Development and characterization of expressed sequence tags for the turkey (Meleagris gallopavo) genome and comparative sequence analysis with other birds

Twenty-one randomly selected clones from a turkey (Meleagris gallopavo) pituitary complementary DNA (cDNA) library were sequenced to develop expressed sequence tags (ESTs) for this economically important avian species whose genome is among the least understood. Primers specific for the ESTs were used to produce amplicons from the genomic DNA of turkey, chicken (Gallus gallus), guinea fowl (Numidia meleagris), pigeon (Columba domestica), and quail (Corturnix japonica). The amplicons were sequenced and analyzed for sequence variation within- and similarity among-species and with GenBank database sequences. The proportion of shared bases between the turkey sequence and the consensus sequence from each of the other species ranged from 72% to 93% between turkey and pigeon and quail and between turkey and chicken, respectively. The total number of single nucleotide polymorphisms (SNPs) observed ranged from 3 in quail to 18 in chicken out of 4898 and 5265 bases analyzed, respectively. The most frequent nucleotide variation observed was a C-->T transition. Linkage analysis of one such SNP in the backcross progeny of the East Lansing reference DNA panel, localized TUS0005, the chicken sequence derived from primers specific for turkey TUT2E EST, to chromosome 4. The ESTs reported, as well as the SNPs may provide a useful resource for ongoing efforts to develop high utility genome maps for the turkey and chicken. The primers described can also be used as a tool in future investigations directed at further understanding the biology of the guinea fowl, pigeon and quail and their relatedness to the turkey.     

Survey of a cDNA library from the turkey (Meleagris gallopavo).

Genome characterization and analysis is an imperative step in identifying and selectively breeding for improved traits of agriculturally important species. Expressed sequence tags (ESTs) represent a transcribed portion of the genome and are an effective way to identify genes within a species. Downstream applications of EST projects include DNA microarray construction and interspecies comparisons. In this study, 694 ESTs were sequenced and analyzed from a library derived from a 24-day-old turkey embryo. The 437 unique sequences identified were divided into 76 assembled contigs and 361 singletons. The majority of significant comparative matches occurred between the turkey sequences and sequences reported from the chicken. Whole genome sequence from the chicken was used to identify potential exon-intron boundaries for selected turkey clones and intron-amplifying primers were developed for sequence analysis and single nucleotide polymorphism (SNP) discovery. Identified SNPs were genotyped for linkage analysis on two turkey reference populations. This study significantly increases the number of EST sequences available for the turkey.     

Amplification of sequence tagged sites in five avian species using heterologous oligonucleotides

Short of a complete genomic DNA sequence, sequence tagged sites (STSs) have emerged as major genomic reagents for the genetic analysis of little-studied ecologically and agriculturally important organisms. Here, we report STS developed for the turkey (Meleagris gallopavo), guinea fowl (Numidea meleagris), Japanese quail (Coturnix coturnix) and pigeon using primers specific for reference DNA sequences of two chicken (Gallus gallus) genes, aggrecan (agc1) and type X collagen (col10). Additional STSs were also developed for turkey, quail and chicken using primers specific for the human apobec-1 gene. The total length of the STSs developed was 5990, 2522, 4127, 1539 and 6600 bp for the turkey, guinea fowl, Japanese quail, pigeon and chicken, respectively. Based on splice site consensus GT and AG sequences, four of the seven agc1-based chicken STS appear to contain introns. The human gene-based STSs showed no significant sequence identity with the reference GenBank sequences. Maximum likelihood, maximum parsimony and neighbour-joining analysis of an agc1-based STS that was common to all five species showed phylogenetic relationships consistent with those previously defined using mitochondria DNA sequences and nuclear gene restriction maps. Additionally, several putative single nucleotide polymorphisms (SNPs) were detected within the STSs, including eight in the turkey, two in the quail, and two in the chicken when multiple sequences were evaluated from each species. This report describes new STSs that are resources for genetic and physical mapping and genome analysis within and among avian species. These resources should further aid in our understanding of the biology of agriculturally important but little-studied guinea fowl and turkey. This revised version was published online in July 2006 with corrections to the Cover Date.     

Using the chicken genome sequence in the development and mapping of genetic markers in the turkey (Meleagris gallopavo)

The efficacy of employing the chicken genome sequence in developing genetic markers and in mapping the turkey genome was studied. Eighty previously uncharacterized microsatellite markers were identified for the turkey using BLAST alignment to the chicken genome. The chicken sequence was then used to develop primers for polymerase chain reaction where the turkey sequence was either unavailable or insufficient. A total of 78 primer sets were tested for amplification and polymorphism in the turkey, and informative markers were genetically mapped. Sixty-five (83%) amplified turkey genomic DNA, and 33 (42%) were polymorphic in the University of Minnesota/Nicholas Turkey Breeding Farms mapping families. All but one marker genetically mapped to the position predicted from the chicken genome sequence. These results demonstrate the usefulness of the chicken sequence for the development of genomic resources in other avian species.     

A 41–42 bp tandemly repeated sequence isolated from nuclear envelopes of chicken erythrocytes is located predominantly on microchromosomes

To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41–42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.     

Comparative analysis of microsatellite loci in chicken and turkey.

Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.     

Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms

DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3′ ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.     

Whole genome SNP discovery and analysis of genetic diversity in Turkey (Meleagris gallopavo)

ABSTRACT: BACKGROUND: The turkey (Meleagris gallopavo) is an important agricultural species and the second largest contributor to the world's poultry meat production. Genetic improvement is attributed largely to selective breeding programs that rely on highly heritable phenotypic traits, such as body size and breast muscle development. Commercial breeding with small effective population sizes and epistasis can result in loss of genetic diversity, which in turn can lead to reduced individual fitness and reduced response to selection. The presence of genomic diversity in domestic livestock species therefore, is of great importance and a prerequisite for rapid and accurate genetic improvement of selected breeds in various environments, as well as to facilitate rapid adaptation to potential changes in breeding goals. Genomic selection requires a large number of genetic markers such as e.g. single nucleotide polymorphisms (SNPs) the most abundant source of genetic variation within the genome. RESULTS: Alignment of next generation sequencing data of 32 individual turkeys from different populations was used for the discovery of 5.49 million SNPs, which subsequently were used for the analysis of genetic diversity among the different populations. All of the commercial lines branched from a single node relative to the heritage varieties and the South Mexican turkey population. Heterozygosity of all individuals from the different turkey populations ranged from 0.17-2.73 SNPs/Kb, while heterozygosity of populations ranged from 0.73-1.64 SNPs/Kb. The average frequency of heterozygous SNPs in individual turkeys was 1.07 SNPs/Kb. Five genomic regions with very low nucleotide variation were identified in domestic turkeys that showed state of fixation towards alleles different than wild alleles. CONCLUSION: The turkey genome is much less diverse with a relatively low frequency of heterozygous SNPs as compared to other livestock species like chicken and pig. The whole genome SNP discovery study in turkey resulted in the detection of 5.49 million putative SNPs compared to the reference genome. All commercial lines appear to share a common origin. Presence of different alleles/haplotypes in the SM population highlights that specific haplotypes have been selected in the modern domesticated turkey.     

The mitochondrial genome sequence and molecular phylogeny of the turkey, Meleagris gallopavo

The mitochondrial genome (mtGenome) has been little studied in the turkey ( Meleagris gallopavo ), a species for which there is no publicly available mtGenome sequence. Here, we used PCR-based methods with 19 pairs of primers designed from the chicken and other species to develop a complete turkey mtGenome sequence. The entire sequence (16 717 bp) of the turkey mtGenome was obtained, and it exhibited 85% similarity to the chicken mtGenome sequence. Thirteen genes and 24 RNAs (22 tRNAs and 2 rRNAs) were annotated. An mtGenome-based phylogenetic analysis indicated that the turkey is most closely related to the chicken, Gallus gallus , and quail, Corturnix japonica . Given the importance of the mtGenome, the present work adds to the growing genomic resources needed to define the genetic mechanisms that underlie some economically significant traits in the turkey.     

Expressed sequence tags for the chicken genome from a normalized 10-day-old white leghorn whole-embryo cDNA library. 3. DNA sequence analysis of genetic variation in commercial chicken populations.

Single nucleotide polymorphisms (SNPs) have emerged as a major class of DNA markers with the advantage of permitting the development of high-density genetic maps adequate for quantitative trait loci (QTL) identification by linkage-disequilibrium analysis. Here we describe results of a relatively high-depth survey of chicken broiler and layer populations for SNPs in targeted genomic regions of chicken expressed sequence tag (EST) sites. The sequences scanned, representing the composite sequence of 12 amplified fragments for a total of 6489 bp, were randomly distributed, occurring on six different chromosomes or linkage groups in the chicken genome. Although one of the genomic DNA sequences did not match the reference cDNA sequence, another contained an intron that separated two putative exons. The number of SNPs observed within each of the 12 EST-targeted genomic regions ranged from 0 to 10 for a total of 44 and a frequency of 0.7%. About 70% of the polymorphisms were shared between layer and broiler populations. The average heterozygosity within the populations ranged from 0.15 to 0.48, with the layer populations showing the higher heterozygosity. SNPs and oligonucleotides described will provide a resource for genetic analysis in commercial chicken populations. The data appear to indicate that the relative frequency of SNPs in the targeted regions scanned is higher than the frequency reported for any of the other regions scanned to date in other eukaryotic genomes. Additionally, the results suggest that the use of DNA pools may offer an efficient approach to SNP detection in chickens, as has been shown in other vertebrates.     

Female-specific DNA sequences in the chicken genome

Eight in silico W-specific sequences from the WASHUC1 chicken genome assembly gave female-specific PCR products using chicken DNA. Some of these fragments gave female-specific products with turkey and peacock DNA. Sequence analysis of these 8 fragments (3077 bp total) failed to detect any polymorphisms among 10 divergent chickens. In contrast, comparison of the DNA sequences of chicken with those of turkey and peacock revealed a nucleotide difference every 25 and 28 bp, respectively. Radiation hybrid mapping verified that these amplicons exist only on chromosome W. The homology of 6 W-specific fragments with chromo-helicase-DNA-binding gene and expressed sequenced tags from chicken and other species indicate that these fragments may have or have had a biological function. These fragments may be used for early sexing in commercial chicken and turkey flocks.     

Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA

Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67 bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.     

An integrated and comparative genetic map of the turkey genome

An integrated genetic linkage map was developed for the turkey (Meleagris gallopavo) that combines the genetic markers from the three previous mapping efforts. The UMN integrated map includes 613 loci arranged into 41 linkage groups. An additional 105 markers are tentatively placed within linkage groups based on two-point LOD scores and 19 markers remain unlinked. A total of 210 previously unmapped markers has been added to the UMN turkey genetic map. Markers from each of the 20 linkage groups identified in the Roslin map and the 22 linkage groups of the Nte map are incorporated into the new integrated map. Overall map distance contained within the 41 linkage groups is 3,365 cM (sex-averaged) with the largest linkage group (94 loci) measuring 533.1 cM. Average marker interval for the map was 7.86 cM. Sequences of markers included in the new map were compared to the chicken genome sequence by 'BLASTN'. Significant similarity scores were obtained for 95.6% of the turkey sequences encompassing an estimated 91% of the chicken genome. A physical map of the chicken genome based on positions of the turkey sequences was built and 36 of the 41 turkey linkage groups were aligned with the physical map, five linkage groups remain unassigned. Given the close similarities between the turkey and chicken genomes, the chicken genome sequence could serve as a scaffold for a genome sequencing effort in the turkey.     

Polymorphic microsatellites developed by cross-species amplifications in common pheasant breeds

Genetic variability was analysed in two common breeds of pheasant (Phasianus colchicus L. 1758) by means of cross-species amplifications of microsatellite loci: 154 chicken, Gallus gallus and 32 turkey, Meleagris gallopavo, primers were tested for amplification of pheasant DNA. Thirty-six primers (25 specific for chicken and 11 for turkey) amplified pheasant DNA. Fifteen markers yielded specific products and were tested for polymorphism. Eight of them (55%) were polymorphic, with an average polymorphism of two alleles per locus. Specific polymerase chain reaction (PCR) products were sequenced; repeats were found in 11 of the 15 markers, although only two loci showed the same repeat and could be homologous to chicken ones.     

Expressed sequence tags for the chicken genome from a normalized 10-day-old White Leghorn whole embryo cDNA library: 1. DNA sequence characterization and linkage analysis

Expressed sequence tags (ESTs) provide a rapid and reliable method for gene discovery as well as a resource for the large-scale analysis of gene expression of known and unknown genes. Here we describe a normalized cDNA library developed from a 10-day-old White Leghorn chicken whole embryo. The utility of the library was evaluated by partial sequencing of 99 randomly selected insert-containing clones and the analysis of EST-targeted genomic regions for single nucleotide polymorphisms (SNPs) in the East Lansing chicken reference DNA mapping panel. Using stringent match criteria of percent identity of 80 or higher across a length of 50 or more bases, 46 ESTs matched database sequences including previously reported Gallus gallus genes. Thirty-seven of the 50 primer pairs developed from 50 unique ESTs amplified a single fragment. The size of the 37 amplicons ranged from 276 to 693 bp for a total of 17,508 and an average of 473. About 70% of the SNPs detected were either G-->A or C-->T transition. The number of SNPs detected within the amplicons from EST-targeted genomic regions ranged from 0 to 4 for a total of 65 and a frequency of about 1 every 470 bases. About 35% of the amplicons contained only 1 SNP, while 19% had 4 SNPs. Using the SNPs that were informative in the East Lansing reference panel, 17 ESTs were mapped on the East Lansing chicken genetic map. The ESTs described, as well as the nucleotide variants identified within the EST-targeted genomic regions, represent significant resources for genome analysis in the chicken.     

Characterization of the turkey MHC chromosome through genetic and physical mapping

Previous studies in the chicken have identified a single microchromosome (GGA16) containing the ribosomal DNA (rDNA) and two genetically unlinked MHC regions, MHC-B and MHC-Y. Chicken DNA sequence from these loci was used to develop PCR primers for amplification of homologous fragments from the turkey (Meleagris gallopavo). PCR products were sequenced and overgo probes were designed to screen the CHORI 260 turkey BAC library. BAC clones corresponding to the turkey rDNA, MHC-B and MHC-Y were identified. BAC end and subclone sequencing confirmed identity and homology of the turkey BAC clones to the respective chicken loci. Based on subclone sequences, single-nucleotide polymorphisms (SNPs) segregating within the UMN/NTBF mapping population were identified and genotyped. Analysis of SNP genotypes found the B and Y to be genetically unlinked in the turkey. Silver staining of metaphase chromosomes identified a single pair of microchromosomes with nucleolar organizer regions (NORs). Physical locations of the rDNA and MHC loci were determined by fluorescence in situ hybridization (FISH) of the BAC clones to metaphase chromosomes. FISH clearly positioned the rDNA distal to the Y locus on the q-arm of the MHC chromosome and the MHC-B on the p-arm. An internal telomere array on the MHC chromosome separates the B and Y loci.     

Characterization of a new repetitive sequence that is enriched on microchromosomes of turkey

We cloned and characterized a new highly repetitive, species-specific DNA sequence from turkey (Meleagris gallopavo). This repeat family, which accounts for approximately 5% of the turkey genome, consists of a 41 bp repeated element that is present in tandem arrays longer than 23 kb. In situ hybridization to turkey metaphase chromosomes (2n=80) demonstrated that this sequence was located primarily on certain microchromosomes: approximately one-third of the 66 microchromosomes showed a positive signal. With respect to the macrochromosomes, hybridization was seen only in a pericentric position on nos. 2 and 3. The turkey microchromosome (TM) sequence shares motifs (alternating A_3–5 and T_3–5 clusters separated by 6–8 bp) that have been found previously in other avian tandemly repeated elements, e.g. a chicken microchromosome sequence, and W (female) chromosome-specific sequences of chicken and turkey. However, the TM sequence does not cross-hybridize under moderately stringent conditions with these other sequence. The spread and amplification of related repetitive sequence elements on microchromosomes and W chromosomes is discussed.by E.R. Schmidt     

Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping

DNA sequencing has markedly changed the nature of biomedical research, identifying millions of polymorphisms along the human genome that now require further analysis to study the genetic basis of human diseases. Among the DNA-sequencing platforms available, Pyrosequencing has become a useful tool for medium-throughput single nucleotide polymorphism (SNP) genotyping, mutation detection, copy-number studies and DNA methylation analysis. Its 96-well genotyping format allows reliable results to be obtained at reasonable costs in a few minutes. However, a specific biotinylated primer is usually required for each SNP under study to allow the capture of single-stranded DNA template for the Pyrosequencing assay. Here, we present an alternative to the standard labeling of PCR products for analysis by Pyrosequencing that circumvents the requirement of specific biotinylated primers for each SNP of interest. This protocol uses a single biotinylated primer that is simultaneously incorporated into all M13-tagged PCR products during the amplification reaction. The protocol covers all steps from the PCR amplification and capture of single-stranded template, its preparation, and the Pyrosequencing assay itself. Once the correct primer stoichiometry has been determined, the assay takes around 2 h for PCR amplification, followed by 15-20 min (per plate) to obtain the genotypes.