Strong correlation between cross-amplification success and genetic distance across all members of 'True Salamanders' (Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite loci

The unpredictable and low cross-amplification success of microsatellite loci tested for congeneric amphibian species has mainly been explained by the size and complexity of amphibian genomes, but also by taxonomy that is inconsistent with phylogenetic relationships among taxa. Here, we tested whether the cross-amplification success of nine new and 11 published microsatellite loci cloned for an amphibian source species, the fire salamander (Salamandra salamandra), correlated with the genetic distance across all members of True Salamanders (genera Chioglossa, Lyciasalamandra, Mertensiella and Salamandra that form a monophyletic clade within the family of Salamandridae) serving as target species. Cross-amplification success varied strongly among the species and showed a highly significant negative relationship with genetic distance and amplification success. Even though lineages of S. salamandra and Lyciasalamndra have separated more than 30 Ma, a within genus amplification success rate of 65% was achieved for species of Lyciasalamandra thus demonstrating that an efficient cross-species amplification of microsatellite loci in amphibians is feasible even across large evolutionary distances. A decrease in genome size, on the other hand, paralleled also a decrease in amplified loci and therefore contradicted previous results and expectations that amplification success should increase with a decrease in genome size. However, in line with other studies, our comprehensive dataset clearly shows that cross-amplification success of microsatellite loci is well explained by phylogenetic divergence between species. As taxonomic classifications on the species and genus level do not necessarily mirror phylogenetic divergence between species, the pure belonging of species to the same taxonomic units (i.e. species or genus) might be less useful to predict cross-amplification success of microsatellite loci between such species.     

PERMANENT GENETIC RESOURCES: Cross-family amplification: microsatellites isolated from Macropodidae are polymorphic in Potoroidae

The superfamily Macropodoidea consists of two families - the Macropodidae and Potoroidae. Cross-species amplification and polymorphism of microsatellite loci is widely recognized within the macropodid family; however, the success of macropodid loci in potoroid species has not been as widely published. In this study, we tested the amplification and polymorphism of 17 cross-species microsatellite loci isolated from macropodids and potoroids in Bettongia lesueur (a potoroid). Success varied between loci and was not predicted by genetic distance from the species of isolation.     

High degree of transferability of 86 newly developed zebra finch EST-linked microsatellite markers in 8 bird species

High-resolution analysis for population genetic and functional studies requires the use of large numbers of polymorphic markers. The recent increase of available genetic tools is facilitated by the use of publicly available expressed sequence tag (EST) sequence databases that are a valuable resource for identifying gene-linked markers. In the present study, we applied bioinformatics analyses to identify microsatellite markers present in EST sequences from a zebra finch (Taeniopgia guttata) EST database and we explore the success of cross-species amplification of EST-linked microsatellite markers in 7 passerine and 1 nonpasserine species. Eighty-six zebra finch EST-linked microsatellite loci were screened for polymorphism revealing a high amplification success rate and adequate levels of polymorphism (33.3-51%) for relatively closely related species, whereas success decreased in the most distantly related species to zebra finch. EST-linked microsatellites appear to be more highly transferable between taxa than anonymous microsatellites as they revealed higher amplification and polymorphism success between different families indicating that they will be a useful source of gene-linked polymorphic markers in a broad range of avian species.     

A wide-range survey of cross-species microsatellite amplification in birds

The possibility to perform cross-species microsatellite amplification in birds was surveyed by analysing sets of primers developed from the swallow and the pied flycatcher genomes on a panel of 48 different bird species. In total, 162 cases (species/marker combinations) of heterologous amplification were recorded. Ten amplification products were sequenced and all were found to be true homologues of the original loci. There was a significant and negative relationship between microsatellite performance and evolutionary distance between the original species and the tested species. As a rough indicator of expected cross-species microsatellite performance we estimate that 50% of markers will reveal polymorphism in a species with a DNA-DNA hybridization δT_mH value of 5 separating it from the original species. This corresponds to a divergence time of = 11 million years before present for passerine birds. The established relationship between performance and evolutionary distance agrees very well with data obtained from some mammalian species. The proportion of polymorphic loci among those markers that amplified decreased with increasing genetic distance, suggesting that few long repeats are preserved during evolution. One of the swallow markers, HrU2, amplified a specific product in all species analysed and will thus allow access to nuclear sequence data over a broad range of species. The only predictor of cross-species performance was the amount of non-specific amplification seen in the original species. An analysis of 10 species from within the family Hirundinidae with the swallow primers consistently revealed extensive polymorphism with average probabilities of identical genotypes ranging from 6 times 10^-4 to 6 times 10^-7. There were distinct allele frequency differences between the Hirundinidae species and we envisage that microsatellite cross-species amplification will be a useful tool in phylogeny construction and in species identification.     

Comparison of genetic diversity at microsatellite loci in near-extinct and non-endangered species of Mexican goodeine fishes and prediction of cross-amplification within the family

The cross-utility of 12 microsatellite loci (including nine newly developed loci) amongst the viviparous subfamily, the Mexican Goodeinae, was assessed, examining both the probability of amplification and the potential incidence of null alleles from tests of F _IS. Genetic diversity was relatively high in comparison to other freshwater species. Amplification success was not correlated with genetic distance between microsatellite source and target species, but taxa that were more distantly related were less likely to be cross-polymorphic for microsatellite loci. On average, species that were cross-polymorphic were separated by a genetic distance of 15% at the cytochrome oxidase I locus, while those that were monomorphic were separated by 19%. There was no evidence that null alleles become more frequent at greater source-target genetic distance.     

Cross-species amplification of microsatellite primers in passerine birds

Developing species specific microsatellite primers can be avoidedby using existing markers which amplify across species. However,for passerines, such cross-species markers are mostly lackingand few guidelines exist for selecting them from the wide rangeof existing markers. Here cross-species amplification tests of 40microsatellite primers in 13 passerine species show an increasein probability of amplification and polymorphism with decreasingphylogenetic distance. Primers which successfully amplified inmany species had a higher chance to be polymorphic. However,since the amplification success, across a broad range of species,of particular primersets remains difficult to predict it iscrucial to identify such markers empirically. Here we describesuch widely applicable bird (passerines) microsatellite markers.     

Utility of pairwise mtDNA genetic distances for predicting cross-species microsatellite amplification and polymorphism success in fishes

In many studies involving microsatellites cross-species amplification, primers designed for one (source) species are used to amplify homologous loci in related (target) species. However, it is not clear how closely related the species must be to attain significant success. Genetic divergence is a clear and easy way to assess similarity between species and provides an accurate measure of their evolutionary distance. Eight Mediterranean target species of the family Serranidae were analysed using twelve primers developed for Serranus cabrilla. Additionally, two mitochondrial genes (12S rRNA and 16S rRNA) were chosen on the basis of their extensive use in phylogenetic and evolutionary analyses to compute genetic divergence between the species. Significant negative correlations were found between genetic divergence and both cross-species amplification and maintained polymorphism of microsatellite markers, which could be generalized by gathering information from different fish studies. The success of obtaining amplifiable and polymorphic microsatellite loci can be a priori approximated knowing the mtDNA genetic divergence between a given source and target species using our inferred regression equations. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microsatellites of <Emphasis Type="BoldItalic">Laminaria digitata</Emphasis> tested in <Emphasis Type="BoldItalic">Lessonia nigrescens</Emphasis>: Evaluation and improvement of cross amplification between kelps of two different families

The use of primers designed originally to amplify DNA for one species in a different one can save time and resources, particularly for microsatellite loci. Microsatellite amplification improvements across two kelp families are reported, where loci originally described in Laminaria digitata (Laminariaceae) were tested in Lessonia nigrescens) was observed in two localities affected by massive mortality events. Nei’s distances among five populations presented similar patterns to those of 30 multilocus dominant loci (RAPD) evaluated in the same localities. Although some success might be achieved in cross-species microsatellite amplifications, the strong mutations detected between these two Laminarian families suggests that better results of cross-amplifications should be expected at much lower taxonomic levels. Thus, although more expensive, construction of new gene libraries is strongly recommended.     

Reverse ascertainment bias in microsatellite allelic diversity in owls (Aves, Strigiformes)

A rich source of markers may be overlooked by screening for polymorphism in the source species only. We screened 129 microsatellite loci isolated from the powerful owl (Ninox strenua) against two closely related species; Ninox connivens and Ninox novaeseelandiae. From the screening effort 20 polymorphic markers were isolated, including six loci which were originally discarded as they were monomorphic in the source species. Further cross-species amplification of all 20 loci across species from two families, Strigidae and Tytonidae, revealed unusually high levels of polymorphism within closely related species, and limited success within phylogenetically distant species. Routine screening of multiple species during the marker development phase can yield a wider range of polymorphic markers which can subsequently enhance cross-species amplification attempts.     

Development of nine polymorphic microsatellite markers for the phytoparasitic nematode Xiphinema index, the vector of the grapevine fanleaf virus

We report isolation, characterization and cross-species amplification of nine microsatellite loci from the phytoparasitic nematode Xiphinema index, the vector of grapevine fanleaf virus. Levels of polymorphism were evaluated in 62 individuals from two X. index populations. The number of alleles varies between two and 10 depending on locus and population. Observed heterozygosity on loci across both populations varied from 0.32 to 0.857 (mean 0.545). The primers were tested for cross-species amplification in three other species of phytoparasitic nematodes of the Xiphinema genus. These nine microsatellite loci constitute valuable markers for population genetics and phylogeographical studies of X. index.     

A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene

Mitochondrial cytochrome b (cytb) is among the most extensively sequenced genes to date across the vertebrates. Here, we employ nearly 2,000 cytb gene sequences from GenBank to calculate and compare levels of genetic distance between sister species, congeneric species, and confamilial genera within and across the major vertebrate taxonomic classes. The results of these analyses parallel and reinforce some of the principal trends in genetic distance estimates previously reported in a summary of the multilocus allozyme literature. In particular, surveyed avian taxa on average show significantly less genetic divergence than do same-rank taxa surveyed in other vertebrate groups, notably amphibians and reptiles. Various biological possibilities and taxonomic "artifacts" are considered that might account for this pattern. Regardless of the explanation, by the yardstick of genetic divergence in this mtDNA gene, as well as genetic distances in allozymes, there is rather poor equivalency of taxonomic rank across some of the vertebrates.     

Isolation and identification of eight microsatellite loci in the Cherokee darter (Etheostoma scotti) and their variability in other members of the genera Etheostoma, Ammocrypta, and Percina

The Cherokee darter Etheostoma scotti is a federally threatened fish endemic to the Etowah River system of northwest Georgia. In order to analyse the population structure and genetic diversity of this fish, eight tetranucleotide microsatellite genetic markers were developed. The marker set was applied to 13 additional darter species to test cross-species amplification and polymorphism. Successful amplification was obtained for all eight loci in each of the 13 other species of darters, with between seven and eight polymorphic loci per species.     

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non-passerine species. The genotyping of four individuals per species revealed that 24-76% (mean 48%) of loci were polymorphic in the passerines and 18-26% (mean 21%) in the non-passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71-85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy-Weinberg proportions. This new set of microsatellite markers is of higher cross-species utility than any set previously designed. The loci described are suitable for a range of applications that require polymorphic avian markers, including paternity and population studies. They will facilitate comparisons of bird genome organization, including genome mapping and studies of recombination, and allow comparisons of genetic variability between species whilst avoiding ascertainment bias. The costs and time to develop new loci can now be avoided for many applications in numerous species. Furthermore, our method can be readily used to develop microsatellite markers of high utility across other taxa.     

Development of 21 polymorphic microsatellite markers for the black-banded sea krait, <Emphasis Type="Italic">Laticauda semifasciata</Emphasis> (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species

The genus Laticauda (Reptilia: Elapidae), commonly known as sea kraits, is venomous marine amphibious snakes distributed throughout the south and southeast Asian islands and mostly found in coastal waters. To facilitate genetic studies, we have developed microsatellite loci for L. semifasciata using the 454 GS-FLX pyrosequencing technique. A total of 65,680 sequences containing a minimum of five repeat motifs were identified from 451,659 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5%) produced strong PCR products, of which 21 were polymorphic among 36 samples of L. semifasciata. All loci exhibited high genetic variability, with an average of 7.38 alleles per locus, and the mean observed and expected heterozygosities were 0.73 and 0.76, respectively. The cross-species amplification of these loci in two laticaudine species, L. colubrina and L. laticaudata, revealed a high transferability (78.6%) and polymorphism (59.5%) of the loci. Our work demonstrated the utility of next-generation 454 sequencing as the rapid and cost-effective method for development of microsatellite markers. The high level of polymorphism in these microsatellite loci will be useful for the detection of population subdivision and the study of migration, gene flow, relatedness and philopatry of L. semifasciata and other laticaudine species.     

Eight novel tetranucleotide and five cross-species dinucleotide microsatellite loci for the ornate chorus frog (Pseudacris ornata)

We describe the cloning and characterization of eight novel tetranucleotide microsatellite loci in the ornate chorus frog (Pseudacris ornata). We also screened 26 loci from GenBank that were isolated from other Pseudacris species and obtained consistent product from five of these dinucleotide loci. All loci are polymorphic. In our sample of 26 frogs from a natural population, polymorphism ranged from 1 to 22 alleles per locus with expected heterozygosities ranging from 0 to 0.958. These loci enable high-resolution studies of P. ornata. Moreover, cross-species amplification success suggests they will also be useful for other chorus frog species.     

Microsatellite variation among divergent populations of stalk-eyed flies, genus Cyrtodiopsis

Microsatellite primers are often developed in one species and used to assess neutral variability in related species. Such analyses may be confounded by ascertainment bias (i.e. a decline in amplification success and allelic variability with increasing genetic distance from the source of the microsatellites). In addition, other factors, such as the size of the microsatellite, whether it consists of perfect or interrupted tandem repeats, and whether it is autosomal or X-linked, can affect variation. To test the relative importance of these factors on microsatellite variation, we examine patterns of amplification and allelic diversity in 52 microsatellite loci amplified from five individuals in each of six populations of Cyrtodiopsis stalk-eyed flies that range from 2.2 % to 11.2% mitochondrial DNA sequence divergence from the population used for microsatellite development. We find that amplification success and most measures of allelic diversity declined with genetic distance from the source population, in some cases an order of magnitude faster than in birds or mammals. The median and range of the repeat array length did not decline with genetic distance. In addition, for loci on the X chromosome, we find evidence of lower observed heterozygosity compared with loci on autosomes. The differences in variability between X-linked and autosomal loci are not adequately explained by differences in effective population sizes of the chromosomes. We suggest, instead, that periodic selection events associated with X-chromosome meiotic drive, which is present in many of these populations, reduces X-linked variation.     

Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags

The construction of high-density linkage maps for use in identifying loci underlying important traits requires the development of large numbers of polymorphic genetic markers spanning the entire genome at regularly spaced intervals. As part of our efforts to develop markers for rainbow trout (Oncorhynchus mykiss), we performed a comparison of allelic variation between microsatellite markers developed from expressed sequence tag (EST) data and anonymous markers identified from repeat-enriched libraries constructed from genomic DNA. A subset of 70 markers (37 from EST databases and 33 from repeat enriched libraries) was characterized with respect to polymorphism information content (PIC), number of alleles, repeat number, locus duplication within the genome and ability to amplify in other salmonid species. Higher PIC was detected in dinucleotide microsatellites derived from ESTs than anonymous markers (72.7% vs. 54.0%). In contrast, dinucleotide repeat numbers were higher for anonymous microsatellites than for EST derived microsatellites (27.4 vs.18.1). A higher rate of cross-species amplification was observed for EST microsatellites. Approximately half of each marker type was duplicated within the genome. Unlike single-copy markers, amplification of duplicated microsatellites in other salmonids was not correlated to phylogenetic distance. Genomic microsatellites proved more useful than EST derived microsatellites in discriminating among the salmonids. In total, 428 microsatellite markers were developed in this study for mapping and population genetic studies in rainbow trout.     

High-throughput microsatellite marker development in two sparid species and verification of their transferability in the family Sparidae

Recently, 454 sequencing has emerged as a popular method for isolating microsatellites owing to cost-effectiveness and time saving. In this study, repeat-enriched libraries from two southern African endemic sparids (Pachymetopon blochii and Lithognathus lithognathus) were 454 GS-FLX sequenced. From these, 7370 sequences containing repeats (SCRs) were identified. A brief survey of 23 studies showed a significant difference between the number of SCRs when enrichment was performed first before 454 sequencing. We designed primers for 302 unique fragments containing more than five repeat units and suitable flanking regions. A fraction (<11%) of these loci were characterized with 18 polymorphic microsatellite loci (nine in each of the focal species) being described. Sanger sequencing of alleles confirmed that size variation was because of differences in the number of tandem repeats. However, a case of homoplasy and sequencing errors in the 454 sequencing were identified. These newly developed and four previously isolated loci were successfully used to identify polymorphic markers in nine other economically important species, representative of sparid diversity. The combination of newly developed markers with data from previous sparid cross-species studies showed a significant negative correlation between genetic divergence to focal species and microsatellite transferability. The high level of transferability we described (48% amplification success and 32% polymorphism) suggests that the 302 microsatellite loci identified represent an excellent resource for future studies on sparids. Microsatellite marker development should commonly include tests of transferability to reduce costs and increase feasibility of population genetics studies in nonmodel organisms.     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Cross-species amplification of Eucalyptus microsatellite loci

This study examined the interspecific amplification of nuclear microsatellite loci developed mainly for eucalypts in the subgenus Symphyomyrtus across five species within the second most speciose subgenus, subgenus Eucalyptus. A set of eight to 10 loci, depending on taxon, have been identified that are highly variable and easily scored. The successful transfer of microsatellite loci to these eucalypt species sidesteps the expensive and time-consuming development of species-specific microsatellite libraries. This primer set will enable the examination and cross-species comparison of the genetic resources of commercially and ecologically important members of the subgenus Eucalyptus.