Microsatellite DNA loci for Western Hemlock [Tsuga heterophylla (Raf.) Sarg]

Polymorphic microsatellite loci were developed for Western Hemlock [Tsuga heterophylla (Raf.) Sarg], a prominent forest tree species in Western North America. Microsatellite‐enriched libraries were screened for (CA)_n dinucleotide repeats from which 33 positive clones were sequenced. Polymerase chain reaction (PCR) primers for 16 microsatellite loci were prepared and tested against DNA from unrelated Western Hemlock trees. The 12 most informative microsatellite loci are reported here. From four to 22 alleles per locus were observed, with an average expected heterozygousity of 0.799.     

Cloning and characterization of microsatellite loci in channel catfish, Ictalurus punctatus

Short tandem repeat (microsatellite) loci were cloned from the channel catfish, Ictalurus punctatus , genome for use as molecular markers for genetic improvement of this important agricultural species. Plasmid clones containing catfish genomic DNA inserts were identified, by hybridization with tandem repeat DNA probes, and sequenced using automated laser fluorescence. A feral population of catfish displayed levels of heterozygosity greater than 0·7 for 13 of 22 loci and heterozygosity greater than 0·5 for 20 of 22 loci. Allelic polymorphism ranged from three to 17 alleles per locus in the feral population. Populations of domestic, farm-raised catfish and a research strain displayed levels of heterozygosity similar to the feral population. Non-invasive tissue sampling provided abundant material for the polymerase chain reaction-based genotype assay. The microsatellite loci will be useful in the molecular characterization and genetic improvement of channel catfish populations.     

Development and characterization of microsatellite loci in the endangered oyster mussel Epioblasma capsaeformis (Bivalvia: Unionidae)

Primers for 10 polymorphic microsatellite loci were developed and characterized for the endangered oyster mussel Epioblasma capsaeformis from the Clinch River, Tennessee. Microsatellite loci also were tested in four other populations or species. Amplification was successful for most loci in these closely related endangered species or populations; therefore, a high level of flanking sequence similarity was inferred for this group of species and populations. Allelic diversity ranged from nine to 20 alleles/locus, and averaged 13.6/locus. This study demonstrated the feasibility of using polymerase chain reaction (PCR) primers to amplify microsatellite loci across freshwater mussel species to conduct population genetics studies.     

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.     

Isolation of microsatellite loci in the pollinating fig wasp of Ficus hispida, Ceratosolen solmsi

Microsatellite loci were isolated for Ceratosolen solmsi , pollinator of the dioecious Ficus hispida. We developed nine polymorphic microsatellite loci based on the method of polymerase chain reaction isolation of microsatellite arrays (PIMA). Enrichment of genomic libraries was performed by random amplified polymorphic DNA (RAPD). A subset of 38 positive clones was sequenced; 15 clones showed microsatellite loci. We tested 15 designed primer pairs and nine of them produced polymorphic amplification in 48 individual wasps collected from different fruits of the dioecious host fig Ficus hispida in China. Among the 48 individuals, 49 alleles were obtained at the nine loci. The observed heterozygosity ranged between 0.357 and 0.634.     

Stock composition in North Atlantic populations of whiting using microsatellite markers

A size-selected library constructed from DNA of the whiting Merlangius merlangus was screened. From about 3200 recombinant clones, 43 microsatellite loci were detected. Thirteen were sequenced in full. Primers were designed from the sequence of the flanking regions for six loci and used to test the allelic variability at these loci using the polymerase chain reaction (PCR). In addition, five primer pairs developed for the stickleback and another seven for cod were tested. Only six primer pairs revealed at least three alleles per locus. The three useful loci Gmo2, Mmer- UEAW01 and Mmer- UEAW02, had 14–23 alleles per locus in 370 samples. Estimates of genetic structure (φ) were not statistically significant. However, estimates of genetic differentiation ( F _st) were significantly different from zero. Heterogeneity χ²-analysis of allele frequencies among populations suggested relatively low levels of differentiation among samples. Significantly different allele frequency distributions were found for Borgensfjord and northern and southern North Sea samples for at least one locus, and between the latter samples for Mmer -UEAW02 and Gmo2 . There were significant excesses of homozygotes in all samples, over expectation for randomly mating populations in Hardy-Weinberg equilibrium. The estimated frequencies of null alleles were 14.3%, for Mmer -UEAW01, 10.2% for Mmer- UEAW02 and 11.6% for Gmo2 . This result calls for a careful interpretation of the significance of these microsatellite data.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

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.     

Isolation and characterization of microsatellites in the Asian walking catfish Clarias macrocephalus

Microsatellite loci were characterized in the walking catfish, Clarias macrocephalus, random clones from a small genomic library using a (GT)₁₅ probe. Primers for DNA amplifications using polymerase chain reaction (PCR) were designed and synthesized for 23 loci. Twelve loci were polymorphic, with the number of alleles ranging from two to 13 alleles per locus. Developed microsatellite primers should prove useful for population studies and genetic mapping of the walking catfish.     

Dinucleotide microsatellite loci isolated from flowering dogwood (Cornus florida L.)

We report eight variable dinucleotide microsatellite loci cloned from flowering dogwood (Cornus florida L.) using a biotin enrichment protocol. Degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR) was used to generate a population of DNA fragments, from which adenine‐cytosine dinucleotide (AC) and adenine‐guanine dinucleotide (AG) repeats were captured using biotinylated probes and streptavidin coated magnetic particles. The captured fragments were cloned into plasmids, and the plasmid library was screened for microsatellites using a simple PCR technique. Selected plasmids were sequenced, and PCR primers were designed and optimized using a thermal‐gradient thermocycler. The loci reported are highly variable with an average of 9.25 allele per locus and an average heterozygosity of 0.84.     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Development of microsatellite markers in Acer capillipes

Acer capillipes is an insect‐pollinated tree species that grows in temperate regions of Japan. We isolated seven polymorphic microsatellite loci from this species using a dual‐suppression polymerase chain reaction (PCR) technique. The number of alleles per locus ranged from two to 10, and the expected heterozygosity ranged from 0.042 to 0.828. Cross‐species amplification from 14 other Acer species was successful for the majority of the isolated loci, suggesting that these loci may be useful for the characterization of other maple species.     

Isolation and Characterization of Four Microsatellite Loci in the Tidewater Goby (Eucyclogobius newberryi)

The first microsatellite loci for the endangered tidewater goby Eucyclogobius newberryi are described. A partial tidewater goby genomic library was constructed in pUC19 plasmid. Microsatellite-containing clones were isolated, sequenced, and amplified. Out of 12 positive clones sequenced, 3 were polymorphic and proved useful in a preliminary genetic screen of 3 well-separated populations (50 individuals each) along the central California coast. The number of alleles per locus ranged from 2 to 3. This initial study suggests a population bottleneck had occurred in one of the populations. Received May 30, 2000; accepted September 14, 2000.     

Polymorphic microsatellite DNA markers from the Oriental Fruit Fly Bactrocera dorsalis (Hendel)

Six polymorphic microsatellite loci are isolated from the Oriental fruit fly Bactrocera dorsalis (Hendel), an agricultural pest in Asia, including Taiwan. To assess their potential utility as high‐resolution genetic markers, polymerase chain reaction (PCR) primers, amplification conditions, and an automated fluorescence detection protocol were developed. In analyses of 71 individual flies from six different areas of Taiwan, allele numbers ranged from five to 25 were detected for each locus. The observed heterozygosity ranged between 0.268 and 0.737 among these loci. No linkage disequilibrium was found. These microsatellite markers have potential utility to population structure and gene flow studies of B. dorsalis (Hendel).     

Characterization of porcine polymorphic microsatellite loci

Twenty-seven (CA)_n and two (GA)_n microsatellite clones were isolated out of a size-selected genomic pig library. These were sequenced and the number of uninterrupted dinucleotides was found to range from 12 to 26. Flanking primers were chosen for 11 dinucleotide repeats and optimal conditions for polymerase chain reaction (PCR) amplifications were established. Different microsatellite loci were amplified simultaneously by combining primer sets. Related and unrelated pigs were screened for length polymorphisms of the different microsatellite loci. The polymorphic information content (PIC) of these loci ranged between 0.62 and 0.83. Segregation studies in pig reference families established Mendelian inheritance. Locus S0022 was found to be X-linked.     

Highly Variable Microsatellites in the California Market Squid Loligo opalescens

Attempts to study the genetic population structure of cephalopods are impeded by the low levels of genetic variation in these species. We have developed polymerase chain reaction (PCR) primers for six hypervariable microsatellite markers in order to analyze the molecular population structure in the Californian market squid Loligo opalescens. Each of these genomic loci has been cloned and fully sequenced. Here we report the sequence and properties of the six PCR primer sets for the amplification of hypervariable microsatellites. Heterozygosity levels in six squid samples from different locations are high for all loci tested. Received February 17, 1999; Accepted March 10, 1999     

Isolation of microsatellite markers from Pinus densiflora Sieb. et Zucc. using a dual PCR technique

We developed seven microsatellite loci from Pinus densiflora using a dual polymerase chain reaction (PCR) technique. Of 186 clones from a library based on suppression PCR, 127 contained microsatellite sequences. Of these, 43 candidates were determined sequences of both flanking regions, and 16 regions from this group were chosen as development markers. Seven of these primer pairs successfully amplified polymorphic single loci among 83 resistant trees against pine wood nematode. The observed heterozygosity of the seven microsatellite markers ranged from 0.247 to 0.843. Mendelian inheritance was confirmed using megagametophytes.     

Complex mutation at a microsatellite locus in sturgeons: Acipenser sinensis, A. schrenckii, A. gueldenstaedtii and A. baerii

Cross‐species amplifications of microsatellite locus Spl‐106, which was originally screened from the genome of shovelnose sturgeon (Scaphirhynchus platorynchus) with a perfect TAGA repeat motif, were carried out in four other species of the genera Acipenser. A total of 34 polymerase chain reaction (PCR) products representing 16 different alleles of this locus was sequenced. Sequence analysis results showed that besides the number changes of repeat units, many mutational events, such as single‐base substitutions and various insertion/deletion (indels) occurred not only at species level but also at individual level, even among the different alleles within the same individual. The repeat motifs varied from perfect (TAGA)n array to perfect compound (TAAA)m (GAAA)n and perfect or imperfect compound (TAAA)m (TAGA)n (TAAA)x arrays in different species and different individuals. The evolution dynamics of this locus in sturgeons was inferred in that it may evolve from a single perfect to different perfect or imperfect compounds.     

Dinucleotide Repeat Loci Contribute Highly Informative Genetic Markers to the Human Chromosome 2 Linkage Map

Microsatellite repeat loci can provide informative markers for genetic linkage. Currently, the human chromosome 2 genetic linkage map has very few highly polymorphic markers. Being such a large chromosome, it will require a large number of informative markers for the dense coverage desired to allow disease genes to be mapped quickly and accurately. Dinucleotide repeat loci from two anonymous chromosome 2 genomic DNA clones were sequenced so that oligonucleotide primers could be designed for amplifying each locus using the polymerase chain reaction (PCR). Five sets of PCR primers were also generated from nucleotide sequences in the GenBank Database of chromosome 2 genes containing dinucleotide repeats. In addition, one PCR primer pair was made that amplifies a restriction fragment length polymorphism on the TNP1 gene (Hoth and Engel, 1991). These markers were placed on the CEPH genetic linkage map by screening the CEPH reference DNA panel with each primer set, combining these data with those of other markers previously placed on the map, and analyzing the combined data set using CRI-MAP and LINKAGE. The microsatellite loci are highly informative markers and the TNP1 locus, as expected, is only moderately informative. A map was constructed with 38 ordered loci (odds 1000:1) spanning 296 cM (male) and 476 cM (female) of chromosome 2 compared with 306 cM (male) and 529 cM (female) for a previous map of 20 markers.     

Polymorphic microsatellite repeats are not conserved between Leishmania donovani and Leishmania major

Thirteen sets of polymerase chain reaction (PCR) primers were designed to amplify microsatellite loci identified in the genome sequence of Leishmania major. Polymorphisms were detected in L. major at all loci. In Leishmania donovani only two of these loci were informative for classification purposes with this data set. The PCR products of all loci from one L. donovani strain were sequenced and it was found that the number of repeats in the microsatellite loci were either substantially reduced with respect to L. major or absent altogether. Consequently it is unlikely to be possible to use the genome sequence of L. major to identify polymorphic microsatellite loci in other Leishmania species.