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Biochemical and genomic studies of dilated cardiomyopathy in the turkey 

Kwaku Gyenai, doctoral candidate  

As a major cause of heart failure, dilated cardiomyopathy (DCM) causes significant losses in both time and in the financial resources devoted to preventing the mortality and morbidity caused by heart disease that could result from DCM. In both humans and animal models, DCM can be either spontaneous (idiopathic) or stress-induced by environmental factors. The etiology of both types remains very little understood. Understanding the biochemical and molecular basis of either DCM may be helpful for the development of tools to reduce the incidence and severity of heart disease. As a consensus animal model for DCM, the domesticated turkey, Meleagris gallopavo , provides a resource that has been used to evaluate factors that influence stress-induced cardiomyopathy. Using experience we have gained in our long-term turkey genomics research, we propose to evaluate genomic and biochemical factors that may be responsible for differences we have detected among turkey strains for the incidence of stress-induced DCM. Specific aims of the project include:

  1. Identify the genetic basis (qualitative or quantitative) of the turkey's response to stress that induces DCM using distinct populations consisting of parental strains, backcrosses, F2s and F3s;
  2. Search, using candidate genes and genome-wide scans and gene expression profiling, for nuclear and mitochondrial genes that control stress-induced DCM in the turkey;
  3. Evaluate differences in oxidative stress among DCM-affected and unaffected birds to determine if the turkey's response to furazolidone is influenced by its oxidative stress level or vice versa that may be correlated with mitochondrial DNA mutations.

In carrying out these aims, we will test the hypothesis that genetic factors, including mitochondria DNA mutations, are responsible for differences among turkey's in their response to stress that induces DCM. This project will provide resources that will significantly aid in our understanding of the biochemical and genetic factors that underlie DCM. Additionally, it will facilitate the identification and validation of the genes that have been reported to influence DCM.

Genome Analysis of the Budgerigar: an Avian model for aging

Xiaojing Guan, doctoral candidate

The free radical theory of aging, which postulates an association between oxidative stress and longevity, remains without a consensus. We propose to use body-weight-matched birds of varying longevities to determine the association, if any, of level of oxidative stress and average life span. The primary hypothesis that we are testing is that differences among avian species for longevity are due to oxidative stress which also leads to differences among birds in the mutation load of the mitochondria. The validity of this hypothesis will be tested through two specific aims: 1. Evaluate age-associated changes in oxidative stress in four long- and short-lived birds; 2. Evaluate age-associated changes in mutations of the mitochondrial genome of the four species. The first specific aim is being conducted by using four biomarkers that have been used as indices of in vivo oxidative stress in animals: plasma uric acid, glutathione (total, oxidized, and reduced), thiobarbituric Acid-Reactive Substances, and malondialdehyde in whole blood collected in immature, adult and post-reproductive birds including the budgerigar, guinea fowl, quail, and turkey. To accomplish specific aim two, we will re-sequence mitochondrial DNA from birds of different ages within and among species to identify variants that may include insertions, deletions, and single nucleotide polymorphisms. The rationale for this work is that the diversity observed among birds for maximum life span is due to differences in age-associated changes in the mitochondrial genome. The long-term goal of my lab is to develop appropriate avian models for studying the genetic basis of longevity and to use these as a major non-mammalian resource for understanding the relationship between oxidative stress and decreased mitochondrial function. The longitudinal studies in each of the four avian species could provide evidence about the relationship, if any, between avian species differences in longevity and oxidative stress. The proposed work may also provide evidence from birds on recent observations in mice about the effect of oxidative stress on age-associated mtDNA mutations.

Candidate DNA Markers Associated With Primary Antibody Response to Sheep Red Blood Cells in the Chicken, Gallus gallus

Tuoyu "Leo" Geng

As both an important agricultural and biomedical trait, humoral or antibody-mediated immunity remains very little understood at the molecular level. Here, we describe a resource population that was used to identify candidate markers associated with antibody response to sheep red blood cells (anti-SRBC). In the population, the parental lines resulted from 28 th generations of divergent selection for high and low anti-SRBC titers were used to generate backcross and F2 progeny through a series of reciprocal matings. A total of 555 random primers were used in the preliminary screening on two individual and one pooled DNA samples each parental line for DNA markers. A two-step process was used for additional validation of association with immune response to SRBC: 48 samples from each line as well as a backcross population of 64 birds. Six random primers were identified to produce line-specific fragments, seven of which were converted into sequence characterized amplified region markers for single nucleotide polymorphism analysis. The genotype distributions of the 7 markers were significantly different (P<0.005) between the parental lines in the SNP-based analysis. Three of the DNA markers including VTC02, VTC04, and VTC06 showed significant association (P<0.05) with anti-SRBC titers in backcross population. The candidate markers and the resource population will be useful in efforts to understand the genetic control of humoral immune response and to develop breeding programs with marker-assisted selection for antibody production in animals.

Candidate Gene Expression Analysis of Toxin-Induced Dilated Cardiomyopathy in the Turkey ( Meleagris gallopavo)

Kuan-chin Lin, MS

ABSTRACT Dilated cardiomyopathy ( DCM ), a heart disease, affects many vertebrates including humans and poultry. The disease can be either idiopathic ( IDCM ) or toxin-induced ( TIDCM ). Though genetic and other studies of IDCM are extensive, the specific etiology of TIDCM is still unknown. Here, we compared mRNA levels of cardiac troponin T (cTnT) and phospholamban (PLN) in turkeys affected and unaffected by TIDCM. Cardiac TnT and PLN were chosen because their altered expression has been observed in IDCM-affected birds. A total of 72 birds, 44 TIDCM-affected and 28 -unaffected, were used in the quantitative reverse transcription polymerase chain reaction. Differences between affected and unaffected turkeys in the mRNA levels of both cTnT and PLN were significant only for cTnT. The sequence of the turkey PLN showed significant similarity at the nucleotide level to the reference chicken sequence and those of other species. In addition to implicating cTnT in TIDCM, the present work describes a partial turkey PLN coding sequence that could be useful for future studies.

Microsatellite Marker-Based Genetic Analysis of Relatedness Among Commercial and Heritage Turkeys (Meleagris gallopavo)

Davida Kamara, MS/Genome PREP scholar

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 among commercial and non-commercial chicken breeds, while the nature of the genetic relatedness among commercial and heritage turkeys remain unknown. 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.

Isolation and characterization of microsatellite markers from the budgerigar, Melopsittacus undulatus

Davida Kamara, MS/Genome PREP scholar

The budgerigar, Melopsittacus undulatus , belongs to a group of birds that are among the most common captive-held avian species that are also most threatened. Budgies are also a valuable resource as model species for many important vertebrate characteristics including auditory and aging. Several microsatellite-enriched genomic libraries were developed using standard protocols. Twelve of 20 primer-pairs developed from microsatellite containing sequences were codominant and polymorphic when tested on 21 birds from three different states. The number of alleles detected ranged from two to four with the expected and observed heterozygosities of 0.09 – 0.61 and 0.00 – 0.85, respectively. The genomic libraries and the primers described here will be useful in characterizing budgies.