Development of microsatellite loci in Pacific herring (Clupea pallasi)

Fifteen polymorphic di‐, tri‐ and tetranucleotide microsatellite markers were developed in Pacific herring (Clupea pallasi) to aid in the delineation of population structure of herring in British Columbia. Twelve of the microsatellite loci were analysed in over 4000 wild herring. Expected heterozygosities ranged 0.73–0.95, and only two loci significantly deviated from Hardy–Weinberg equilibrium.     

Isolation and characterization of tri‐ and tetranucleotide microsatellite loci in the Asian elephant,Elephas maximus

Asian elephants (Elephas maximus) are an endangered species. Their future survival depends on intensive conservation and management, based on in‐depth knowledge of particular populations. Molecular genetic methods, especially microsatellite analysis through noninvasive sampling, provides an effective means of obtaining such information. The use of tri‐ and tetranucleotide microsatellite markers is advantageous in noninvasive sampling through dung analysis. Here we describe the isolation and characterization of five tri‐ and tetranucleotide markers in the Asian elephant. All five loci were found to be polymorphic in a sample of 20 Asian elephants from Sri Lanka.     

Microsatellites loci isolated in the freshwater fish Brycon hilarii

We describe seven microsatellite loci, including tetra‐, tri‐ and dinucleotides, isolated from Brycon hilarii, which is a migratory fish inhabiting the Paraguay River basin (Brazil) and is highly valued for its meat quality. Three to eight alleles per locus were detected and the expected heterozygosity ranged from 0.31 to 0.81. Positive results were obtained with cross‐amplification in five other species of Brycon. These microsatellites provide a potential tool for wild population and aquaculture studies of B. hilarii and other species of the genus.     

Polymorphic microsatellite loci for the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) and some remarks on their isolation

Five polymorphic tri‐ and tetranucleotide microsatellite loci suitable for population genetic analysis were identified in the cotton bollworm Helicoverpa armigera from two partial phagemid genomic libraries enriched for microsatellite inserts. The overall microsatellite‐cloning efficiency in H. armigera is 2.5%, which is approximately eightfold lower than that for the gadoid fishes (20%) employing the same enrichment protocol, supporting the notion of a relative low frequency of microsatellite sequences in lepidopteran genomes. In addition, a large proportion of cloned microsatellite sequences turned out to be repetitive DNA, thus further increasing the difficulty of developing such markers in butterflies and moths.     

Isolation and characterization of microsatellite markers in Fraser fir (Abies fraseri)

We describe the isolation and characterization of 14 microsatellite loci from Fraser fir (Abies fraseri). These markers originated from cloned inserts enriched for DNA sequences containing tandem di‐ and tri‐nucleotide repeats. In total, 36 clones were selected, sequenced and evaluated. Polymerase chain reaction (PCR) primers for 14 of these sequences consistently produced simple PCR profiles and were found to be polymorphic among 13 Fraser fir samples. In addition, more than half of these loci were found to amplify a wide range of samples from several Abies taxa.     

Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

Fifty‐two polymorphic microsatellite loci in the rust fungus,Cronartium quercuum f.sp. fusiforme

We report on 52 microsatellite markers for use in Cronartium quercuum f.sp. fusiforme. The markers were developed from di‐, tri‐, and tetranucleotide repeat‐enriched genomic libraries. In 46 isolates collected from two natural populations in the southeastern USA, the number of alleles per locus ranged from two to 20 (mean 6.94) with gene diversity values ranging from 0.043 to 0.933 (mean 0.537). The markers should prove highly useful for genetic ‘fingerprinting’ of single‐spore isolates commonly used in host–pathogen gene interaction studies, as marker loci for linkage mapping studies, and for examining fine‐scale population genetic structure in natural populations of the fungus.     

Ten novel microsatellite loci characterized for a remarkably widespread fish: Galaxias maculatus (Galaxiidae)

Ten polymorphic microsatellite markers (five tetra‐, one compound tetra‐, one octa‐ and three dinucleotides) were isolated and characterized for Galaxias maculatus, a fish species widely distributed in the Southern Hemisphere. Markers were tested in 89 individual samples from a single location and the number of alleles ranged between 2 and 28. Observed and expected heterozygosities ranged from 0.103 to 0.910 and 0.098 to 0.935 respectively. No evidence was detected for either linkage disequilibrium (P‐values > 0.05 for each locus pair) or deviations from HWE (P‐values > 0.05 for every loci).     

Development of microsatellite loci in pinto abalone (Haliotis kamtschatkana)

Twelve novel di‐, tri‐ and tetranucleotide microsatellite loci to the pinto abalone (Haliotis kamtschatkana) are described. Over 400 individuals were analysed at each microsatellite locus. Observed heterozygosities ranged from 0.44 to 0.93, and numbers of alleles from 20 to 63. Six of the loci contained excesses in homozygosity indicative of inbreeding, nonrandom mating, population admixture, or null alleles.     

Development and characterization of eight new microsatellite markers for the haremic sandperch,Parapercis cylindrica (family Pinguipedidae)

Eight di‐, tri‐ and tetranucleotide microsatellite markers were developed for the haremic sandperch Parapercis cylindrica using a linker‐ligated, magnetic bead enrichment protocol. Screening of at least 17 individuals showed these markers to be polymorphic with observed heterozygosity ranging from 0.381 to 1.000 (mean = 0.742) and the numbers of alleles ranging from three to 18. The average polymorphic information content for these eight loci was 0.723. These markers may be used for parentage studies aimed at exploring the complex mating strategies employed by this haremic coral reef fish and will be valuable for population genetic studies.     

Characterization of microsatellite loci in the creek chub (Semotilus atromaculatus)

Thirty‐two polymorphic microsatellite loci (29 novel, 3 cross‐species) are characterized in the creek chub, Semotilus atromaculatus, a widespread and abundant North American minnow. Novel repeat‐containing sequences were isolated using a standard microsatellite enrichment procedure. A set of di‐, tri‐, and tetranucleotide markers with allele numbers ranging from two 16 are now available for estimating relatedness within and among populations in this ecologically important species.     

Characterization of 18 new microsatellite loci in Atlantic cod (Gadus morhua L.)

Eighteen new microsatellite loci consisting of 10 di‐, 5 tri‐, 2 tetra‐ and 1 heptanucleotide repeats are introduced for the Atlantic cod (Gadus morhua L.). All loci were co‐amplified in two polymerase chain reactions (plus two previously published microsatellites) and all products were typed clearly. The number of alleles per locus ranged from six (PGmo130) to 45 (PGmo76) and the observed heterozygosity ranged from 0.356 (PGmo130) to 0.957 (PGmo95). All loci except one followed Hardy–Weinberg expectations. Genetic linkage disequilibrium analysis between all pairs of loci did not yield any significant values.     

Isolation and characterization of 27 polymorphic microsatellite loci for the common snook,Centropomus undecimalis

Here we describe 32 di‐, tri‐ and tetranucleotide microsatellite loci isolated by PIMA, a polymerase chain reaction (PCR)‐based procedure, for the common snook (Centropomus undecimalis). Five loci were monomorphic, and the remaining loci averaged 6.7 alleles per locus in a sample of 45 common snook. For polymorphic loci, expected heterozygosities ranged from 0.02 to 0.91 (mean = 0.538). Significant departures from Hardy–Weinberg equilibrium expectations occurred in two loci. Exact tests for genotype disequilibrium gave evidence for linkage at one pair of loci. Many cross‐species primer assays yielded PCR fragments of the expected size for 11 species of Centropomus and two species of the confamilial genus Lates.     

Isolation and characterization of microsatellite DNA loci in Aquilegia sp.

We obtained a microsatellite‐enriched genomic library isolated from the tissue of a single columbine (Aquilegia sp.) plant taken from a southwestern USA natural population. The primers developed for these microsatellite loci performed consistently in polymerase chain reactions and yielded multiallelic genotypes with relatively high observed heterozygosities. We describe polymerase chain reaction primers and conditions to amplify 16 unique, codominant di‐, tri‐ and tetra‐nucleotide microsatellite DNA loci so that other population biology researchers using columbine natural populations as a model system may benefit.     

Microsatellites in the striped ground crickets,Allonemobius (Orthoptera: Gryllidae)

Polymorphic di‐, tri‐ and tetranucleotide repeats were examined in Allonemobius to determine whether they could serve as useful markers in studies of sperm precedence, population genetics and hybrid zone structure. Ten microsatellite DNA loci were sufficiently polymorphic to be used for paternity tests and showed no evidence of linkage disequilibrium or deviations from Hardy–Weinberg equilibrium in Allonemobius socius. Nine of 10 of these microsatellites can be amplified from three other Allonemobius species, suggesting that these markers will have widespread utility in this ground cricket genus.     

Development and characterization of six new microsatellite markers for the silver‐ or gold‐lipped pearl oyster,Pinctada maxima (Pteriidae)

Six di‐, tri‐ and tetranucleotide microsatellite loci were developed for the silver‐ or gold‐lipped pearl oyster Pinctada maxima using a linker‐ligated, magnetic bead enrichment protocol. Based on a minimum of 134 Indonesian pearl oyster samples, number of alleles and observed heterozygosity at each locus ranged from six to 17 alleles and from 0.172 to 0.813 (mean = 0.448), respectively. Mean polymorphic information content for the six loci was 0.562. These loci should be very useful in DNA parentage analyses and population differentiation of P. maxima in Australia and Indonesia.     

Development of sequence‐tagged microsatellite site markers for chickpea (Cicer arietinum L.)

Microsatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence‐tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)‐amplified fragments. Thirty‐five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross‐species transferability of the sequence‐tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra‐ and interspecific genetic linkage maps.     

Identification of polymorphic microsatellite loci in the mud crab Scylla serrata (Brachyura: Portunidae)

Five microsatellite loci are identified and characterized from the genome of Scylla serrata, a widespread and commercially important species of coastal marine crab. The loci were detected by randomly screening for di‐ and tri‐nucleotide repeat units within a partial genomic library developed for the species. The five loci consist of dinucleotide repeats and are both co‐dominant and polymorphic within the species. A sample (N = 36) of S. serrata from one Australian population has an average observed heterozygosity of 0.875 and provides no evidence of either linkage among loci or significant deviation from random mating expectations across loci. PCR products for each of the five loci were also observed from a small sample of three other species within the Scylla genus. These markers may provide genetic information that will be useful for both aquaculture and studies of natural populations of the genus.     

Screening of ESTs from Mytilus for the detection of SSR markers in Mytilus californianus

A set of expressed sequence tag‐simple sequence repeat (EST‐SSR) markers for the genus Mytilus was developed through bioinformatic mining of the GenBank public database. A total of 33 782 EST sequences from GenBank were downloaded and screened for di‐, tri‐ and tetranucleotide, with 1274 EST containing SSR markers. Nine microsatellite markers were characterized in Mytilus californianus with a number of alleles per locus ranging from two to six, and total observed and expected heterozygosities ranging from 0.490 to 0.730 and from 0.510 to 0.860 respectively. Cross‐species amplification was achieved in several other species, confirming the usefulness of these markers in Mytilus genetics.     

Novel tetranucleotide microsatellite markers from the Del Norte salamander (Plethodon elongatus) with application to its sister species the Siskiyou Mountain salamander (P. stormi)

Eleven tetranucleotide microsatellite loci were developed for the Del Norte salamander (Plethodon elongatus). The loci were variably polymorphic, ranging from two to 20 alleles per locus, with expected heterozygosities ranging from 0.07 to 0.86. The loci also amplified in a congener, the Siskiyou Mountain salamander (P. stormi). The microsatellite loci will be used to assess the utility of highly polymorphic markers to assay within‐ and between‐species differentiation between these two closely related species.