laboratory. NY. Chicken genomics and development.
- Gyenai, K. B., and Smith E. J., 2005. Genetic analysis of dilated cardiomyopathy in the turkey, Meleagris gallopavo. Abstract. Poster. 25th Annual MANRRS Conference, Pittsburgh, PA March 27-31.
- Gyenai, K.B., and Smith E. J., 2005. Genetic analysis of dilated cardiomyopathy in the turkey, Meleagris gallopavo. Abstract. Oral. Southern Poultry Science Society Annual Meeting. January, 2005. Atlanta, GA.
- Smith, E.J., Geng, T., Kamara, D. Lin, K.C. and Hartman, S. 2005. Avian genomes: resources for biomedical research. Current Genomics 6: 75-80.
- Smith, E.J., L. Shi, and Z. Tu, 2005. Gallus gallus aggrecan gene-based phylogenetic analysis of selected avian taxonomic groups. Genetica 124:23-32.
- Lin, K.C. and Edward J. Smith, 2005. Candidate genes expression profile for dilated cardiomyopathy in the turkey, Meleagris gallopavo. Abstract. Poster. Second Annual Research Symposium, Edward Via College of Osteopathic Medicine, Blacksburg, Virginia.
- Amenkhienan, E., and Edward Smith. 2005. In silico analysis of alternative splicing in Gallus gallus genes controlling circadian rhythm. Abstract. Poster. Multicultural Academic Opportunities Program Summer Research Symposium. Blacksburg, VA.
- Guan, X., and Edward J. Smith. 2005. Comparative evaluation of oxidative stress in birds that differ in longevity. Abstract. Poster. Second Annual Research Symposium, Edward Via College of Osteopathic Medicine, Blacksburg, Virginia.
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Progress 10/01/03 to 09/30/04
Outputs
Knowledge of the relationships among commercial and non-commercial turkey strains is currently negligible, but is potentially useful for genetic improvement of this economically important poultry species. The genetic relatedness of 6 non-commercial domesticated turkey strains to two commercial populations has been investigated using genomic reagents previously described from our lab. The commercial strains were from two independent major turkey breeding companies and are designated as Commercial 1 and Commercial 2. The non-commercial turkey strains analyzed included Royal Palm, Bourbon Red, Narragansett, Blue Slate, Spanish Black and Buff. Three marker systems were used including a single nucleotide polymorphism-based polymerase chain reaction-restriction fragment length polymorphism, multiple DNA fragments from randomly amplified polymorphic DNA analyses, and two alleles from microsatellite or simple sequence repeat analyses. Pair-wise genetic distance estimates
between strains ranged from 0.001 to 0.361 between Commercial 2 and Buff and between Bourbon Red and Commercial 1, respectively. Together, the data suggest that Spanish Black and Royal Palm belong to a separate clade, while Bufff forms another with Commercial 1, Commercial 2 and Bourbon Red. The current investigations provide, for the first time, information about the relatedness of non-commercial domesticated turkeys to commercial populations. The data could be useful in commercial turkey breeding programs that involve introgression of novel genes to improve the performance of birds including disease resistance.
Impacts
DNA markers derived from the turkey genetic map have given us the ability to determine if the phenotypic differences that breeders observe among different strains have a biological basis. This information is of interest to diverse industry groups, the public as well as the American Livestock Breed Conservancy.
Publications
- Smith, E. J., Geng T, Long E, Sponenberg DP, Pierson FW, Larsen CT, and Gogal R, 2004. Molecular analysis of the relatedness of five domesticated turkey strains. Biochemical Genetics, in Press.
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Progress 10/01/02 to 09/30/03
Outputs
The turkey remains one of the few economically important livestock and poultry species whose genome is very little understood. Along with others, our efforts at dveloping a useful turkey map and thus the tools for efficient genetic analysis continues. With our collaborators, we mapped new microsatellite markers and tested the utility of the turkey markers in revealing chicken genetic information. As reported in the manuscript in press, twenty linkage groups (including the Z chromosome) containing 74 markers have been established, along with 37 other unassigned markers. This map will lay the foundations for further genetic mapping and the identification of genes and quantitative trait loci in this economically important species. Genome comparisons, based on genetic maps, with related species such as the chicken would then also be possible. All primer information, polymerase chain reaction (PCR) conditions, allele sizes and genetic linkage maps can be viewed at
http://roslin.thearkdb.org/. The DNA is also available on request through the Roslin Institute. An additional turkey work has involved using the map-based markers establish the genetic relatedness of non-commercial domesticated turkeys. Previously, our analyses included five strains: the royal palm, narragansett, blue slate, bourbon red, and spanish black. We have now added commercial strains and the buff. These analyses continue to show distinctness of only a few of the birds. Only royal palm, for example, appears to have a within-strain genetic similarity that is higher than between strains in pair-wise comparisons. These data now form the baseline for evaluating the effect of genetic distance on heterosis for reproductive traits in the turkey.
Impacts
DNA markers derived from the turkey genetic map have given us the ability to determine if the phenotypic differences that breeders observe among different strains have a biological basis. This information is of interest to diverse industry groups as well as the American Livestock Conservancy.
Publications
- Burt, D.W., Morrice, D.R., Sewalem, A., Smith, J, Paton, I.R., Smith, E.J., Bentley, J., and Hocking, P.M, 2004. Preliminary linkage map of the turkey (Meleagris gallopavo) based on microsatellite markers. Animal Genetics (in Press).
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Progress 10/01/01 to 09/30/02
Outputs
The turkey is one of only a few agriculturally important animal and poultry species for which there is no genetic linkage map. In addition, our understanding of the genetic differences among domesticated turkey strains of diverse origins and backgrounds and among wild and commercial turkeys remains negligible. We have developed a first-generation linkage map for the turkey using a diverse set of markers previously described. We also investigated use of previously described genomic reagents for turkeys and chickens to distinguish among five turkey varieties including Blue Slate, Bourbon Red, Narragansett, Royal Palm, and Spanish Black and two commercial populations. The molecular analyses, all involving PCR methods, were microsatellite-, sequence tagged site-, and RAPD-based. Evidence from these analyses suggests that the Royal Palm and Narragansett are more closely related to one another while more distantly related to the other three traditional varieties.
Impacts
The comparative genome analysis work of the turkey varieties provides genomic resources, including three primer-pairs each specific for a unique microsatellite-containing sequence, which may contribute to the burgeoning efforts of wildlife biologists to understand the population histories of wild turkey strains including subspecies of wild turkey. When combined with phenotypic information, the data developed from the molecular analysis may help us understand the value of non-commercial turkey varieties in the on-going turkey genome analysis efforts and mapping as well as to resource population development for QTL mapping. Also, gene mapping, QTL identification, and comparative mapping studies in the turkey are now possible with the availability of a first-generation linkage map.
Publications
- E. K. Latch, E. J. Smith, and O. E. Rhodes, Jr. 2002. Isolation and characterization of microsatellite loci in wild and domestic turkeys (Meleagris gallopavo). Molecular Ecology Notes 2:176-178.
- David R. Morrice, Asheber Sewalem, Jacqueline Smith, Ian R. Paton, Ed J. Smith, Paul M. Hocking, James Bentley, Steve Lerne and David W. Burt, 2003. Preliminary linkage map of the Turkey (Meleagris gallopavo) using microsatellite markers developed from an enriched genomic library. Animal Genetics, in Press.
- Edward J. Smith, Elizabeth C. Long, Padma Billam, F. William Pierson, D. Phillip Sponenberg, Calvert Larsen, Robert Gogal, 2002. Molecular analysis of relatedness among five turkey varieties, Poultry Science, Abstract of presentation at 91st Annual Meeting.
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