Comparative analysis of the informativeness of ISSR markers for estimating genetic diversity of horse breeds

The informativeness of six different types of ISSR markers was compared in order to evaluate genetic diversity of Mongolian, Buryat and Tuvinian horse breeds (Equus caballus). The dinucleotide-based ISSR primers ((GA)₉C, (AG)₉C, and (CA)₉G) proved to be of little use to examine genetic polymorphism among horses due to the low number of amplified and polymorphic fragments. The polymorphism information content (PIC) for the dinucleotide-based ISSR primers was 2.88 times lower than that for trinucleotide-based ISSR markers. Among the trinucleotide-based ISSR markers, CAC ISSR marker was also found to be noninformative, due to the low number of polymorphic loci identified. The highest values of the polymorphism information content (PIC) were obtained for two types of ISSR markers, based on (GAG)₆C (PIC, 3.61) and on (ACC)₆G (PIC, 2.50). Thus, the results of the present study showed that GAG and ACC ISSR markers were most informative for evaluation of genetic diversity among horses.     

Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites

Ten (TG)_n positive clones, isolated from an equine genomic library and sequenced, contained 12–19 uninterrupted TG repeats. Primers for polymerase chain reaction (PCR) were synthesized and nine of these (TG)_n loci (HTG7-15) were successfully amplified and utilized in this study together with five previously reported equine microsatellite loci (HTG2-6). The PCR products were analysed by polyacrylamide gel electrophoresis followed by automated laser fluorescence detection or autoradiography. All microsatellites showed polymorphism and stable Mendelian inheritance. Differences in microsatellite variability between horse breeds were detected. A linkage analysis comprising HTG2-15, one coat colour gene and 16 genetic blood markers enabled addition of HTG2 to linkage group U2 and a new linkage group (U6) was established comprising the loci HTG7 and HTG12. Close linkage was excluded within a set of eight microsatellites. The estimated probability of exclusion in four breeds for a parentage test based on these eight loci varied between 0.96 and 0.99.     

Two multiplex sets of eight and five microsatellite markers for the European corn borer,Ostrinia nubilalis Hübner (Lepidoptera: Crambidae)

Primer sequence and polymorphism data are presented for 13 microsatellite loci isolated from the European corn borer moth, Ostrinia nubilalis, as part of a project to construct a linkage map for the two pheromone strains. Experimental conditions are described for polymerase chain reaction (PCR) multiplexing, which allows genotyping in two electrophoresis runs of eight and five markers each. In a sample of 27 individuals coming from one European locality, the number of alleles per locus ranged from one to 12, and gene diversity from 0 to 0.859. Seven loci showed a deficit of heterozygotes. Eleven loci cross‐amplify in the related Ostrinia furnacalis.     

Isolation and characterization of microsatellite markers in Elliot's pheasant (Syrmaticus ellioti)

We isolated eight microsatellite loci from genomic DNA in Elliot's pheasant Syrmaticus ellioti using streptavidin‐coated magnetic beads. In the analyses of 53 individuals sampled, these loci displayed polymorphism varying from six to nine alleles per locus and observed heterozygosities ranging from 0.174 to 0.430. The results suggested that these novel microsatellite markers could become useful molecular tools for genetic studies of S. ellioti.     

Characterization of microsatellite loci in polyploid Lavatera assurgentiflora ssp. assurgentiflora and ssp. glabra (Malvaceae)

Lavatera assurgentiflora (Malvaceae) is one of four species of the genus Lavatera native to California and Baja California. Two geographically defined subspecies are recognized: L. a. assurgentiflora on the northern islands and L. a. glabra on the southern islands. We isolated nine polymorphic microsatellite loci that amplify in both subspecies of L. assurgentiflora. Substantial levels of polymorphism were observed at many of the loci. Four loci exhibited more than 10 alleles, polymorphism information content ranged from 0.4 to 0.8, and up to six alleles were found in some individuals, supporting reports that these taxa are hexaploid. All loci also amplified in Lavatera lindsayi from Guadalupe Island, and we anticipate that they will cross‐amplify in other California Lavatera species as well.     

Characterization of polymorphic microsatellite markers for the Carnaby's cockatoo (Calyptorhynchus latirostris) and related black cockatoo species

Microsatellite loci were isolated from Carnaby's black cockatoo (Calyptorhynchus latirostris: Aves), a highly valued, endangered, and endemic species of bird from Western Australia. This study describes three dinucleotide and one tetranucleotide microsatellite loci for which the primers produced clear and polymorphic amplification patterns with between two and nine alleles and moderate levels of variability. Two additional dinucleotide markers which were monomorphic in the Carnaby's cockatoo were able to amplify and were polymorphic in two other species of black cockatoo, greatly increasing the utility of these markers.     

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.     

Isolation and characterization of microsatellite markers from Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch), and cross‐amplification in the closely related species crucian carp,Carassius auratus auratus (Linnaeus)

We report the development of 20 microsatellite markers for Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch). Nineteen out of 20 showed polymorphism with alleles ranging from two to 14. These loci were screened to amplify the closely related species crucian carp, Carassius auratus auratus (Linnaeus) and all of them can amplify DNA with the size similar to the former. The origin of silver crucian carp is in issue and the population genetic structure is still unclear. Microsatellite markers isolated from the silver crucian carp and their utility in the crucian carp will be useful for these researches.     

Isolation of simple and compound polymorphic tetranucleotide microsatellites for the neotropical leaflitter frog Eleutherodactylus ockendeni (Leptodactylidae)

Few studies of population structure and genetic diversity exist for frogs in the Amazon of South America, an area renowned for exceptionally high species richness. We isolated seven highly variable tetranucleotide microsatellite loci for the neotropical leaflitter frog, Eleutherodactylus ockendeni using an enrichment method. Three of the repeats are simple, three are compound and one is imperfect. We screened all loci with 175 individuals from one geographical area in the upper Napo of Ecuador and found high polymorphism in all loci (> 14 alleles/locus). These markers are suitable for population genetics studies of E. ockendeni and perhaps other leaflitter frogs of the same genus.     

Isolation and characterization of microsatellite loci in the Japanese pipistrelle (Pipistrellus abramus)

We describe the first set of ten microsatellite markers isolated in Pipistrellus abramus. The number of alleles per locus ranged from 7 to 13. The observed and expected heterozygosities values ranged from 0.486 to 0.971 and from 0.752 to 0.876, respectively. Three loci revealed significant departure from Hardy–Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These informative microsatellite markers will be a powerful molecular tool for studying the population genetic structure of P. abramus, as well as other species of this genus.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium tamarense (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) are currently a serious problem from an economic and food hygiene point of view throughout the world. We isolated 13 polymorphic microsatellite loci from this species. These loci provided microsatellite markers with high polymorphism ranging from four to 15 alleles per locus and gene diversity between 0.632 and 0.974. The markers are available for more detailed investigations of genetic structure and gene flow of A. tamarense populations.     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.     

Identification and characterization of 10 microsatellite primers for the calanoid freshwater copepods Eudiaptomus gracilis and E. graciloides using enriched genomic libraries

The calanoid copepods Eudiaptomus gracilis and Eudiaptomus graciloides are widespread among European lakes. We constructed enriched genomic libraries for both species in order to isolate and characterise microsatellite markers. The obtained 7 polymorphic microsatellite‐loci for E. gracilis and 3 for E. graciloides are the first for any freshwater copepod. They display an expected heterozygosity ranging from 0.60 to 0.96 and 0.63 to 0.94 and observed heterozygosity ranging from 0.53 to 0.87 and 0.57 to 0.87, respectively. We were not able to cross‐amplify the isolated loci across species, indicating long divergence among both congeneric species despite morphological similarity.     

Isolation and characterization of microsatellite DNA loci from the golden cuttlefish,Sepia esculenta Hoyle (Cephalopoda)

The first microsatellite markers were isolated from the golden cuttlefish, Sepia esculenta Hoyle. Eleven primer sets were designed to amplify the marker sequences via polymerase chain reaction. The 45–50 individuals from one wild population in the coastal waters of Ehime Prefecture, Japan were used to screen polymorphism in the 11 microsatellite loci. All the microsatellite loci were polymorphic, with the range of alleles from seven to 27 per locus. The observed and expected heterozygosities ranged from 0.380 to 0.980 and from 0.654 to 0.940, respectively. These marker loci except for one locus showing significant deviation from Hardy–Weinberg equilibrium will be useful for the assessment of genetic variation and population structure of this species.     

Development of microsatellite markers in ginkgo (Ginkgo biloba L.)

Ginkgo biloba L. is one of the oldest unevolved tree species on Earth. We isolated five polymorphic microsatellite loci from G. biloba using a dual‐suppression polymerase chain reaction technique. These loci provided microsatellite markers with high polymorphism ranging from three to 13 alleles per locus. The observed and expected heterozygosities ranged from 0.667 to 0.952 and from 0.640 to 0.897, respectively. The markers will contribute to research on the conservation, genetic diversity and mating patterns of G. biloba.     

Eight highly polymorphic microsatellite loci for the Australian gecko Heteronotia binoei

Eight highly polymorphic dinucleotide microsatellite loci were developed for the Bynoe's gecko, Heteronotia binoei. Across the species as a whole, expected heterozygosities for the loci range from 0.59 to 0.92, with observed numbers of alleles ranging from 13 to 27. All eight loci successfully amplify in each of the three most widespread sexual chromosome races of Heteronotia binoei, and with the exception of one locus in one race all are polymorphic. All eight loci also amplify in hybrid parthenogenetic Bynoe's geckos, in several other sexual chromosome races, and in related Heteronotia species.     

Polymorphic microsatellite markers for the Socotran endemic herb Begonia socotrana

Six polymorphic microsatellite markers have been developed to examine population structure and outcrossing rates in the narrow‐range endemic Begonia socotrana. Only two of the markers amplify products in its recently discovered sister species B. samhaensis. All of the loci amplify in winter‐flowering Begonia hybrids derived from B. socotrana, revealing little polymorphism and demonstrating the narrow genetic base of the material used in their production.     

Development and characterization of microsatellite markers to study population genetic diversity of Przewalski's naked carp Gymnocypris przewalskii in China

Two populations of Przewalski's naked carp Gymnocypris przewalskii, 30 individuals per population, were screened for 10 microsatellite loci. Moderate allele variation was found in these loci with two to eight alleles per locus. The expected and observed heterozygosity ranged from 0·019 to 0·805 and from 0·160 to 0·575, respectively.     

Isolation and characterization of microsatellites in Echinochloa (L.) Beauv. spp.

Five primer pairs were developed that amplify microsatellite loci in three agronomically important Echinochloa (L.) Beauv. species: E. colona (L.) Link, E. crus‐galli (L.) Beauv. and E. crus‐pavonis (Kunth) Schultes. The microsatellites were tested on 24 individuals representing three species collected in rice fields from different geographical regions and revealed 3–7 alleles per microsatellite. Gene diversity [1 ? Σp_ij²] for four polymorphic loci within E. crus‐galli ranged from 0.12 to 0.61. Alleles at a fifth locus were useful in discriminating the species. The microsatellites should provide useful markers for intraspecific diversity studies and aid classification of species within this complex genus.     

Isolation and characterization of di‐ and tetranucleotide microsatellite loci in the yellow‐spotted night lizard Lepidophyma flavimaculatum (Squamata: Xantusiidae)

We report the development of 17 di‐ and tetranucleotide microsatellite markers in the Yellow‐Spotted Night Lizard Lepidophyma flavimaculatum. Levels of heterozygosity ranged from 0 to 100%. Several loci also amplify in other Lepidophyma species and several species of the sister genus Xantusia. High levels of variation at some loci indicate that they will be useful for parentage assessment and population genetic studies, whereas the 15 loci that amplify across multiple Lepidophyma species suggest these will be useful in determining the origin of parthenogenetic populations.