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.     

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.     

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.     

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.     

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.     

Development and mapping of ten porcine microsatellite markers

Thirty (TG)_n microsatellite clones were isolated from a pig genomic library, sequenced, and tested for their suitability to detect polymorphism on a panel of animals by means of the polymerase chain reaction. Ten of these clones were developed into suitable markers and subsequently segregation of these markers was determined in the five PiGMaP reference pedigrees. A linkage analysis was performed on these 10 microsatellites together with 365 other loci that have been typed on these reference families. Eight of the microsatellites have been mapped to eight different linkage groups that have been previously assigned to different chromosomes (chromosomes 1, 6, 7, 9, 14, 15, 17 and 18). Of the remaining two markers, one is X-linked and the other shows no linkage. The number of alleles detected by these microsatellites, in the reference pedigrees, varied from six to sixteen and the heterozygosity varied from 42 to 85% in the 26 unrelated founder animals of these reference pedigrees.     

Characterization and linkage mapping often sheep microsatellite markers derived from a sheep x hamster cell hybrid

A cosmid library has been constructed from a sheep x hamster cell hybrid containing sheep chromosome t₁, rob (6;24). Clones containing sheep DNA were identified by hybridizing to a total sheep genomic DNA probe. Small fragments (<500 bp) containing (AC)_n microsatellites were subcloned and sequenced. Ten microsatellite markers were characterized and six were mapped back to chromosomes 6 and 24. The remaining microsatellites mapped to chromosome 26, which was shown to be present in a small proportion of cells of the cell line.     

Pig microsatellites isolated from cosmids revealing polymorphism and localized on chromosomes

One of the most widely studied simple sequences in the mammalian genome is the (TG)_n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)₁₀ probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)_n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.     

Isolation and mapping of polymorphic microsatellites in cattle

Summary
A partial plasmid library with bovine genomic inserts of about 500 basepairs was screened with a (dC-dA)_n-(dG-dT)_n oligonucleotide probe for the repeated nucleotide motif (CA)_n. Eleven positive clones (0.3% of all colonies screened) were discovered and were subsequently isolated and sequenced. Eight microsatellite loci were analysed, one with eight alleles, one with seven alleles, three with six alleles, one with three alleles and two with two alleles. Six of these microsatellites were mapped by PCR-analysis of a panel of somatic hybrid lines.     

FHF-2 in the turkey (Meleagris gallopavo)

A cDNA clone homologous to the fibroblast growth factor homologous factor (FHF-2) was isolated and sequenced from the turkey (Meleagris gallopavo). The DNA sequence of the turkey was almost identical to that of the chicken (99% similarity) differing at only 8 of 770 nucleotides in the coding region resulting in a single amino acid difference between these poultry species. The 3'UTR of the turkey FHF-2 gene was 445 nucleotides in length and included an imperfect CT microsatellite (ms) repeat. The sequence of the 3'UTR was amplified from genomic DNA of the chicken and found to be highly conserved differing at only three nucleotides when compared to the turkey. Length of the CT repeat was indifferent in a sample of 52 turkeys (monomorphic) however, the number of CT repeats was greater in the turkey than in the chicken. No inter-individual polymorphism was detected in multiple sequences of the 3'UTR of the FHF-2 gene in the turkey. Based on comparison of the turkey and chicken sequences, the mutation rate for coding and associated non-coding (3'UTR) regions of FHF-2 are approximately equal.     

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.     

Developing microsatellite markers from cDNA: a tool for adding expressed sequence tags to the genetic linkage map of the chicken

A chicken embryonic cDNA library was screened with a (TG)₁₃ probe in order to develop polymorphic microsatellite markers. The redundancy of the embryonic cDNA library with a chicken brain cDNA library, which was used for microsatellite development in a previous study, was extremely high. Of the 300 (TG)₁₃ positive clones, only 80 were unique for the embryonic cDNA library. Still, nine expressed sequences derived from the embryonic cDNA library were mapped in the Wageningen (WAU) resource population. In addition seven microsatellite markers from the chicken brain cDNA library, which were monomorphic or unlinked in the two international reference families in the previous study, were also mapped in the WAU population. Three of the 16 mapped chicken expressed sequence tags (ESTs) showed relatively high percentages of sequence similarity to sequences found in other species. As two of these genes, RAB6 and ZFX/ZFY, have been mapped in humans, they contribute to the comparative map of the chicken.     

Highly polymorphic microsatellite markers in poultry

Microsatellite markers have been established for a large number of species, but up till now very few polymorphic microsatellite markers have been reported in poultry. We have isolated 34 polymorphic chicken microsatellite markers of the poly (TG) type. The number of repeats varied from 9 up to 33. Often, other repeats such as poly (T) or poly (GAA) were present adjacent to the poly (TG) repeat. Polymerase chain reaction amplification of the microsatellites resulted in detection of three or more alleles in a test panel of five different animals for 75% of the microsatellites. Segregation of five microsatellite markers has been tested in a small family.     

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.     

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 42 highly polymorphic bovine microsatellite markers

We have isolated 42 highly polymorphic microsatellite (GT/CA)_n markers from Japanese black cattle Wagyu ( Bos taurus ). Forty-one of the markers were assigned to bovine autosomes with lod scores > 6, through linkage analyses performed on the International Bovine Reference Family Panel (IBRP). The remaining marker showed X-linked inheritance. These markers exhibited an average heterozygosity value of 0.67 with between four and 17 alleles on the IBRP.     

Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific library

A genomic DNA library was produced from flow-sorted rabbit chromosome 1 and enriched for fragments containing CA-repeats. Clones containing CA-repeats were identified and primers for amplification of the microsatellite were developed after sequencing the clone. The degree of polymorphism was tested in rabbits from different breeds. This approach identified 12 microsatellite markers which could be used for studying linkage relationships in the progeny of an F(2)-intercross: (AX/JUxIIIVO/JU) F(2), and two backcrosses: (OS/JxX/J)X/J and (WH/JxX/J)X/J. Seven of these markers were mapped on chromosome 1.