Microsatellite DNA loci from the Diamondback terrapin (Malaclemys terrapin)

We describe polymerase chain recation (PCR) primers and conditions to amplify one dinucleotide and five tetranucleotide microsatellite DNA loci isolated from the Diamondback terrapin (Malaclemys terrapin). The PCR primers were tested on 21 terrapins from Cape Romain, SC, USA. The microsatellite primers developed yielded a high number of alleles (8–14) and high observed heterozygosities (0.57–1.0).     

Tetranucleotide microsatellite DNA loci from the dollar sunfish (Lepomis marginatus)

We describe polymerase chain reaction (PCR) primers and conditions to amplify one dinucleotide and eight tetranucleotide microsatellite DNA loci isolated from the dollar sunfish (Lepomis marginatus). The PCR primers were tested on 20 or more individuals from five populations. The dollar sunfish microsatellite primers developed yielded a high number of alleles (4 to 14 per locus), and high observed heterozygosities (0.500–0.857).     

Development of microsatellite DNA loci from the wood stork (Aves,Ciconiidae, Mycteria americana)

We isolated 11 polymorphic microsatellite loci for wood stork (Mycteria americana). Polymerase chain reaction (PCR) primers and conditions are described for the amplification of five dinucleotide, one trinucleotide and five tetranucleotide microsatellite loci. The PCR primers were tested on two wood stork populations, Fazenda Ipiranga, Mato Grosso, Brazil (n = 11) and Tamiami West, Everglades, Florida, USA (n = 20). The primers yielded two to four alleles per locus, an observed heterozygosity of 0.0–0.727 and a polymorphic information content of 0.048–0.604. The low level of polymorphism for these markers is consistent with previous studies of this species.     

Tetranucleotide,trinucleotide, and dinucleotide loci from the bobcat (Lynx rufus)

We describe primers and polymerase chain reaction (PCR) conditions to amplify four dinucleotide, one trinucleotide, and three tetranucleotide microsatellite DNA loci from the bobcat (Lynx rufus). The primers were tested on 22 individuals collected from a population located within southwestern Georgia (USA). The primer pairs developed in this study yielded an average of 7.4 alleles per locus (range four to 10), an average observed heterozygosity of 0.60 (range 0.40 to 0.76), and an average polymorphic information content of 0.70 (range 0.51 to 0.78).     

Polymorphic microsatellite DNA amplification customized for chimpanzee paternity testing

DNA segments containing GT/AC dinucleotide repeats in the chimpanzee (Pan troglodytes) genome were screened. Thirteen transformedE. coli colonies were identified with the (GT)₁₀ probe to have chimpanzee DNA fragments containing (GT)_n repeats. These potentially polymorphic (variable n) DNA segments were sequenced. Primers for the polymerase chain reaction (PCR) amplifying these DNA segments were designed. Six pairs of primers yielded polymorphic PCR products. Three of them revealed considerable length polymorphisms and heterozygosities in a group of captive chimpanzees. For studies on chimpanzees in the wild and in captivity, these primers should be useful for paternity testing, for investigating genetic variations, and for improving the genetic maintenance of breeding colonies. The strategy adopted in the present study to obtain PCR primers amplifying polymorphic microsatellite DNA segments may well be applicable to almost all eukaryotic organisms.     

Characterization of microsatellite DNA loci for the southern flying squirrel (Glaucomys volans)

Polymerase chain reaction primers for microsatellite DNA loci (one dinucleotide, four tetranucleotide and two compound) and the conditions necessary to amplify each are described for the southern flying squirrel (Glaucomys volans). These primers were tested on 22 or more individuals from a population at the Savannah River Site in South Carolina. These microsatellite primers yielded a high allelic diversity (6–22 alleles/locus), and moderate to high observed heterozygosities (0.318–0.826). Primers developed for the northern flying squirrel (Glaucomys sabrinus) were also tested for use on G. volans, with only two successful cross amplifications from the seven loci.     

Chimpanzee microsatellite PCR primers applied to paternity testing in a captive colony

Previously designed primers for the polymerase chain reaction (PCR) amplifying microsatellite DNA segments containing GT/AC dinucleotide repeats in the chimpanzee (Pan troglodytes) genome were used for paternity testing in a breeding colony in captivity. Combinations of three PCR primers identified the fathers of all the tested 40 chimpanzees born in an eight-year period. The results suggested: (1) a positive (though not conclusive) correlation between male rank and number of offspring; (2) choice of mating partners by the female rather than by the male; and (3) absence of stable mating pairs over the years. For studies of chimpanzees in captivity and in the wild, these primers should be useful for paternity testing, for investigating genetic variations, and for improving genetic maintenance of breeding colonies.     

Short microsatellite DNA markers for the red fox (Vulpes vulpes)

Seven short microsatellite loci (< 165 bp) were characterized for red foxes for the amplification of degraded DNA extracted from historical samples. Following polymerase chain reaction (PCR) using primers developed in the domestic dog, red fox‐specific primers were designed within the flanking regions. The number of detected alleles ranged between six and 15 alleles and the expected heterozygosities ranged between 0.67 and 0.92. No deviations from Hardy–Weinberg equilibrium were detected for any of the markers.     

Microsatellite genotyping by primer extension and MALDI-ToF mass spectrometry

A novel method for genotyping microsatellite alleles using primer extensions and mass spectrometry analysis has been developed. Following PCR amplification of the target region, a genotyping primer, with its 3′ end directly flanking the microsatellite repeats, was extended by a mixture of dNTPs complementary to the nucleotides composing the microsatellite. The length and molecular weight of extended primers vary with the number of repeats present in the allele(s) under examination. The weights of extension products were determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) and used to identify genotypes on the basis of differential primer extension. This is a platform that is not gel based and is amenable to multiplexing and automation. The technique enables identification of heterozygous progeny in which alleles differ by a single trinucleotide repeat. The method is illustrated by genotyping a polymorphic microsatellite identified in an intron of the barleyMlo gene.     

Microsatellite loci isolated from narrow‐leaved cattail Typha angustifolia

We present 11 dinucleotide microsatellite DNA loci isolated from the narrow‐leaved cattail (Typha angustifolia) and describe conditions for their amplification. The PCR primers were tested on at least 20 individuals of Typha angustifolia and T. latifolia from two Ukrainian populations per species. The primers amplify loci with relatively high numbers of alleles (averaging 7.22 and 4.95 alleles per locus in T. angustifolia and T. latifolia, respectively), and polymorphic information content (averaging 0.61 and 0.46 in T. angustifolia and T. latifolia, respectively).     

Tetranucleotide microsatellite loci from eastern bluebirds Sialia sialis

We describe primers and polymerase chain reaction (PCR) conditions to amplify 18 tetranucleotide microsatellite DNA loci in eastern bluebirds (Sialia sialis). The primers were tested using individuals from two study sites in Georgia and South Carolina. Among individuals from the Georgia population (n = 23), the primer pairs developed in this study yielded an average of 6.6 alleles per locus (range 2–12), an average observed heterozygosity of 0.56 (range 0.24–0.96) and an average polymorphic information content of 0.65 (range 0.3–0.86). Among individuals from the South Carolina population (n = 19), the primer pairs yielded an average of 5.8 alleles per locus (range 2–9), an average observed heterozygosity of 0.56 (range 0.05–0.86) and an average polymorphic information content of 0.63 (range 0.29–0.83).     

Sequence characterization of microsatellites in diploid and polyploid Ipomoea

 The objectives of the present study were to evaluate the inheritance and nucleotide sequence profiles of microsatellite genetic markers in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.] and its putative tetraploid and diploid ancestors, and to test possible microsatellite mutation mechanisms in polyploids by direct sequencing of alleles. Sixty three microsatellite loci were isolated from genomic libraries of I. batatas and sequenced. PCR primers were designed and used to characterize microsatellite loci in two hexaploid I. batatas populations, a tetraploid Ipomoea trifida population, and a diploid I. trifida population. Nine out of the sixty three primer pairs tested yielded a clearly discernible, heritable banding pattern; five showed Mendelian segregation. All other primer pairs produced either smeared banding patterns, which could not be scored, or no bands at all in I. batatas. All of the primers which produced discernible banding patterns from I. batatas also amplified products of similar size in tetraploid and diploid I. trifida accessions. The sequence analysis of several alleles in the three species showed differences due to mutations in the repeat regions consistent with small differences in the repeat number. However, in some cases insertions/deletions and base substitutions in the microsatellite flanking regions were responsible for polymorphisms in both polyploid and diploid species. These results provide strong empirical evidence that complex genetic mechanisms are responsible for SSR allelic variation in Ipomoea. Four I. batatas microsatellite loci showed polysomic segregation fitting tetraploid segregation ratios. To our knowledge this is the first report of segregation ratios for microsatellites markers in polyploids. Received: 4 January 1999 / Accepted: 4 January 1999     

Characterization of seven polymorphic microsatellite loci in the Common Loon (Gavia immer)

We describe polymerase chain reaction (PCR) primers and conditions to amplify seven microsatellite DNA loci isolated from the Common Loon (Gavia immer). The PCR primers were tested on 83 individuals from 10 locations in North America, including breeding, migration stopover, and wintering areas. Between two and seven alleles were observed to segregate at the seven microsatellite loci, with observed heterozygosities ranging from 0.048 to 0.695.     

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.     

Cross‐species amplification among peromyscines of new microsatellite DNA loci from the oldfield mouse (Peromyscus polionotus subgriseus)

We describe polymerase chain reaction (PCR) primers and conditions to amplify 11 microsatellite DNA loci isolated from the oldfield mouse (Peromyscus polionotus subgriseus). These were tested for amplification using nine species and subspecies maintained at the Peromyscus Genetic Stock Center, with an average success rate of 65% and two loci amplifying in all species. Polymorphism was tested within the P. polionotus subgriseus and the recently obtained P. maniculatus sonorensis colonies. P. p. subgriseus had modest numbers of alleles per locus (1–4), whereas P. m. sonorensis had many alleles per locus (5–10) and high expected heterozygosities (0.625–0.878).     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

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     

A refined panel of 42 microsatellite loci to universally genotype catarrhine primates

1. Microsatellite genotyping is an important genetic method for a number of research questions in biology. Given that the traditional fragment length analysis using polyacrylamide gel or capillary electrophoresis has several drawbacks, microsatellite genotyping‐by‐sequencing (GBS) has arisen as a promising alternative. Although GBS mitigates many of the problems of fragment length analysis, issues with allelic dropout and null alleles often remain due to mismatches in primer binding sites and unnecessarily long PCR products. This is also true for GBS in catarrhine primates where cross‐species amplification of loci (often human derived) is common.
2. We therefore redesigned primers for 45 microsatellite loci based on 17 available catarrhine reference genomes. Next, we tested them in singleplex and different multiplex settings in a panel of species representing all major lineages of Catarrhini and further validated them in wild Guinea baboons (Papio papio) using fecal samples.
3. The final panel of 42 microsatellite loci can efficiently be amplified with primers distributed into three amplification pools.
4. With our microsatellite panel, we provide a tool to universally genotype catarrhine primates via GBS from different sample sources in a cost‐ and time‐efficient way, with higher resolution, and comparability among laboratories and species.

     

Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves

Aims: The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves. Methods and Results: Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the β-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves. Significance and Impact of the Study: DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.     

Isolation and characterization of microsatellite loci in Fragaria

This study reports the development and characterization of 20 microsatellite primer pairs in wild strawberry Fragaria vesca. One hundred primers were obtained from an AC‐enriched library developed in the cultivar ‘Ilaria’. A set of eight F. vesca genotypes was used to detect the polymorphism resulting in an average of 7.0 alleles, an average observed heterozygosity of 0.32 and an average expected heterozygosity of 0.73. Nineteen (95%) of the primers also amplified the cultivated octoploid strawberry Fragaria×ananassa.