Identifying Y-chromosomal diversity by long-template PCR

Comparing Y-chromosomal and mitochondrial haplotype variation is a promising approach to independently investigate paternal and maternal evolutionary histories in wild mammal populations. However, the difficulty of developing male-specific genetic markers, because of its distinctive genetic architecture and the general low level of polymorphisms observed on the Y chromosome, hampers usually an effective application of this approach. Here, we present a further method of the established Y chromosome conserved anchored tagged sequences strategy to develop Y-chromosomal markers by screening introns of male-specific region (MSY) genes for sequence polymorphisms. By applying long-template PCR using target species-specific primers, adequate sequence information of several kb in size can be obtained. We applied this method in the snow vole (Chionomys nivalis) and obtained 12.4 kb of male-specific sequence data for nine males representing four populations in the Swiss Alps. A total of 28 single nucleotide polymorphisms, four indels (> 1 bp) and one polymorphic microsatellite were identified in introns of the SMCY and DBY genes. Based on this information, we developed a Y-chromosomal genotyping assay and identified four different paternal lineages within one local snow vole population. The method we present is straightforward and as such will probably be suitable to detect adequate Y-chromosomal diversity in a wide range of mammalian species.     

Isolation of Y chromosome-specific microsatellites in the horse and cross-species amplification in the genus Equus

Y chromosome polymorphisms such as microsatellites or single nucleotide polymorphisms represent a paternal counterpart to mitochondrial DNA (mtDNA) for evolutionary and phylogeographic studies. The use of Y chromosome haplotyping in natural populations of species other than humans is still hindered by the lack of sequence information necessary for polymorphism screening. Here we used representational difference analysis (RDA) followed by a screen of a bacterial artificial chromosome (BAC) library for repetitive sequences to obtain polymorphic Y-chromosomal markers. The procedure was performed for the domestic horse (Equus caballus) and we report the first six Y-chromosomal microsatellite markers for this species. Three markers were also useful for haplotyping taxa of the zebra/ass lineage. Y-chromosomal microsatellite markers show a single haplotype in the domestic horse, whereas notable variation has been observed in the other members of the genus Equus.     

New, Male-Specific Microsatellite Markers from the Human Y Chromosome

Seven novel microsatellite markers have been developed from a new cosmid library constructed from flow-sorted human Y chromosomes. These microsatellites are tetranucleotide GATA repeats and are polymorphic among unrelated individuals. Five of the seven markers are male-specific, with no PCR product being generated from female DNA. One marker produces male-specific, polymorphic PCR products but occasionally produces a much larger, invariant product from female DNA. The remaining marker is polymorphic in both males and females with many shared alleles between the sexes. This report of six new, male-specific markers doubles the number of tetranucleotide markers that are currently available for the human Y chromosome. These new markers will be valuable where nonrecombining, gender-specific DNA markers are desired, including forensic investigations as well as studies of populations and their evolutionary histories.     

Y chromosome conserved anchored tagged sequences (YCATS) for the analysis of mammalian male-specific DNA

Y chromosome haplotyping based on microsatellites or single nucleotide polymorphisms has recently proven to be a powerful approach for evolutionary studies of human populations, and also holds great promise for the studies of wild species. However, the use of the approach is hampered in most natural populations by the lack of Y chromosome markers and sequence information. Here, we report the large-scale development of Y chromosome conserved anchor tagged sequence (YCATS) markers in mammals by a polymerase chain reaction screening approach. Exonic primers flanking 48 different introns of Y-linked genes were developed based on human and mouse sequences, and screened on a set of 20 different mammals. On average about 10 introns were amplified for each species and a total of 100 kb of Y chromosome sequence were obtained. Intron size in humans was a reasonable predictor of intron size in other mammals (r2 = 0.45) and there was a negative correlation between human fragment size and amplification success. We discuss a number of factors affecting the possibility of developing conserved Y chromosome markers, including fast evolution of Y chromosome sequences due to male-biased mutation and adaptive evolution of male-specific genes, dynamic evolution of the Y chromosome due to being a nonrecombining unit, and homology with X chromosome sequences.     

Phylogenetic assessment of introns and SINEs within the Y chromosome using the cat family felidae as a species tree

The cat family Felidae was used as a species tree to assess the phylogenetic performance of genes, and their embedded SINE elements, within the nonrecombining region of the Y chromosome (NRY). Genomic segments from single-copy X-Y homologs SMCY, UBE1Y, and ZFY (3,604 bp) were amplified in 36 species of cat. These genes are located within the X-degenerate region of the NRY and are thought to be molecular "fossils" that ceased conventional recombination with the X chromosome early within the placental mammal evolution. The pattern and tempo of evolution at these three genes is significant in light of the recent, rapid evolution of the family over approximately 12 Myr and provides exceptional support for each of the eight recognized felid lineages, as well as clear diagnostic substitutions identifying nearly all species. Bootstrap support and Bayesian posterior probabilities are uniformly high for defining each of the eight monophyletic lineages. Further, the preferential use of specific target-site motifs facilitating SINE insertion is empirically supported by sequence analyses of SINEs embedded within the three genes. Target-site insertion is thought to explain the contradiction between intron phylogeny and results of the SMCY SINE phylogeny that unites distantly related species. Overall, our data suggest X-degenerate genes within the NRY are singularly powerful markers and offer a valuable patrilineal perspective in species evolution.     

Molecular characterization of a bovine Y-specific DNA sequence conserved in taurine and zebu breeds.

The identification of new bovine male-specific DNA sequences is of great interest because the bovine Y chromosome remains poorly characterized in terms of physical and genetic maps. Since taurine and zebu Y chromosomes are structurally different, the identification of Y-specific sequences present in both sub-species is particularly important: these sequences are of evolutionary significance and can be broadly used for embryo sexing. In this work, we initially used the random amplified polymorphic DNA (RAPD) technique to search for male-specific sequences present as monomorphic markers in genomic DNA from zebu and taurine bulls. A male-specific RAPD band was found to be present and highly conserved in both sub-species, as demonstrated by Southern blotting, fluorescent in situ hybridization (FISH) and DNA sequencing. In a previous work, a pair of primers derived from this marker was successfully used in taurine and zebu embryo sexing.     

Identification of new microsatellite markers in Panax ginseng

Microsatellites, also called simple sequence repeats (SSR), are very useful molecular genetic markers commonly used in crop breeding, species identification and linkage analysis. In the present study, we constructed a microsatellite-enriched genomic library of Panax ginseng, and identified 251 novel microsatellite sequences. Tri-nt repeat units were the most abundant (46.6%), followed by di-nt repeats (35.5%). The (AG)n motif was most common (23.1%), followed by the (AAC)n motif (22.3%). From the genotyping of 94 microsatellites using marker-specific primer sets, we identified 11 intraspecific polymorphic markers as well as 14 possible interspecific polymorphic markers differing between P. ginseng and P. quinquefolius. The exact allele structures of the polymorphic markers were determined and the alleles were named. This study represents the first report of the bulk isolation of microsatellites by screening a microsatellite-enriched genomic library in P. ginseng. The microsatellite markers could be useful for linkage analysis, genetic breeding and authentication of Panax species.     

Waste not,want not: Microsatellites remain an economical and informative technology for conservation genetics

Comparisons of microsatellites and single‐nucleotide polymorphisms (SNPs) have found that SNPs outperform microsatellites in population genetic analyses, questioning the continued utility of microsatellites in population and landscape genetics. Yet, highly polymorphic markers may be of value in species that have reduced genetic variation. This study repeated previous analyses that used microsatellites with SNPs developed from ddRAD sequencing in the black‐capped vireo source‐sink system. SNPs provided greater resolution of genetic diversity, population differentiation, and migrant detection but could not reconstruct parentage relationships due to insufficient heterozygosities. The biological inferences made by both sets of markers were similar: asymmetrical gene flow from source sites to the remaining sink sites. With the landscape genetic analyses, we found different results between the two molecular markers, but associations of the top environmental features (riparian, open habitat, agriculture, and human development) with dispersal estimates were shared between marker types. Despite the higher precision of SNPs, we find that microsatellites effectively uncover population processes and patterns and are superior for parentage analyses in this species with reduced genetic diversity. This study illustrates the continued applicability and relevance of microsatellites in population genetic research.     

Genomic heterogeneity in the density of noncoding single-nucleotide and microsatellite polymorphisms in Plasmodium falciparum

The density and distribution of single-nucleotide polymorphisms (SNPs) across the genome has important implications for linkage disequilibrium mapping and association studies, and the level of simple-sequence microsatellite polymorphisms has important implications for the use of oligonucleotide hybridization methods to genotype SNPs. To assess the density of these types of polymorphisms in P. falciparum, we sampled introns and noncoding DNA upstream and downstream of coding regions among a variety of geographically diverse parasites. Across 36,229 base pairs of noncoding sequence representing 41 genetic loci, a total of 307 polymorphisms including 248 polymorphic microsatellites and 39 SNPs were identified. We found a significant excess of microsatellite polymorphisms having a repeat unit length of one or two, compared to those with longer repeat lengths, as well as a nonrandom distribution of SNP polymorphisms. Almost half of the SNPs localized to only three of the 41 genetic loci sampled. Furthermore, we find significant differences in the frequency of polymorphisms across the two chromosomes (2 and 3) examined most extensively, with an excess of SNPs and a surplus of polymorphic microsatellites on chromosome 3 as compared to chromosome 2 (P=0.0001). Furthermore, at some individual genetic loci we also find a nonrandom distribution of polymorphisms between coding and flanking noncoding sequences, where completely monomorphic regions may flank highly polymorphic genes. These data, combined with our previous findings of nonrandom distribution of SNPs across chromosome 2, suggest that the Plasmodium falciparum genome may be a mosaic with regard to genetic diversity, containing chromosomal regions that are highly polymorphic interspersed with regions that are much less polymorphic.     

Bovine Y chromosome microsatellite polymorphisms

Thirty-eight bovine Y chromosome (BTAY) microsatellites (MS) were assessed for polymorphisms in DNA samples obtained from 17 unrelated bulls. Thirty-three of these microsatellites are new and were used for the construction of a first generation radiation hybrid map for BTAY (Liu et al., 2002). Five MS had been previously reported and were used as positive controls. Fourteen out of 38 MS were found to be polymorphic; the remaining 24 were uninformative among the animals tested. The number of hemizygous loci per MS within individual ranged from two to over 20. Seven MS presented smear- or ladder-like bands, a unique feature for Y chromosome multi-copy hemizygous MS loci. The locus length variance, within individual, ranged from 2 to 42 bp corresponding to the MS with the minimum and maximum number of loci observed, respectively. Within the 14 polymorphic MS, the five pseudoautosomal MS, on average, were more polymorphic (35.3%) than the nine Y-specific MS (19.6%). Haplotypes resulting from combinations of these polymorphic loci will provide a powerful tool for future studies on the origin of domestic cattle and the evolution of bovid species.     

Isolation and characterization of 70 novel microsatellite markers from ostrich (Struthio camelus) genome.

Microsatellite markers are widely used in linkage mapping, parentage testing, population genetic studies, and molecular evolution studies in many agricultural species, while only a limited number of ostrich (Struthio camelus) microsatellites have been isolated. Thus, we constructed a random small-insert genomic library and a microsatellite-enriched library containing CA repeats. Fourteen clones containing CA repeats were isolated from 3462 clones in the non-enriched library by radioactive screening and 248 positive clones were isolated from 300 sequenced clones from the enriched library by PCR screening. After the enrichment procedures, the proportion of clones containing CA repeats was raised to 78.8%, compared with 0.4% in the non-enriched libraries, indicating that the enrichment value approaches 200 fold, which decreased the time and cost of cloning. The number of complete simple CA repeats in these positive clones ranged from 5 to 29. The primers for 94 of these microsatellites were developed and used to detect polymorphisms, of which 61 loci exhibited length polymorphisms in 17 unrelated ostrich individuals. The new polymorphic microsatellite markers we have identified and characterized will contribute to the ostrich genetic map, parentage testing, and comparative genomics between avian species.     

Y-specific microsatellite polymorphisms in a range of bovid species

At least five dinucleotide (CA)n microsatellite repeat arrays have been assigned to the bovine Y-chromosome, with one marker (INRA124) shown to be polymorphic. We describe here the assessment of a panel of four Y-specific microsatellite markers for polymorphism in a range of cattle and related species. It was possible to amplify all the markers in the animals sampled and all showed variation. Three of the microsatellite loci (INRA124, INRA189 and BM861) displayed putative taurine- and zebu-specific alleles which can be useful indicators of male-mediated gene flow in hybrid populations. In the future these microsatellites, in combination with other Y-specific markers should provide a high-resolution Y haplotype system for evolutionary studies in both domesticated cattle and other related species.     

Handling marker-marker linkage disequilibrium: pedigree analysis with clustered markers

Single-nucleotide polymorphisms (SNPs) are rapidly replacing microsatellites as the markers of choice for genetic linkage studies and many other studies of human pedigrees. Here, we describe an efficient approach for modeling linkage disequilibrium (LD) between markers during multipoint analysis of human pedigrees. Using a gene-counting algorithm suitable for pedigree data, our approach enables rapid estimation of allele and haplotype frequencies within clusters of tightly linked markers. In addition, with the use of a hidden Markov model, our approach allows for multipoint pedigree analysis with large numbers of SNP markers organized into clusters of markers in LD. Simulation results show that our approach resolves previously described biases in multipoint linkage analysis with SNPs that are in LD. An updated version of the freely available Merlin software package uses the approach described here to perform many common pedigree analyses, including haplotyping and haplotype frequency estimation, parametric and nonparametric multipoint linkage analysis of discrete traits, variance-components and regression-based analysis of quantitative traits, calculation of identity-by-descent or kinship coefficients, and case selection for follow-up association studies. To illustrate the possibilities, we examine a data set that provides evidence of linkage of psoriasis to chromosome 17.     

Comparing RADseq and microsatellites for estimating genetic diversity and relatedness — Implications for brown trout conservation

The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction‐site‐associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout (Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as F_ST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half‐ and full‐siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual‐level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness and individual‐level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.     

A comprehensive survey of human Y-chromosomal microsatellites

We have screened the nearly complete DNA sequence of the human Y chromosome for microsatellites (short tandem repeats) that meet the criteria of having a repeat-unit size of > or = 3 and a repeat count of > or = 8 and thus are likely to be easy to genotype accurately and to be polymorphic. Candidate loci were tested in silico for novelty and for probable Y specificity, and then they were tested experimentally to identify Y-specific loci and to assess their polymorphism. This yielded 166 useful new Y-chromosomal microsatellites, 139 of which were polymorphic, in a sample of eight diverse Y chromosomes representing eight Y-SNP haplogroups. This large sample of microsatellites, together with 28 previously known markers analyzed here--all sharing a common evolutionary history--allowed us to investigate the factors influencing their variation. For simple microsatellites, the average repeat count accounted for the highest proportion of repeat variance (approximately 34%). For complex microsatellites, the largest proportion of the variance (again, approximately 34%) was explained by the average repeat count of the longest homogeneous array, which normally is variable. In these complex microsatellites, the additional repeats outside the longest homogeneous array significantly increased the variance, but this was lower than the variance of a simple microsatellite with the same total repeat count. As a result of this work, a large number of new, highly polymorphic Y-chromosomal microsatellites are now available for population-genetic, evolutionary, genealogical, and forensic investigations.     

Multiple mono- and polymorphic Y-linked copies of the SRY HMG-box in microtidae.

Sex determination in mammals is controlled by SRY (sex-determining region of the Y chromosome), a single-copy gene located on the Y-specific region. Several exceptions to this rule have been described: some rodent species present Y-specific multiple copies (either mono- or polymorphic) of this gene, and two Ellobius species and one Tokudaia species determine sex without a Y chromosome or the SRY gene. Recently, we have described multiple polymorphic copies of the SRY gene in both males and females of the vole species Microtus cabrerae. The female location and the presence of stop codons in some copies from males and females also suggest that they are nonfunctional copies of this gene (pseudogenes). We have investigated the SRY HMG-box in nine species of the family Microtidae; we report here the presence, in eight of these species, of multiple mono- or polymorphic copies of the SRY gene located on the Y chromosome.     

Karyotypic conservatism in the suborder Feliformia (Order Carnivora)

Multidirectional comparative chromosome painting was used to investigate the karyotypic relationships among representative species from three Feliformia families of the order Carnivora (Viverridae, Hyaenidae and Felidae). Complete sets of painting probes derived from flow-sorted chromosomes of the domestic dog, American mink, and human were hybridized onto metaphases of the spotted hyena (Crocuta crocuta, 2n = 40) and masked palm civet (Paguma larvata, 2n = 44). Extensive chromosomal conservation is evident in these two species when compared with the cat karyotype, and only a few events of chromosome fusion, fission and inversion differentiate the karyotypes of these Feliformia species. The comparative chromosome painting data have enabled the integration of the hyena and palm civet chromosomes into the previously established comparative map among the domestic cat, domestic dog, American mink and human and improved our understanding on the karyotype phylogeny of Feliformia species.     

Y-chromosome analysis

After initially slow progress in identifying DNA polymorphisms on the non recombining part of the human Y chromosome, a large number of polymorphic markers are now available to define individual or population-specific haplotypes or haplogroups. Among them the Y-chromosomal STRs (Short Tandem Repeats) have been increasingly used over the past few years for the study of human evolution as well as for human identification in forensic case-work and paternity testing. After a brief summary of the features of such markers, this paper deals with some applications of the Y-STR haplotyping.     

Differentiating salmon populations at broad and fine geographical scales with microsatellites and single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs) are appealing genetic markers due to several beneficial attributes, but uncertainty remains about how many of these bi-allelic markers are necessary to have sufficient power to differentiate populations, a task now generally accomplished with highly polymorphic microsatellite markers. In this study, we tested the utility of 37 SNPs and 13 microsatellites for differentiating 29 broadly distributed populations of Chinook salmon ( n  = 2783). Information content of all loci was determined by In and     , and the top 12 markers ranked by In were microsatellites, but the 6 highest, and 7 of the top 10     ranked markers, were SNPs. The mean ratio of random SNPs to random microsatellites ranged from 3.9 to 4.1, but this ratio was consistently reduced when only the most informative loci were included. Individual assignment test accuracy was higher for microsatellites (73.1%) than SNPs (66.6%), and pooling all 50 markers provided the highest accuracy (83.2%). When marker types were combined, as few as 15 of the top ranked loci provided higher assignment accuracy than either microsatellites or SNPs alone. Neighbour-joining dendrograms revealed similar clustering patterns and pairwise tests of population differentiation had nearly identical results with each suite of markers. Statistical tests and simulations indicated that closely related populations were better differentiated by microsatellites than SNPs. Our results indicate that both types of markers are likely to be useful in population genetics studies and that, in some cases, a combination of SNPs and microsatellites may be the most effective suite of loci.     

A radiation hybrid map for the bovine Y Chromosome

Screening a bovine Y Chromosome-specific DNA library resulted in 34 new microsatellites, six of which mapped to the pseudoautosomal region (PAR), and 28 localized to the Y-specific region. These microsatellites, together with 23 markers previously mapped to the bovine Y Chr, were scored on a 7000-rad cattle–hamster radiation hybrid (RH) panel. Retention frequency of individual markers ranged from 18.5% to 76.5% with an average of 48.4%. Markers with high retention frequency (>55%) were found to exist in multiple copies on the Y Chr. Thirteen markers were placed on the PAR RH map with the AmelY gene proximal to the pseudoautosomal boundary and 46 markers, including Sry and Tspy gene, on the Y-specific region of the RH map. The microsatellites developed and mapped in this work will be useful for comparative mapping of cattle, sheep, and goat, studying the origin, evolution, and migration of bovidae species and provide an initial platform to develop a high-resolution map of the Y Chr and positional cloning of Y-specific genes.