MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Characterization of a Cardiac Complementary Deoxyribonucleic Acid Library from the Turkey (Meleagris gallopavo) K. M. Mendoza,* W. Chiang,† G. M. Strasburg,† and K. M. Reed* *Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul 55108; and †Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824 ABSTRACT Although the domestic turkey (Meleagris gallopavo) is a valuable agricultural commodity, genetic studies on this species lag behind those of other agricultural species. In this study, we examined expressed sequence tags (EST) from a turkey cardiac cDNA library constructed from 4 birds representing 2 developmental stages. A collection of 3,937 EST sequences were sequenced and analyzed for gene annotation and sequence variation. Clustering of sequences resulted in 353 contigs and 874 singletons (1,227 putative transcripts). All EST
sequences were compared by BLASTN to the chicken whole genome sequence and to Ensembl and National Center for Biotechnology Information databases. The majority of significant matches correspond to genes found in the chicken. Sequence polymorphisms were identified in 310 contigs, 64 where the minor allele was observed to be present in more than 1 sequence. This study gives species-specific insight into the cardiac transcriptome of turkeys and provides resources for future studies of cardiac function.
Key words: turkey, cardiac muscle, expressed sequence tag, round heart, cardiomyopathy 2008 Poultry Science 87:1165–1170 doi:10.3382/ps.2007-00518
INTRODUCTION Diseases, both infectious and genetic, significantly affect the efficiency of poultry production. In the United States, the total annual costs of disease prevention, vaccination, and medical treatments are billions of dollars per year (USDA, 1997). Dilated cardiomyopathy [DCM or round heart (RH)] is a prevalent syndrome in some commercial turkeys (Roberson, 2005). Round heart is characterized by dilated and poorly contracting cardiac chambers (Magwood and Bray, 1962; Sautter et al., 1968; Jankus et al., 1972; Genao et al., 1996). Affected birds show marked weight loss by 2 to 4 wk of age, and in some commercial settings, mortality of 1.5 to 3% is observed (Frame et al., 1999). In an industry estimated at $3 billion annually (US Poultry and Egg Association; http://poultryegg.org), production losses in turkey due to RH can be significant. The etiology for RH disease is still not well characterized. Environmental factors such as altitude, diet, rapid growth, and viral infection can affect disease prevalence (Czarnecki, 1984; Julian et al., 1992). A DCM condition similar to spontaneous RH can be induced in turkeys by
©2008 Poultry Science Association Inc. Received December 20, 2007. Accepted February 24, 2008. 1 Corresponding author:
[email protected]
oral administration of high doses of furazolidone (Jankus et al., 1972). There is also a genetic predisposition to RH in that flocks with a high incidence of the disorder (greater than 70%) have been selectively bred (Hunsaker, 1971; Staley et al., 1981; Pierpont et al., 1985). These selected birds were also more susceptible to the effects of furazolidone than nonselected controls (Staley et al., 1978). A genetic approach to investigating this disease is clearly warranted. The focus of turkey genomics has been on development of an integrated genetic linkage map with comparative alignment to the chicken genome (Reed et al., 2007a). Although this is an important accomplishment, genomic research in the turkey still lags behind other agriculturally valuable species, such as the chicken (Gallus gallus). Comparative genomics provides researchers with valuable resources based on the synteny between genomes. However, regardless of the degree of homology between their genomes, many transcripts may be unique to each species (Boardman et al., 2002). In addition, genes may exhibit duplicate loci, differential expression, and alternate splice variants at varying developmental stages (Chaves et al., 2003), making species-specific sequences invaluable. Several genes including phospholamban (PLN), cardiac troponin T (TNNT2; Biesiadecki et al., 2002), and titin (Hein et al., 1994) are suggested to influence RH in turkeys. The identification and functional analysis of gene products is maximized by the development of species-specific expressed sequence tags (EST). Expressed sequence tags
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represent the transcribed portion of the genome and identify only genes that are expressed in the selected tissue. Species-specific sequencing and EST sequence identification is a crucial first step in downstream expression studies including microarray or quantitative PCR analysis, as well as interspecies comparisons for in silico mapping. Previous studies in the chicken included cDNA from precardiac mesoendoderm cells not mature cardiac tissue (Afrakhte and Schultheiss, 2004). This is the first study examining the mature cardiac gene transcripts of an avian species. The purpose of our study was to identify genes naturally expressed in cardiac tissue of turkeys (unknown RH etiology) and to catalog a suite of associated single nucleotide polymorphisms (SNP). Of primary interest is the identification of turkeyspecific sequences for downstream applications in studying RH disease.
MATERIALS AND METHODS A cDNA library was constructed from cardiac tissues collected by Sandra Velleman (Ohio State University, Columbus) following an approved Institutional Animal Care and Use Committee protocol from turkey lines developed by Karl Nestor at the Ohio Agricultural Research and Development Center (Ohio State University). Samples included 2 birds from the F1 line (selected for increased 16wk body weight) and 2 from the RBC2 line (random bred control). To maximize the variety of expressed sequences, birds from 2 developmental stages were used (1 d and 16 wk posthatch) representing different developmental time points. Tissues were collected and preserved from killed individuals within 5 min of death, snap-frozen, then stored at −80°C until use.
Library Construction Total RNA was extracted from the tissues using the TRI reagent (Molecular Research Center, Cincinnati, OH) following the protocols of the manufacturer. Ribonucleic acid quality was analyzed by the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA), to ensure RNA quality. PolyA+ selection was performed on the total RNA by the PolyATract mRNA Isolation System (Promega, Madison, WI) according to the protocols of the manufacturer. The library was constructed from 4 to 5 g of mRNA using CloneMiner cDNA library construction kit (Invitrogen, Carlsbad, CA) following the protocols of the manufacturer. Superscript II RT was used to generate first strand cDNA with Biotin-attB2-Oligo (dT) primer. Second strand cDNA was synthesized from the first strand cDNA using T4 DNA polymerase, creating blunt-ended, doublestranded cDNA. The adaptor aatB1 was ligated to the 5′ end of the cDNA. Excess primers, adaptors, and small cDNA fragments were removed by size-fractioned column chromatography. A recombination reaction was performed to ligate the aatB-flanked cDNA molecules to the aatP-donor vector pDONR222 (Invitrogen). This ligation was transformed into ElectroMAX DH10B T1 phage-resis-
tant cells. The resulting cDNA library titer was estimated at 2.0 × 107 cfu/mL. Template generation for sequencing was performed by rolling circle amplification at the University of Washington High-Throughput Genomics Unit (Seattle).
Sequence Analysis Single-pass 5′ clone sequences were generated from the cardiac cDNA library at the High-Throughput Genomics Unit, University of Washington (Seattle). All sequences were trimmed of pDONR222 vector and low-quality tails then deposited in GenBank (EX717061-EX719678 and ES216654-ES217501). Mitochondrial DNA (mtDNA) sequences were identified by comparison with the chicken whole mtDNA sequence (GenBank NC_001323) and were removed from the project. In addition, sequences with reads of less than 50 bp were eliminated. The remaining sequences were assembled into contigs using Sequencher software (Gene Codes Corp., Ann Arbor, MI) using 98% match and 50-bp minimum overlap parameters. All contigs and singletons were compared with the chicken whole genome sequence (WGS; build 2.1) by BLASTN search using Ensembl Genome browser. Sequences that returned overlapping positions in the chicken WGS were then manually examined for sequence overlap and further contig assembly. The resulting contigs and singletons were then compared with the GenBank National Center for Biotechnology Information nonredundant (nr) database by BLASTN. Gene ontology annotations for the cardiac sequences were retrieved by BLASTN searches of the Ensembl chicken cDNA database. For this comparison, an E-value cutoff of 0.001, bit score cutoff of 100, and positives cutoff of 80 were used. Assembled contigs were examined for the presence of SNP and insertion-deletions (INDELS). Initially, Sequencher was used to generate a list of all polymorphisms by setting the contig consensus sequence to inclusivity to generate a reference sequence, with subsequent comparison of the contig sequences to the reference by plurality. Each SNP identified by Sequencher was then individually verified by examination of the trace files.
RESULTS A total of 3,937 single-pass sequences were generated from the cardiac cDNA library. Average length of the trimmed sequence reads was 745 bp. Mitochondrial DNA, incorporated into the library as an artifact of the construction process, had the largest representation in the library with 917 sequences comprising 23.3% of the total sequence reads. After removal of mtDNA sequences and low-quality short sequences, 2,620 sequences remained and were considered as high quality for further analysis. Primary assembly of sequences resulted in 356 tentative consensus sequences and 921 single sequences (singletons) for a total of 1,277 predicted distinct transcripts. Tentative consensus (TC) lengths ranged from 118 to
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2,253 bp (average 750 bp) and contained an average of 5 EST sequences (range 2 to 125). All TC and singletons were compared with sequences in GenBank by BLASTN searches of the nr database using default parameters. Significant E-value scores (≥1.0 × 10−4) were returned for 345 of the 356 TC and 791 of the 921 singletons. Among the TC, 256 hits were to previously annotated genes, 81 to unannotated EST sequences, and 8 hits to chicken bacterial artificial chromosome clone sequences. Eleven TC represent novel sequences with no significant hits in the National Center for Biotechnology Information nr database. Among the singletons, 641 hits were to previously annotated genes, 150 to unannotated EST sequences, and 130 were novel sequences. The majority of significant matches among TC and singletons occurred with chicken EST sequences (1,056 of 1,136 queries, 92.9%). The 4 genes most highly represented in the cardiac library sequences are α cardiac actin (125 sequences), β globulin (85 sequences), α globin (80 sequences), and myosin light chain 2 (56 sequences). Other important cardiac-specific sequences also represented in the EST collection include C protein, LIM protein, phospholamban, troponin I, and troponin T. The assembled distinct transcript set (1,277; 356 TC and 921 singletons) was compared with the chicken WGS by BLASTN. A total of 833 distinct transcripts consisting of 2,052 EST sequences had significant matches to putative homologous sites on 30 of the assembled chicken chromosomes (Table 1). The number of matches ranged from 121 on GGA1 to 4 on GGA16. Of the aligned transcripts, roughly half (446) occurred on macrochromosomes (GGA1 to 9 and Z) with the remainder aligned to microchromosomes. These data provide in silico predictions of the chromosomal location for polymorphisms (SNP) identified in this project (see below). The remaining sequences had no significant match at the selected cutoff E-value, did not identify a unique homologous region within the chicken sequence, or were assigned to sequence contigs not yet assigned to chromosomes. A comparative genomics approach was then used to refine the number of unique genes represented by the EST data. Based on the position of sequences in the chicken genome, a second contig assembly was performed by reexamining TC and singletons with overlapping or adjacent hits. This resulted in a reduction by 50 in the distinct transcript set (1,227; 353 TC and 874 singletons). The 353 TC from the second assembly were then examined for sequence polymorphisms (SNP). Among the TC, a total of 310 polymorphisms were identified, with an average of 1 per 746 bp. The majority of polymorphisms (275 of 310) were single substitutions (SNP), but 35 INDELS were also identified. As expected, transition substitutions were the most commonly observed (195 of 275), followed by transversions (80 of 275). The number of individuals included in any SNP discovery effort will directly influence the frequency at which minor alleles may be observable for a given sequence depth and estimates of minor allele frequency are more accurate for TC with greater sequence depth. Because the cDNA library
Table 1. Comparative distribution of turkey tentative consensus sequences and single expressed sequence tags (EST) in the chicken whole genome sequence with significant BLASTN hits to assigned chromosomes Chicken chromosome GGA1 GGA2 GGA3 GGA4 GGA5 GGA6 GGA7 GGA8 GGA9 GGA10 GGA11 GGA12 GGA13 GGA14 GGA15 GGA16 GGA17 GGA18 GGA19 GGA20 GGA21 GGA22 GGA23 GGA24 GGA25 GGA26 GGA27 GGA28 GGAZ Total
Transcriptional units
EST, n
104 70 49 54 48 26 34 23 38 26 14 26 27 24 33 4 16 15 26 16 20 18 21 9 11 23 13 16 29 833
372 122 87 86 227 31 56 59 47 50 30 62 42 133 117 16 27 21 87 44 39 22 32 25 14 61 37 56 50 2,052
was constructed from cardiac tissue of 4 individuals (8 possible alleles), the minor allele frequency cutoff at which 1 individual in the library carries at least 1 copy of the minor allele is 0.125 (1/8). Minor allele frequencies for 263 polymorphisms occurred at frequencies of greater than 0.125. Sixty-four of the polymorphisms (56 SNP and 8 INDELS) in 39 TC were recurrent in that they were observed in more than 1 sequence in a contig (Table 2). Examples of cardiac-specific candidate genes for RH that contained recurrent SNP were phospholamban, troponin I, and troponin T. When sequences were translated, 41 of the recurrent polymorphisms occurred within coding regions, with 34 representing synonymous substitutions and 7 being nonsynonymous substitutions. Twenty of the polymorphisms occurred in untranslated regions (5′ or 3′). Examination of TC with multiple SNP such as adenosine 5′-triphosphate synthase γ (4 SNP with 3 haplotypes) and ferritin (9 SNP with 4 haplotypes) suggests the cardiac EST sequences will provide a starting point for identification of additional sequence variants. Of particular interest is TNNT2, which is a candidate gene for cardiomyopathy (Biesiadecki et al., 2002; Osterziel and Perrot, 2005). Although not all TNNT2 transcripts are of sufficient length to include all of the polymorphic sites, we detect a minimum of 3 SNP haplotypes for this gene in the cardiac library (Table 3). Four SNP were found in the TNNT2 transcripts: 2 C/T transitions in exon 1 (untrans-
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Table 2. Summary data for recurrent polymorphisms [single nucleotide polymorphisms and insertions-deletions (INDELS)] identified in the tentative consensus (TC) sequences from the turkey cardiac cDNA library1 Gene
EST, n
MAF
Sequence
Type
GenBank Accession
Actin, α, cardiac muscle 1 (ACTC1) Actin, α, skeletal muscle 1(ACTA1)
8 18 18 20 20 20 14 11 16 10 10 10 6 6 11 7 14 15 15 14 14 14 14 14 14 11 11 12 79 78 80 6 6 6 9 9 8 9 43 53 4 9 7 7 5 7 5 5 8 8 6 4 4 6 6 6 14 6 11 15 15 17 5 5
0.375 0.222 0.278 0.400 0.100 0.400 0.429 0.182 0.188 0.200 0.200 0.200 0.333 0.333 0.364 0.429 0.429 0.267 0.267 0.286 0.286 0.143 0.357 0.214 0.214 0.182 0.182 0.500 0.367 0.026 0.100 0.333 0.500 0.500 0.444 0.444 0.500 0.444 0.233 0.189 0.500 0.222 0.429 0.429 0.400 0.429 0.400 0.400 0.375 0.375 0.333 0.500 0.500 0.333 0.333 0.333 0.143 0.333 0.364 0.466 0.466 0.118 0.400 0.400
CTAAYAACG ACAGacagCCAG CCAGRGCCG AGGCYCCCC CCGGYGATG ACAAYGTCC CCTAYAACA GAGAWCCGC TTTTRGGAG GGCAYTGTA GAACRCATG TAGCRAATA AGAGcagGCTT GTGGaagCCTG GCACYTACA CGCGYGTGG CCGCRTAGC AATTYGAGA TCAARAACA CCGCYGCCT ACCGYCACC ACCASAGCA TCTCtcGGCA AGCTSTAYG TSTAYGCCT CCGTYCCGC TCCCRCAAC TGCGYGGCC CGAGRCCCT GCATRGCAR CAGCWATTG AGGAYGGCT AGCTRGAGG AGCTYCTGA CTGARGAAC CAATYGAAG AAGGMGGAA TGAGYTGCT CCAGYACCT CTGAYCCAG TCACRGACT AGCTKCCAC TGGGRGACC AGAARCTGG GGGGYTGGA AAAARGGTG AGCGYGGTG GGTGSTGGT TGAGRCACA AACAYGGAA GGAGRAACG GCCTMTCGG TCCTYGGTG GCAAMAART AMAARTGCA TTTTtGGGG CCCCMCCTC CACCYACCG TCGGYACAG AGCASCAGA GAAGaagGTGG AGGARGAAG CTGGSCCGC TGACYCGAG
S NT S S S S S NS S S S S T T NT S NT S S NT NT NT NT S S NT S S NS NT NT S S S S S S S NT S NT NT S S S S NT NT NS NS NS S S NT NT NT S NT NT NS T S NS S
EX719027, EX717735, EX717735, EX719200, EX717790, EX717790, EX719242, EX719339, EX718923, EX718214, EX718214, EX718214, EX719369, EX719369, EX718348, EX718130, EX717935, EX717453, EX719518, EX717807, EX717102, EX718308, EX717102, EX717102, EX717102, EX717296, EX717296, EX718153, EX718342, EX717948, EX719660, EX717932, EX717268, EX717268, EX718864, EX718864, EX719579, EX717170, EX717940, EX717940, EX718912, EX718912, EX717742, EX717742, EX719378, EX719329, EX717876, EX717876, EX717250, EX717250, EX718321, EX719294, EX719294, EX717459, EX717459, EX717459, EX719089, EX718005, EX717582, EX717880, EX718758, EX717926, EX718499, EX718499,
Apolipoprotein A-I (APOA1) Adenosine 5′-triphosphate synthase α subunit (ATP5A1) Adenosine 5′-triphosphate synthase γ 9 (ATP5C1) Cys-Cys-His-Cys-type zinc finger (CNBP) Cysteine-rich protein 2 (CRIP2) Desmin (DES, LOC395906) Eukaryotic translation elongation factor 1, α 2 (EEF1A2) Ferritin, heavy polypeptide 1 (FTH1)
Guanine nucleotide binding protein (GNB2L1) Hemoglobin, α 1 (HBA1) Hemoglobin, β (HBB) Jun-binding protein (LOC396411) Polymerase I and transcript release factor (PTRF) Myosin heavy polypep 6 (MYH6)
Myosin, light chain 2, regulatory, cardiac, slow (MYL2) Phospholamban, cardiac (PLB) Ribosomal protein L10a (RPL10A) Ribosomal protein L17 (RPL17) Ribosomal protein S6 (RPS6) Ribosomal protein S28 (LOC768930) SH3 domain binding glutamic acid-rich (SH3BGR) Selenoprotein W, 1 (SEPW1) Solute carrier family 16, member 1 (SLC16A1) Transcribed locus, moderately similar to XP_001114370.1 Troponin I, cardiac (TNNI3) Troponin T, cardiac (TNNT2)
Ubiquinol, cytochrome c reductase (UQCRC1)
EX718373 EX717310 EX717310 EX719293 EX718211 EX718211 EX718240 EX717424 EX717998 EX719600 EX719600 EX719600 EX719105 EX719105 EX718922 EX718019 EX718186 EX719647 EX717394 EX718842 EX718172 EX718612 EX718172 EX718172 EX718172 EX718653 EX718653 EX718485 EX717920 EX719168 EX717774 EX718500 EX719351 EX719351 EX719537 EX719537 EX719113 EX718137 EX717904 EX717904 EX717672 EX717672 EX717489 EX717489 EX717739 EX718167 EX717608 EX717608 EX719577 EX719577 EX719212 EX718036 EX718036 EX718147 EX718147 EX718147 EX717228 EX717825 EX718758 EX717671 EX717582 EX719250 EX717620 EX717620
GGA 5 3
24 Z 1 12 8 7 20 5
16 14 1 UN 27 1
15 3 26 Z Z 28 1 UN 26 NSS NSS 26
12
1 For each polymorphism, the corresponding gene, number of aligned sequences surveyed, minor allele frequency (MAF), and chromosomal location within the chicken genome (GGA) are given. Sequence polymorphisms are denoted by underlining with INDELS in lower case. Variable nucleotide abbreviations are as follows: K = G or T; M = A or C; R = A or G; S = C or G; W = A or T; and Y = C or T. Substitution types include S = synonymous; NS = nonsynonymous, NT = nontranslated and T = translated (INDELS). Location in the chicken genome for each TC is based on BLASTN to the whole genome sequence (build 2.1).
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TURKEY CARDIAC GENES Table 3. Variation of the cardiac troponin T (TNNT2) gene and distribution of sequence polymorphisms observed in transcripts of the turkey1 E1 (17) C ? ? ? C C ? T ? ?
E1 (27)
Exon 5 presence
E7 (24)
E7 (34 to 36)
E13 (21)
Sequences, n
T T ? ? C C ? C C C
Y N Y N N Y Y N Y N
C C C C G G G G G G
AAG AAG AAG AAG — — — — — —
G G G G A ? A G G G
2 2 1 2 1 1 1 2 1 2
1 Two developmental isoforms of TNNT2 are denoted by presence or absence of exon 5; embryonic isoform contains exon 5 (Y), whereas it is excluded in the adult isoform (N). Observed single nucleotide polymorphism (SNP) haplotypes and isoforms are indicated with the number of sequences included in each class, nucleotide location of SNP within the exon, denoted in parentheses. A question mark (?) denotes that sequence does not contain a region of SNP.
lated), a nonsynonymous transversion (C/G) in exon 7, and a synonymous transition (A/G) in exon 13. The SNP in exon 7 results in an amino acid substitution (proline ↔ alanine). In addition to these SNP, a 3-nucleotide insertion (single glutamic acid residue) was observed at the end of exon 7 in roughly half of the cDNA sequences. All sequences with this insertion also had the C nucleotide at the SNP locus within exon 7. The cardiac EST sequences contained both developmental isoforms of TNNT2 denoted by the presence or absence of exon 5; embryonic isoform contains exon 5, whereas adult isoform excludes it. The observed SNP variants were not specific for either the embryonic or adult isoforms.
DISCUSSION The turkey has been identified as an effective animal disease model for human heart failure (Genao et al., 1996; Wu et al., 2004). Myocardium of the turkey shares morphologic, functional, and biochemical characteristics with that of humans and other mammals. Increased left ventricle diameter, diminished ejection fraction, systemic hypotension, and left ventricle free wall thinning are all reported in human heart failure, as well as the turkey model (Wu et al., 2004). There are many benefits to this animal model of heart failure, most important being the remarkable physiological and biochemical similarities between human heart failure and turkey RH. However, the biochemical similarities may only be elucidated when the genes important in turkey RH are further defined. Our goal was to create a cardiac-specific turkey cDNA library and to obtain turkey-specific sequences of important cardiac genes. Despite the high frequency of mtDNA sequences in this nonnormalized library, examination of nearly 4,000 EST sequences identified over 1,200 putative transcripts. In addition, a large number of EST sequences expressed in cardiac tissue but with unknown homology were also identified. These included 11 (3.1%) of the 354 contigs and 130 (14.8%) of the 874 singletons. Further work is needed to determine chromosomal location and function of these transcripts.
Single nucleotide polymorphisms are the most common form of polymorphisms between individuals in the genome (Emara and Kim, 2003), and over 300 such polymorphisms were cataloged among the assembled contigs with 64 for which the minor allele was observed to be present in more than one sequence. The SNP haplotypes were easily identified in a small set of sequence contigs containing multiple SNPs. These SNP provide a new source of allelic variants useful in candidate gene studies or genetic and comparative mapping, or both. For example, Paxton et al. (1999, 2005) identified polymorphic DNA sequences (sequence-characterized amplified regions; SCAR) that differed in frequency between RH+ and RH− birds. The genomic location and position of these markers relative to putative cardiac-related genes were determined via the turkey-chicken comparative map (Reed et al., 2007a). Similarly, 25 of the transcribed sequences from the cardiac library have predicted chromosomal locations within 1 Mb of these SCAR markers. These included transcripts of 3 genes (phospholamban, myosin-binding protein c, and creatine kinase M) identified by Reed et al. (2007b) as being associated with the RH SCAR. Sequences corresponding to several important cardiac genes were identified in the turkey including troponin T, a candidate gene for cardiomyopathy. Both the chicken and turkey normally express 2 isoforms of cardiac troponin T (TNNT2), embryonic and adult, that differ in the presence of exon 5 (Cooper and Ordahl, 1985; Biesiadecki et al. 2002; Table 3). In addition, an abnormal splice variant that excludes exon 8 (11 amino acids) is described as being associated with RH in the turkey (Biesiadecki and Jin, 2002; Biesiadecki et al., 2004). This low-molecular weight TNNT2 variant shows altered molecular conformation and binding affinity for troponin I and tropomyosin, as well as an increase in the calcium sensitivity of myosin ATPase (Biesiadecki et al., 2002, 2004). The lowmolecular weight variant is also found in normal birds but is expressed at a significantly higher rate in RH birds. The cardiac library contained transcripts of both the embryonic and adult isoforms; however, low-molecular
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weight exon 8 variants were not observed. The TNNT2 consensus sequence of the turkey was 96.7% similar to that of the chicken with 1 amino acid difference (there is an additional amino acid residue in some turkey sequences). Variable presence of this additional amino acid was originally described by Biesiadecki et al. (2002) and attributed to allelic variation in exon 7. The new turkey sequences identified in this study contain at least 1 isoform of 13 of the 20 DCM candidate genes identified in humans (Osterziel and Perrot, 2005). Six of these gene sequences [α actin, troponin T (isoforms 1 and 2), troponin I, phospholamban, and dystrophin] also contain cataloged SNP. These turkey-specific sequences are currently being used to design gene expression assays to examine differences in gene expression between RH+ and RH− birds.
ACKNOWLEDGMENTS This research was supported by a grant from the Cooperative State Research, Education and Extension Service, USDA (2005–35604–15628).
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