Chromosomal localization and molecular characterization of 53 cosmid-derived bovine microsatellites

Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap. One objective of BovMap is to develop a set of anchored loci for the cattle genome map.Two cosmid libraries were screened with probes corresponding to the (AC) _n microsatellite motif. Positive clones were mapped by FISH, and then a subset was further analyzed by sequencing the region flanking the microsatellite repeat. In total, 58 clones were hybridized with chromosomes and identified loci on 22 of the 31 different bovine chromosomes. Three clones contained satellite DNA. Two or more markers were placed on 12 chromosomes. Sequencing of the microsatellites and flanking regions was performed directly from 43 cosmids, as previously reported (Ferretti et al. Anim. Genet. 25, 209–214, 1994). Primers were developed for 39 markers and used to describe the polymorphism associated with the corresponding loci.     

Characterization, genetic and physical mapping analysis of 36 horse plasmid and cosmid-derived microsatellites

Thirty-six new horse microsatellites (11 from plasmid libraries and 25 from a cosmid library) were isolated and characterized on a panel of four horse breeds. Thirty were found to be polymorphic with heterozygosity levels ranging between 0.20 and 0.87. Twenty-two of the cosmids were physically mapped to R-banded single horse Chromosomes (Chrs) 1, 3, 4, 9, 11, 12, 13, 15, 18, 19, 21, 22, 23 and three to pericentromeric regions. Furthermore, linkage analysis between a selection of 42 DNA markers, including those presented in this study, and 16 conventional markers of the horse hemotype was performed on six paternal half-sib horse families. Five linkage groups were detected, of which four were assigned to Chr 10, 11, 15, and 18. This work increased by one-third the number of published polymorphic DNA markers suitable for horse mapping and approximately doubled the number of known linkage groups. Our cosmids labeled 14 out of the 31 horse autosomes. Moreover, the physical anchoring of part of these markers will orient linkage and synteny groups on the chromosomes and will contribute to their assignment. Received: 17 March 1997 / Accepted: 25 May 1997     

Isolation and characterization of microsatellites from the canine genome

Microsatellite sequences comprising (dC-dA)_n.(dG-dT)_n repeats have been isolated from canine libraries and sequenced. Oligonucleotide primers have been synthesized to the micro-satellite flanking sequences and used in the polymerase chain reaction to amplify those loci from genomic DNA. The degree of polymorphism of each microsatellite was estimated in a set of unrelated dogs. It is concluded that of the 10 loci studied, nine are sufficiently polymorphic to be useful in genetic studies.     

Variability of canine microsatellites within and between different dog breeds

Polymorphic animal microsatellites have proved valuable genetic markers. For this project, the variability of 19 canine microsatellite loci was examined within and between three pure breeds of dog: Greyhounds, Labradors, and German Shepherds. The number of alleles, absolute and relative frequencies, and the statistics that express polymorphism within a breed were determined. The evolutionary relationships among these closely related dog breeds were estimated by genetic distance measures developed for use with microsatellite loci. According to the pairwise genetic distances, Greyhounds and German Shepherds had longer diverse evolutionary histories than Greyhounds and Labradors or Labradors and German Shepherds. Although a few breed-specific alleles were observed, the significant differences between breeds are in their relative frequencies and distribution of the alleles across a locus. None of the three pure dog breeds corresponds to Hardy-Weinberg equilibrium. A considerable reduction in intrapopulation variation was observed within three pure breeds, compared with the population of individuals belonging to 15 dog breeds. This reduction was especially pronounced in the Greyhound breed, which expressed the lowest degree of variation. Intrapopulation variations of Labradors and German Shepherds did not differ significantly, that of Labradors being only slightly higher. The intra-species variation of dogs is lower than in humans, mouse, or rat, but similar to that in domestic animals, probably reflecting similarly high inbreeding coefficients. However, some highly informative loci were common to all dog breeds tested so far. Such population data are necessary for mapping studies and linkage analysis in dogs. Received: 31 July 1996 / Accepted: 21 October 1996     

Characterization of novel microsatellites from Drosophila transversa

We investigated a partial genomic library of Drosophila transversa for microsatellites and developed 12 markers for genetic analyses. This is the first time that microsatellite primers from the quinaria species group have been described. Four loci were cross-amplified in D. phalerata. Nine out of the 12 microsatellite markers developed are likely to be on the X chromosome.     

Two polymorphic microsatellites in a coding segment of the canine androgen receptor gene

A 0.6-kb segment of exon 1 of the canine androgen receptor gene contains two polymorphic CAG tandem repeats which encode strings of glutamine homopolymers. The number of CAGs in each tandem repeat was determined by (1) polymerase chain reaction (PCR) amplification of a gene segment containing both repeats, (2) cleavage between repeats with restriction enzyme EcoO109I and (3) fractionation of the restriction fragments containing individual CAG repeats by denaturing polyacrylamide gel electrophoresis (PAGE). Individual genomic DNA samples from 80 unrelated dogs (53 males plus 27 females for a total of 107 X chromosomes) contained 10–12 CAGs in the 5′ repeats and 10–13 CAGs in the 3′ repeats. Thirteen distinct androgen receptor genotypes were identified. Eleven (or 41%) of the 27 unrelated females were heterozygous in one or both repeat regions, whereas all male samples produced single bands as expected for X chromosome markers. A total of seven distinct haplotypes contributed to the 13 genotypes. The ‘polymorphism information content’ or PIC for this seven-allele X chromosome marker was 0.67.     

A comparative genetic analysis of the Irish greyhound population using multilocus DNA fingerprinting, canine single locus minisatellites and canine microsatellites

Pairwise analysis of Hin fI/33·6 DNA fingerprints from a total of one hundred and fifty-three Irish greyhounds of known pedigree were used to determine band-share estimates of unrelated, first-degree and second-degree relationships. Forty-eight unrelated Irish greyhounds were used to determine allele frequencies for three single-locus minisatellites, and following a preliminary screen, eight of the most polymorphic tetra-nucleotide microsatellites from a panel of 15. The results indicated that both band-share estimates by DNA fingerprinting and microsatellite allele frequencies are highly effective in resolving parentage in this greyhound population, while single-locus minisatellites showed limited polymorphism and could not be used alone for routine parentage testing in this breed. The present study also demonstrated that, to obtain optimal resolution of parentage, sample sets of known pedigree status are required to determine the band-share distribution and/or microsatellite allele frequencies.     

Development of 198 novel EST-derived microsatellites in Eucalyptus (Myrtaceae)

? Premise of the study: Expressed sequence tag (EST)-derived microsatellites were identified in Eucalyptus through screening the GenBank database. The loci were sequence-verified and explored for polymorphism among 20 genotypes. ? Methods and Results: In total, 198 novel microsatellites were developed from 8262 unigenes, with the identity of 73.6-100% to the original sequences and presence of the expected repeat motifs. One hundred and eighty-four markers proved to be polymorphic among 10 E. urophylla and 10 E. tereticornis genotypes, with the number of alleles per locus, observed heterozygosity, and polymorphic information content being 2-17 (mean: 7.11), 0-1.0 (mean: 0.4511), and 0.0940-0.9131 (mean: 0.6571), respectively. ? Conclusions: These markers will be useful for germplasm characterization, genome mapping, and gene tagging for economic traits in the two species examined and may have potential for genetic applications in Eucalyptus.     

Population-scale analysis of human microsatellites reveals novel sources of exonic variation

Using our microsatellite specific genotyping method, we analyzed tandem repeats, which are known to be highly variable with some recognized as biomarkers causative of disease, in over 500 individuals who were exon sequenced in a 1000 Genomes Project pilot study. We were able to genotype over 97% of the microsatellite loci in the targeted regions. A total of 25,115 variations were observed, including repeat length and single nucleotide polymorphisms, corresponding to an average of 45.6 variations per individual and a density of 1.1 variations per kilobase. Standard variant detection did not report 94.2% of the exonic repeat length variations in part because the alignment techniques are not ideal for repetitive regions. Additionally some standard variation detection tools rely on a database of known variations, making them less likely to call repeat length variations as only a small percent of these loci (~ 6000) have been accurately characterized. A subset of the hundreds of non-synonymous variations we identified was experimentally validated, indicating an accuracy of 96.5% for our microsatellite-based genotyping method, with some novel variants identified in genes associated with cancer. We propose that microsatellite-based genotyping be used as a part of large scale sequencing studies to identify novel variants.