Development and characterisation of 20 microsatellite loci isolated from the large bent-wing bat, Miniopterus schreibersii (Chiroptera: Miniopteridae) and their cross-taxa utility in the family Miniopteridae

The large bent-wing bat, Miniopterus schreibersii (Kuhl 1819), has a long history of taxonomic uncertainty and many populations are known to be in a state of decline. Microsatellite loci were developed for the taxonomic and population genetic assessment of the Australian complex of this species. Of the 33 primer sets designed for this research, seven (21%) were deemed suitably polymorphic for population-level analyses of the Australian taxa, with five (71%) of these loci revealing moderate to high levels of polymorphism (PIC = 0.56 to 0.91). The cross-taxa utility of the M. schreibersii microsatellite markers was assessed in the microbat (Chiroptera) family Miniopteridae. Sub-species and species covering the Miniopteridae's global distribution (with the exception of the Middle East) were selected, numbering 25 taxa in total. Amplification was successful for 26 loci, of which 20 (77%) were polymorphic. High cross-taxa utility of markers was observed with amplification achieved for all taxa for between four (20%) and 20 (100%) loci, and polymorphism was considered moderate to high (PIC = 0.47-0.91) for 12 (60%) of these loci. The high cross-taxa utility of the microsatellites reported herein reveal versatile and cost-effective molecular markers, contributing an important genetic resource for the research and conservation of Miniopteridae species worldwide.     

Noncoding mitochondrial loci for corals

We developed five degenerate primer pairs for the amplification and sequencing of two noncoding regions found in the mitochondrial genome of corals. These primers amplify products ranging from 380 to 950 bp, and work in a wide variety of scleractinian taxa from both the Pacific and Caribbean. Based on our initial analysis of ～300 sequences from 13 scleractinian taxa, both these noncoding regions appear to have equivalent levels of variability to the most variable of previously published coral mitochondrial loci, but work in a wider variety of taxa. We believe these primers will be of use to coral biologists studying questions above the level of species; as with other mithochondrial DNA markers in corals, these loci will likely provide little resolution for within‐species studies.     

Exon-primed, intron-crossing (EPIC) loci for five nuclear genes in deep-sea protobranch bivalves: primer design, PCR protocols and locus utility

We describe PCR primers and amplification protocols developed to obtain introns from conserved nuclear genes in deep-sea protobranch bivalves. Because almost no sequence data for protobranchs are publically available, mollusk and other protostome sequences from GenBank were used to design degenerate primers, making these loci potentially useful in other invertebrate taxa. Amplification and sequencing success varied across the test group of 30 species, and we present five loci spanning this range of outcomes. Intron presence in the targeted regions also varied across genes and species, often within single genera; for instance, the calmodulin and β-tubulin loci contained introns with high frequency, whereas the triose phosphate isomerase locus never contained an intron. In introns for which we were able to obtain preliminary estimates of polymorphism levels in single species, polymorphism was greater than traditional mitochondrial loci. These markers will greatly increase the ability to assess population structure in the ecologically important protobranchs, and may prove useful in other taxa as well.     

New polymorphic microsatellite loci,cross‐species amplification and PCR multiplexing in the black aphid,Aphis fabae Scopoli

Aphis fabae includes four morphological cryptic subspecies, which are mostly identified by their partially distinct secondary host range. To determine the extent of gene flow and isolation between these four taxa, we isolated and characterized 12 microsatellite loci from Aphis fabae fabae and tested cross‐species amplification of eight loci from the closely related species Aphis gossypii. Using eight previously described microsatellite loci, we have developed the polymerase chain reaction (PCR) multiplexing of 24 loci, which were separated in tree sets and five PCRs. These sets of microsatellite loci provide high throughput capacity for large‐scale population genetic studies at a minimum cost.     

Transferability of olive microsatellite loci across the genus <Emphasis Type="Italic">Olea</Emphasis>

The transferability of microsatellite markers developed for olive cultivars (Olea europaea L.) has been tested and confirmed in the Olea complex. Thirty two genotypes, belonging to different taxa of the genus Olea, have been analyzed with four olive SSRs. Positive amplifications at all loci were obtained in 13 taxa (at least one accession per species). Sixty seven different alleles have been detected at the four loci analyzed. Polymorphic products have been observed at the inter- and intra-species level. Some SSR loci have shown multiple amplification products in some species. The high number of unique alleles has allowed the unambiguous discrimination of most accessions. Similarity coefficients and relationships among the Olea taxa have been calculated based on SSR amplification results. The reliability of SSRs as markers for intra-species variability evaluation has been confirmed while their use to explore relationships at the inter-species level is discussed, being dependent on the locus analyzed.Communicated by H.F. Linskens     

Expressed sequence tag-simple sequence repeats isolated from Shorea leprosula and their transferability to 36 species within the Dipterocarpaceae

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often transferable to related taxa. Here, we report the development and characterization of EST-SSRs from Shorea leprosula. Fifty-four sequences containing SSRs were identified in 2003 unigenes assembled from 3159 ESTs. Twenty-four EST-SSRs were developed, of which four gave multiple amplifications, five were found to be monomorphic and 15 showed polymorphism, with allele numbers ranging from two to 17 in a single Pasoh Forest Reserve population of 24 individuals. The observed and expected heterozygosities ranged from 0.05 to 0.91 and from 0.16 to 0.93, respectively. Cross-species transferability of the 15 loci to 36 species within Dipterocarpaceae revealed between four and 14 loci that gave positive amplification and 10 loci were found to be transferable to more than 15 species.     

A set of microsatellite markers for population genetics of leopard cat (Prionailurus bengalensis) and cross-species amplification in other felids

We describe the isolation and characterization of novel microsatellite loci from the leopard cat, Prionailurus bengalensis Kerr, 1792 (Family Felidae). Using Illumina HiSeq2500 sequencing technology, we sequenced the leopard cat genome and identified 1.5 million loci of simple sequence repeats with di- to deca-nucleotide motifs. We developed twelve polymorphic markers with tetra-nucleotide motif types after screening 35 loci for amplification and polymorphism. The observed and expected heterozygosities of the markers were 0.438 and 0.423, respectively. The number of alleles per locus ranged from 2 to 7, with a mean polymorphism information content of 0.383. Eleven loci were at Hardy-Weinberg equilibrium and no linkage disequilibrium was detected among any pairs of loci. We tested cross-species amplification of these markers across five other felids (Panthera tigris, P. pardus, P. onca, Acinonyx jubatus, and Felis catus). All loci were transferable to at least one other feline species and four amplified all five species. The microsatellite markers developed in this study will be valuable for estimating ecological parameters of populations and to establish conservation and management strategies for feline species.     

Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.     

Three species of Isotoma (Collembola, Isotomidae) based on morphology, isozymes and ecology

Morphological markers and isozymes were used for identifying three presumed species of the Isotoma genus. Morphological traits separated three taxa of the genus. 10 isozymes determined by at least 11 loci were analysed from each taxon, and 2 loci were taxon-specific, supporting the hypothesis that the three taxa represented three species. The genetic variation found within the taxa measured as fraction of polymorphic loci at the 99% level was 0.82, 0.55 and 0.55 with the corresponding observed heterozygosity 0.15, 0.09 and 0.12. Two populations of the same taxon from Denmark and Norway, respectively, were very closely related. Additional ecological criteria, obtained from a literature survey, also revealed pronounced differences between the three taxa. Due to these facts three distinct species are proposed, namely I. anglicana Lubbock 1862, I. riparia Nicolet 1841 and I. viridis Bourlet 1839.     

A set of polymorphic microsatellite loci for Vriesea gigantea and Alcantarea imperialis (Bromeliaceae) and cross‐amplification in other bromeliad species

Fifteen polymorphic microsatellite markers were isolated and characterized in two species of Bromeliaceae: Vriesea gigantea and Alcantarea imperialis. The number of alleles observed for each locus ranged from three to 16. The loci will be used for studies of the genetic structure of natural populations, reproductive biology, and evolutionary relationships among and within these genera. A cross‐amplification test in 22 taxa suggests that the markers will be useful for similar applications in numerous other bromeliad species.     

Isolation and characterization of twelve polymorphic microsatellite markers in bottlenose dolphins (Tursiops truncatus)

We developed polymerase chain reaction primers for 12 dinucleotide microsatellite loci in the bottlenose dolphin, Tursiops truncatus. Seven markers were obtained after hybridization screening, and five following random genome sequencing. Orthologous positions were computed for nine markers on the bovine genome and for seven on the human genome. The markers are distributed across chromosomes and found in different types of DNA regions. All 12 loci are polymorphic for Tursiops. Five loci were also polymorphic in the related species Stenella frontalis and the more distantly related river dolphin, Inia geoffrensis, indicating these markers will be informative across the Delphinidae and other cetacean taxa.     

A novel set of microsatellite marker loci linkage-mapped in the house musk shrew, Suncus murinus.

Ten microsatellite DNA loci developed for the white-toothed shrew (Crocidura russula) were tested for PCR amplification and for utility in linkage studies in the house musk shrew, Suncus murinus. Four primer pairs successfully yielded PCR amplicons and showed polymorphism between two mutant strains, BAN-kc,oeb and WZ. Cloning and sequencing of the PCR amplicons of all the four loci confirmed the presence of microsatellite sequences. Alleles segregating in an F2 resource population constructed from the two strains ranged between two and five. Linkage analysis of the four loci together with 18 other polymorphic markers and three mutant loci resulted in five linkage groups containing three newly mapped microsatellite loci. This study reports the first microsatellite markers being registered in this species.     

Characterization of 13 microsatellites for Lahontan cutthroat trout (Oncorhynchus clarki henshawi) and cross-amplification in six other salmonids

Thirteen newly developed tri- and tetranucleotide repeat microsatellite markers were developed for Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a threatened subspecies endemic to the Lahontan hydrographic basin in the western USA. These loci are highly polymorphic with five to 30 alleles per locus and observed heterozygosities ranging from 0.4 to 0.7. Cross-species amplification of these markers was most successful in the closely related rainbow trout, Oncorhynchus mykiss, with only three loci amplifying in brown trout, Salmo trutta. Nonoverlapping allelic distributions for many of these loci among the six salmonid species screened suggest these markers may be useful for hybrid determination.     

Conserved Microsatellites in Ants Enable Population Genetic and Colony Pedigree Studies across a Wide Range of Species

Broadly applicable polymorphic genetic markers are essential tools for population genetics, and different types of markers have been developed for this purpose. Microsatellites have been employed as particularly polymorphic markers for over 20 years. However, PCR primers for microsatellite loci are often not useful outside the species for which they were designed. This implies that a new set of loci has to be identified and primers developed for every new study species. To overcome this constraint, we identified 45 conserved microsatellite loci based on the eight currently available ant genomes and designed primers for PCR amplification. Among these loci, we chose 24 for in-depth study in six species covering six different ant subfamilies. On average, 11.16 of these 24 loci were polymorphic and in Hardy-Weinberg equilibrium in any given species. The average number of alleles for these polymorphic loci within single populations of the different species was 4.59. This set of genetic markers will thus be useful for population genetic and colony pedigree studies across a wide range of ant species, supplementing the markers available for previously studied species and greatly facilitating the study of the many ant species lacking genetic markers. Our study shows that it is possible to develop microsatellite loci that are both conserved over a broad range of taxa, yet polymorphic within species. This should encourage researchers to develop similar tools for other large taxonomic groups.     

Characterization of microsatellite loci from Litsea hypophaea (Lauraceae), a tree endemic to Taiwan

? Premise of the study: Microsatellite primers were developed for the endemic tree Litsea hypophaea (Lauraceae) in Taiwan to investigate its genetic diversity and population genetic structure and to investigate species delimitation within Litsea. ? Methods and Results: Fifteen new simple sequence repeat markers were developed from L. hypophaea with a magnetic bead enrichment method. Most loci were also amplified from three closely related species, L. coreana, L. lii, and L. acutivena. The number of alleles and observed and expected heterozygosities across loci varied with a range of 1-25, 0.000-1.000, and 0.000-0.956, respectively. ? Conclusions: The application of these microsatellite markers of L. hypophaea provides a tool for understanding genetic diversity and population differentiation. In addition, interspecific amplification suggests that these markers will also be useful for species identification of related taxa within Litsea in Taiwan.     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

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.     

Polymorphic microsatellites in the Reeves's pheasant developed by cross-species amplification

Cross-species microsatellite amplification is an effective way of obtaining microsatellite loci for closely related taxa in bird species. The Reeves's pheasant, Syrmaticus reevesii, is a vulnerable species endemic to China. To improve population genetics and parentage analysis studies in this species, we obtained nine polymorphic microsatellite markers, in addition to the nine markers previously isolated, from the cross-species amplification of 52 markers. The number of alleles per locus varied between two and 12 with expected heterozygosity ranging from 0.298 to 0.714 (n = 107). The success rates of simulated paternity tests using CERVUS software improved at different confidence levels after adding these markers to the previous ones.