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.     

Polymorphic microsatellites in Buff-throated partridge developed by cross-species amplification

Microsatellite loci for the buff-throated partridge (Tetraophasis szechenyii), an endemic pheasant species of China, are here described for the first time. Twenty-five microsatellite markers from chicken and Japanese quail were tested on buff-throated partridge DNA by means of cross-amplification. Twenty (80%) primers yielded specific products and polymorphisms were tested in a wild population of buff-throated partridge. Twelve (48%) proved to be polymorphic with an average of two alleles per locus. Current results of buff-throated partridge microsatellites loci could be employed in population genetic studies and on other endangered pheasant species.     

Turkey microsatellite DNA loci amplified by chicken-specific primers

Forty-eight primer-pairs complementary to unique DNA sequences flanking chicken (genus Gallus ) genomic (TG)_n microsatellite repeats were previously designed. These primer-pairs were used in the polymerase chain reaction to amplify turkey (genus Meleagris ) genomic DNA loci. Results indicated that the majority (92%) of these primer-pairs generated amplification products in turkey genomic DNA. Hybridization using end-labelled (TG)₈ as a probe showed that, out of 41 primer-pairs tested, only 14 generated an amplification product that also contained a detectable (TG)_n microsatellite repeat when turkey DNA was the template. Among 18 primerpairs tested for polymorphism, using three commercial turkey lines, five were found to exhibit length polymorphism, three of which did not contain a detectable TG repeat. Therefore, a significant portion of chicken microsatellite markers can be useful for genomic mapping and linkage analysis in the turkey, reducing the costs involved in producing turkey-specific microsatellite markers.     

Use of chicken microsatellite markers in turkey: a pessimistic view

Eighty-eight chicken microsatellite markers, previously developed in our laboratory, were tested for their ability to amplify polymorphic fragments using turkey genomic DNA. Amplification products were obtained for 61 chicken microsatellite markers (69.1%) whereas 27 (30.9%) did not give rise to any products, even when different polymerase chain reaction conditions were employed. From the 61 markers that gave a product, only eight showed a length polymorphism while 37 were monomorphic on the three divergent commercial turkey lines used. The remaining 16 markers yielded many unspecific bands and no specific amplification product could be obtained. Five polymorphic and eleven monomorphic products contained a detectable microsatellite repeat. Furthermore, of the markers that detected a polymorphism in turkey, the observed heterozygosity (15–50%) and allelic variation (only 2 in most cases) was very low. Therefore, on the basis of our results, we think that chicken microsatellite markers are not very useful for mapping purposes in turkey.     

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.     

Characterization of 35 novel microsatellite DNA markers from the duck (Anas platyrhynchos) genome and cross-amplification in other birds

In order to study duck microsatellites, we constructed a library enriched for (CA)n, (CAG)n, (GCC)n and (TTTC)n. A total of 35 pairs of primers from these microsatellites were developed and used to detect polymorphisms in 31 unrelated Peking ducks. Twenty-eight loci were polymorphic and seven loci were monomorphic. A total of 117 alleles were observed from these polymorphic microsatellite markers, which ranged from 2 to 14 with an average of 4.18 per locus. The frequencies of the 117 alleles ranged from 0.02 to 0.98. The highest heterozygosity (0.97) was observed at the CAUD019 microsatellite locus and the lowest heterozygosity (0.04) at the CAUD008 locus, and 11 loci had heterozygosities greater than 0.50 (46.43%). The polymorphism information content (PIC) of 28 loci ranged from 0.04 to 0.88 with an average of 0.42. All the above markers were used to screen the polymorphism in other bird species. Two markers produced specific monomorphic products with the chicken DNA. Fourteen markers generated specific fragments with the goose DNA: 5 were polymorphic and 9 were monomorphic. But no specific product was detected with the peacock DNA. Based on sequence comparisons of the flanking sequence and repeat, we conclude that 2 chicken loci and 14 goose loci were true homologous loci of the duck loci. The microsatellite markers identified and characterized in the present study will contribute to the genetic map, quantitative traits mapping, and phylogenetic analysis in the duck and goose.     

Japanese quail microsatellite loci amplified with chicken-specific primers

Forty-eight primer pairs for chicken (Gallus gallus) microsatellite loci were tested in polymerase chain reaction (PCR) amplification of Japanese quail (Coturnix japonica) genomic DNA. Amplification products were obtained from 28 primer-pairs (58.3%) after optimizing the PCR conditions. Eleven (22.9%) of these generated specific products and 17 yielded non-specific amplification products. Eight markers (ADL0037, ADL0038, ADL0142, ADL0143, ADL0206, ADL0315, ADL0366, and HUJ0006) were polymorphic and three were monomorphic (ADL0023, ADL0024, and ADL0257) in four Japanese quail populations. Specific amplification products from each of the 11 PCR primers were sequenced. Seven of the eight polymorphic and one of three monomorphic markers contained simple tandem repeats. Six of these microsatellite loci (ADL0037, ADL0315, ADL0142, ADL0143, ADL0366 and ADL0257) may be homologous to the corresponding chicken loci from which the markers were developed.     

Preliminary linkage map of the turkey (Meleagris gallopavo) based on microsatellite markers

The turkey is an agriculturally important species for which, until now, there is no published genetic linkage map based on microsatellite markers--still the markers most used in the chicken and other farm animals. In order to increase the number of markers on a turkey genetic linkage map we decided to map new microsatellite sequences obtained from a GT-enriched turkey genomic library. In different chicken populations more than 35-55% of microsatellites are polymorphic. In the turkey populations tested here, 43% of all turkey primers tested were found to be polymorphic, in both commercial and wild type turkeys. 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.     

Utility of chicken-specific microsatellite primers for mapping the turkey genome

As part of the University of Minnesota's initiative to map the turkey genome, we are currently evaluating chicken microsatellite loci for use in mapping the turkey genome. To date, 141 primer pairs have been tested for amplification at six different combinations of temperature and MgCl2 concentration. Microsatellite primer pairs from the Chicken Comprehensive Mapping Kit #2, and additional unpublished chromosome 1 and 2 primers were screened. Analyzable PCR products were produced from 78 of the 141 (55%) primer combinations. In the majority of cases (68%), PCR fragments obtained from the turkey were similar in size to respective chicken loci. The presence of dinucleotide repeats (CA/TG repeats) was determined by Southern hybridization with a (TG)15, oligonucleotide probe. Five of 12 (41.63%) turkey fragments hybridized under low stringency conditions. The length of the dinucleotide repeats in the turkey, relative to the chicken sequences, were found to correspond directly with hybridization intensity. Amplification of homologous loci was confirmed by direct sequencing and subsequent alignment of the turkey and chicken sequences. The results of this study indicate that the use of chicken-specific microsatellite primers will rapidly and significantly enhance construction of a genetic map for the turkey.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey, pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey,pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

Genomic analysis of genetic markers associated with inherited cardiomyopathy (round heart disease) in the turkey (Meleagris gallopavo)

In turkeys, spontaneous cardiomyopathy or round heart (RH) disease is characterised by dilated ventricles and cardiac muscle hypertrophy. Although the aetiology of RH is still unknown, the disease can have a significant economic impact on turkey producers. In an initial attempt to identify genomic regions associated with RH, we utilised the chicken genome sequence to target short DNA sequences (sequence-characterised amplified regions, SCARs) identified in previous studies that had significant differences in frequency distribution between RH+ and RH- turkeys. SCARs were comparatively aligned with the chicken whole-genome sequence to identify flanking regions for primer design. Primers from 32 alignments were tested and target sequences were successfully amplified for 30 loci (94%). Comparative re-sequencing identified putative SNPs in 20 of the 30 loci (67%). Genetically informative SNPs at 16 loci were genotyped in the UMN/NTBF turkey mapping population. As a result of this study, 34 markers were placed on the turkey/chicken comparative map and 15 markers were added to the turkey genetic linkage map. The position of these markers relative to cardiac-related genes is presented. In addition, analysis of genotypes at 109 microsatellite loci presumed to flank the SCAR sequences in the turkey genome identified four significant associations with RH.     

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.     

Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

Reticuloendotheliosis virus 60S RNA labeled with (125)I, or reticuloendotheliosis virus complementary DNA labeled with (3)H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and (125)I-labeled viral RNAs and by determination of melting temperatures. In contrast, (125)I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck.     

Cell killing by spleen necrosis virus is correlated with a transient accumulation of spleen necrosis virus DNA.

Spleen necrosis virus productively infects avian and rat cells. The average number of molecules of unintegrated and integrated viral DNA in cells at different times after infection was determined by hybridization and transfection assays. Shortly after infection, there was a transient accumulation of an average of about 150 to 200 molecules of unintegrated linear spleen necrosis virus DNA per chicken, turkey, or pheasant cell. No such accumulation was seen in infected rat cells. Soon after infection there was in chicken cells, but not inturkey, pheasant, or rat cells, also a transient integration of an average of 35 copies of viral DNA per cell. By 10 days after infection, the majority of this integrated viral DNA was lost from the population of infected chicken cells. At the same time, the majority of the unintegrated viral DNA was also lost from infected chicken, turkey, and pheasant cells. The transient cytopathic effect seen in these infected cells also occurred at this time. Late after infection about five copies of apparently nondefective spleen necrosis proviruses were stably integrated at multiple sites in chicken, turkey, pheasant, and rat DNA. These results demonstrate a correlation between the transient accumulation of large numbers of spleen necrosis virus DNA molecules and the transient occurrence of cytopathic effects.     

Allelic variation and genetic linkage of avian microsatellites in a new turkey population for genetic mapping

Efforts to build a comprehensive genetic linkage map for the turkey (Meleagris gallopavo) have focused on development of genetic markers and experimental resource families. In this study, PCR amplification was attempted for 772 microsatellite markers that had been previously developed for three avian species (chicken, quail and turkey). Allelic polymorphism at 410 markers (53.1% of total examined) was determined by genotyping ten individuals (six F1 parents and four grandparents) in a new resource population specifically developed for genetic linkage mapping. Of these 410 markers, 109 (26.6%) were polymorphic in the tested individuals, with an average of 2.3 alleles per marker. Higher levels of polymorphism were found for the turkey-specific markers (61.1%) than for the chicken (22.7%) or quail-specific markers (33.3%). To test the fidelity of the matings, demonstrate the power of these families for linkage analysis, and determine genetic linkage relationships, 86 polymorphic markers were genotyped for up to 224 birds including founder grandparents, parents and F2 progeny. Linkage relationships for many of the chicken markers elucidated in the turkey were comparable to those observed in the chicken. These data demonstrate that the new UMN/NTBF resource population will provide a solid foundation for constructing a comparative genetic map of the turkey.     

Homology between avian oncornavirus RNAs and DNA from several avian species.

3H-labeled 35S RNA from avian myeloblastosis virus (AMV), Rous associated virus (RAV)-0, RAV-60, RAV-61, RAV-2, or B-77(w) was hybridized with an excess of cellular DNA from different avian species, i.e., normal or leukemic chickens, normal pheasants, turkeys, Japanese quails, or ducks. Approximately two to three copies of endogenous viral DNA were estimated to be present per diploid of normal chicken cell genome. In leukemic chicken myeloblasts induced by AMV, the number of viral sequences appeared to have doubled. The hybrids formed between viral RNA and DNA from leukemic chicken cells melted with a Tm 1 to 6 C higher than that of hybrids formed between viral RNA and normal chicken cell DNA. All of the viral RNAs tested, except RAV-61, hybridized the most with DNA from AMV-infected chicken cells, followed by DNA from normal chicken cells, and then pheasant DNA. RAV-61 RNA hybridized maximally (39%) with pheasant DNA, followed by DNA from leukemic (34%), and then normal (29%) chicken cells. All viral RNAs tested hybridized little with Japanese quail DNA (2 to 5%), turkey DNA (2 to 4%), or duck DNA (1%). DNA from normal chicken cells contained only 60 to 70% of the RAV-60 genetic information, and normal pheasant cells lacked some RAV-61 DNA sequences. RAV-60 and RAV-61 genomes were more homologous to the RAV-0 genome than to the genome of RAV-2, AMV, or B-77(s). RAV-60 and RAV-61 appear to be recombinants between endogenous and exogenous viruses.     

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.     

A first-generation map of the turkey genome.

A primary linkage map of the domestic turkey (Meleagris gallopavo) was developed by segregation analysis of genetic markers within a backcross family. This reference family includes 84 offspring from one F1 sire mated to two dams. Genomic DNA was digested using one of five restriction enzymes, and restriction fragment length polymorphisms were detected on Southern blots using probes prepared from 135 random clones isolated from a whole-embryo cDNA library. DNA sequence was subsequently determined for 114 of these cDNA clones. Sequence comparisons were done using BLAST searches of the GenBank database, and redundant sequences were eliminated. High similarity was found between 23% of the turkey sequences and mRNA sequences reported for the chicken. The current map, based on expressed genes, includes 138 loci, encompassing 113 loci arranged into 22 linkage groups and an additional 25 loci that remain unlinked. The average distance between linked markers is 6 cM and the longest linkage group (17 loci) measures 131 cM. The total map distance contained within linkage groups is 651 cM. The present map provides an important framework for future genome mapping in the turkey.