Isolation and characterization of microsatellite markers in Phytophthora ramorum,the causal agent of sudden oak death

We describe specific primers and conditions to amplify two dinucleotide and five trinucleotide microsatellite DNA loci isolated from the oomycete Phytophthora ramorum, the causal agent of sudden oak death. The primer sets were tested on 14–30 isolates from North America and Europe. Seven of 14 loci differentiated between A1 and A2 mating types. All seven loci successfully amplified DNA isolated from infected plant tissue. Four loci may be useful for the diagnosis of P. ramorum because they do not amplify closely related Phytophthora species.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Nuclear microsatellite DNA markers for New Zealand kiwi (Apteryx spp.)

Kiwi (Apterygidae) is an endemic New Zealand avian family comprising five species whose conservation is actively managed. We present five polymorphic microsatellite DNA loci isolated from North Island brown kiwi (Apteryx mantelli). In addition, we demonstrate cross‐amplification, and in some cases, polymorphism, of these microsatellite DNA loci in four other kiwi species. Therefore, these markers may be broadly applicable to conservation genetic studies within this family.     

Development and characterization of novel microsatellite markers for the common earthworm (Lumbricus terrestris L.)

We developed and characterized 10 highly polymorphic microsatellite loci from an SSR‐enriched genomic DNA library of the common earthworm (Lumbricus terrestris L). Characterization of these loci using 32 individuals revealed high levels of genetic diversity, five to 18 alleles per locus and a high observed and expected heterozygosity. These loci will be used for paternity analysis and population genetic studies of the co‐evolution between L. terrestris and its parasites.     

Polymorphic microsatellite DNA markers for Banksia attenuata (Proteaceae)

We developed 11 polymorphic microsatellite DNA markers for an Australian native shrub Banksia attenuata. The number of alleles per locus in 50 individuals varied from five to 18, observed and expected heterozygosities ranged from 0.300 to 0.740 and from 0.537 to 0.918, respectively. Six loci showed no significant deviation from Hardy–Weinberg equilibrium (P > 0.05), and null alleles appear to exist at locus BA‐B1. All loci showed independent inheritance.     

Characterization of microsatellite loci in red wood ants Formica (s. str.) spp. and the related genus Polyergus

Ants are interesting subjects for studies of evolution of altruism. We developed 13 microsatellite loci in a red wood ant Formica (s. str.) yessensis from random amplified polymorphic DNA fragments to study genetic structure within populations and colonies. Five loci bore two to five alleles in both F. (s. str.) yessensis and F. (s. str.) truncorum and two were also polymorphic in a related species, Polyergus samurai. Results suggest that the loci will be useful in evolutionary studies on Formica and Polyergus species.     

Identification of Neurofibromatosis 1 (NF1) Homologous Loci by Direct Sequencing, Fluorescence in Situ Hybridization, and PCR Amplification of Somatic Cell Hybrids

Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1 -homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5′ end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template.     

Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)

The isolation of molecular markers in Asparagus acutifolius, a wild edible plant species, is important to characterize local ecotypes that could be cultivated and preserved. We isolated and characterized polymorphic microsatellite loci from A. acutifolius by constructing and screening an enriched DNA library. Primer pairs were designed for 12 loci. Seven primer pairs worked well during amplification reactions and were tested on a wild population from Pontecagnano (SA), Italy. These loci showed a high level of genetic variability, with the numbers of alleles identified ranging from two to five and observed heterozygosity ranging from 0.20 to 0.73.     

New microsatellite markers in turbot (Scophthalmus maximus) derived from an enriched genomic library and sequence databases

Scophthalmus maximus is an important commercially aquaculture fish species. We tackle the search for new microsatellites using two different approaches: an enriched partial genomic library and a screening of all turbot DNA sequences deposited in GenBank. Out of 15 genomic library derived loci, five gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.13 to 0.91. Out of seven loci derived from database sequences, two amplified successfully with an H_E ranging from 0.56 to 0.63. The average allele number of these microsatellites was 5.7 per locus with a range between two and 15.     

Isolation and characterization of microsatellite loci in Lampsilis abrupta (Bivalvia: Unionidae) and cross‐species amplification within the genus

We have documented the first microsatellites isolated from a unionid and demonstrated that these markers can be useful for surveys of neutral genetic variation in several Lampsilis species. We describe the isolation and characterization of 15 polymorphic microsatellite DNA loci for the endangered unionid Lampsilis abrupta. Among individuals from five collections, allelic diversity ranged from six to 17 alleles and averaged 10.4 alleles per locus. Individual heterozygosity was observed to range from 20.0% to 86.7% and averaged 46.9%. Cross‐species amplification was investigated in nine additional Lampsilis species. A high level of flanking sequence similarity was inferred as 13 of 15 loci amplified in at least seven species.     

Isolation of microsatellites from two species of scleractinian coral

Microsatellites were isolated from two broadcast‐spawning species of scleractinian coral (Platygyra daedalea and Goniastrea favulus) from Australia's Great Barrier Reef. We found 27 microsatellites across both species, although only five loci were polymorphic in each species. Microsatellite loci displayed a wide range of diversity levels with four to 11 alleles per locus (H_O = 0.26–0.91) in P. daedalea and two to seven alleles per locus (H_O = 0.16–0.96) in G. favulus. Most loci showed departures from Hardy–Weinberg equilibrium which may reflect nonrandom mating but may also be related to difficulties associated with coral DNA.     

Microsatellites as DNA markers in Sitka spruce

Nine microsatellite loci were found by screening a genomic DNA library of Sitka spruce (Picea sitchensis) with the four oligonucleotide probes (TG), (CAC), (GATA) and (AT). Pairs of flanking primers were generated for seven microsatellites. Five primer pairs were used to screen up to 58 Sitka spruce clones. The five loci SStg3a, SStg4, SStg4a, SStg4c and SSgataS were found to have 15, 13, 4, 3 and 6 different length alleles respectively, and in using a combination of them almost all 58 Sitka spruce genotypes could be identified. The five primer pairs were successful in amplifying DNA from two other spruce species (Picea albutilia and Picea smithiana), while only one primer pair could amplify DNA from the pine species, Pinus sylvestris and Pinus latifolia. The inheritance of microsatellites in Sitka spruce was co-dominant Mendelian.     

Polymorphic Chromosomal Specificity of Centromere Satellite Families in Arabidopsis halleri ssp. gemmifera

The chromosomal localizations of repetitive DNA clusters (ribosomal DNA and centromere satellites) were analyzed by fluorescent in situ hybridization in five strains of Arabidopsis halleri ssp. gemmifera. All five A. gemmifera strains have three chromosome pairs with 45S (5.8S-16S-26S) rDNA loci, and one pair with both 5S and 45S rDNA loci. These localizations are different from that of A. thaliana. Very unusually, there are three families of centromeric satellite DNAs (pAa, pAge1, and pAge2), and they showed polymorphism among the five strains studied. Overall, we found four different centromere satellite compositions. A plant from Fumuro was heterozygous for the chromosome specificities of centromere satellite families, possibly due to a reciprocal translocation involving centromere regions. Changes of centromeric satellite repeats appear to be rapid and frequent events in the history of A. gemmifera, and seem to occur by exchanging clusters as units.     

Microsatellites for eastern Australian Banksia species

We developed eight primer pairs for Banksia microsatellite markers (five using DNA from Banksia oblongifolia and three from Banksia robur) in order to study the processes of speciation within hybridizing B. oblongifolia, B. robur and Banksia paludosa complex. We genotyped four populations of B. oblongifolia and B. robur, and three of B. paludosa. Numbers of alleles ranged from 1 to 13 across the three species and observed average heterozygosities ranged from 0.000 to 0.833. At least four loci completely discriminated B. robur from B. oblongifolia and three discriminated B. paludosa from B. oblongifolia. Seven of these primers amplified DNA from at least two of three other local species.     

Characterization of nuclear microsatellites in New Caledonian Araucaria species

Thirteen of the world's 19 species of Araucaria are endemic to the Pacific Ocean island of New Caledonia. In order to investigate the evolutionary biological processes underlying the radiation of the genus on the island we have developed a set of nuclear microsatellite loci. Using a membrane enrichment procedure, five loci have been developed (four derived from A. subulata DNA and one from A. rulei DNA) which amplified in A. columnaris. PCR products of the expected size were also produced in a very limited sample of eight other New Caledonian Araucaria species tested.     

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.     

Polymorphic microsatellite loci for eusocial wasps (Hymenoptera: Vespidae)

Hymenopterans are an important model for studying the evolution of cooperation in animal societies. Here, we characterize 19 microsatellite loci, isolated from the common wasp Vespula vulgaris, that can be used to study genetic variation in three genera (seven species) within the Vespidae. The number of alleles in V. vulgaris was moderate, varying from 2 to 14, with expected heterozygosity ranging between 0.04 and 0.93. Eleven loci amplified DNA in V. austriaca and Dolichovespula sylvestris, nine in V. germanica, eight in Vespa crabro and V. rufa, seven in D. media and only five loci could be used for D. norwegica.     

Isolation and characterization of microsatellites in the Southeast Asian river catfish Mystus nemurus

A total of 26 simple sequence repeats were identified using a random amplified polymorphic DNA (RAPD) based technique in the Southeast Asian river catfish Mystus nemurus. We report on the characterization of five polymorphic microsatellite loci in M. nemurus. The average number of alleles per locus was 3.2. These are the first microsatellite loci that have been developed for this species. These markers should prove useful as tools for managing the brood stocks and for future aquacultural development of this species.     

Trinucleotide microsatellite loci for the zebra mussel Dreissena polymorpha,an invasive species in Europe and North America

The high mutation rate at microsatellite loci can supply important demographic information on founder events and range expansion in an invasive species such as the zebra mussel Dreissena polymorpha, following its initial introduction. In order to facilitate studies into the colonization patterns of this species in new habitats in Europe and North America, five trinucleotide microsatellite loci were isolated from a partial DNA library. Allelic diversity at all described loci was high, ranging from 20 to 35 alleles per locus. Homologous loci were not amplified in a second related invasive species, Dreisenna bugensis, using the primers developed here.     

Isolation and characterization of microsatellite loci from the rust pathogen,Melampsora lini

We developed and characterized primers for 11 variable microsatellite loci present in the genome of the flax rust, Melampsora lini. The microsatellite loci were identified by sequencing clones from a library of EcoRI DNA fragments enriched for four simple sequence repeat motifs (AAG, AAT, TC and TG). All 11 primer pairs successfully amplified DNA fragments from a sample of 102 M. lini isolates (98 isolated from Linum marginale and four from Linum usitatissimum), revealing a total of 32 alleles. Allelic diversity at the 11 loci ranged from 0.030 to 0.449.