Microsatellite loci from the Caribbean Fruit Fly,Anastrepha suspensa (Diptera: Tephritidae)

The lack of polymorphic genetic markers suitable for genotyping sperm, eggs, and all life stages of the important agricultural pest, Anastrepha suspensa, have prevented detailed genetic studies of its breeding system, reproductive dynamics, and population dynamics. We describe polymerase chain reaction (PCR) primers and reaction conditions for amplifying nine polymorphic microsatellite DNA loci isolated from this species. The PCR primers were tested on four to five individuals each collected from five geographically distant locations in Florida. Heterozygosity values and the number of alleles per locus varied from 0.11 to 0.89, and from two to 12, respectively.     

Isolation and characterization of 22 polymorphic microsatellite loci for the Barrow's goldeneye (Bucephala islandica)

We describe primers and polymerase chain reaction conditions to amplify 22 microsatellite loci from the Barrow's goldeneye (Bucephala islandica). The primers were tested on 27 individuals from a population breeding in British Columbia, Canada. The developed primer pairs yielded an average of 6.11 alleles per locus (range 2-12), an average observed heterozygosity of 0.70 (range 0.07-0.96) and a polymorphic information content of 0.07-0.88.     

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).     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Isolation of ten tetranucleotide microsatellite loci in the Northern Wheatear (Oenanthe oenanthe)

We isolated 10 polymorphic microsatellite DNA loci from the Northern Wheatear (Oenanthe oenanthe) and optimized these for future studies in population genetics and behavioural ecology. The loci were screened for polymorphism using 107 individuals from one population in Germany. The primers amplified loci with high numbers of alleles ranging from two to 20 alleles per locus.     

Isolation and characterization of 10 microsatellite loci in the magnetic termite,Amitermes meridionalis (Isoptera: Termitidae)

The elongated mounds of the ‘magnetic termite’, Amitermes meridionalis are a prominent feature of the Northern Territory in Australia. They are restricted to habitat patches of seasonally flooded plains which are largely isolated from each other. To investigate the population structure of A. meridionalis, we developed 10 polymorphic microsatellite loci. We tested the variability of the markers on at least 20 individuals from two populations. We found three to 12 alleles per locus with a level of heterozygosity at each locus ranging from 0.05 to 0.74.     

Characterization of polymorphic microsatellite markers for the green turtle (Chelonia mydas)

We describe primers and polymerase chain reaction conditions to amplify 12 microsatellite loci from the green turtle (Chelonia mydas), including one dinucleotide, four trinucleotide and seven tetranucleotide loci. The primers were tested on 78 individuals from a Pacific population nesting in the Hawaiian Islands. The primer pairs developed in this study yielded an average of 8.33 alleles per locus (range of 3-15 alleles), an average observed heterozygosity of 0.668 (range 0.309-0.910), and an average polymorphic information content of 0.647 (range 0.287-0.894).     

Development of microsatellite markers in heterostylous Hedyotis chrysotricha (Rubiaceae)

? Premise of the study: Microsatellite primers were developed for a heterostylous herb, Hedyotis chrysotricha to investigate the effect of habitat fragmentation on its genetic diversity and population structure. ? Methods and Results: Twelve primer sets were developed and their polymorphisms were tested on 47 individuals from two island populations of H. chrysotricha in Thousand Island Lake of China. The number of alleles per locus ranged from five to 10, with an average of seven alleles. Expected heterozygosity per locus ranged from 0.284 to 0.821 and observed heterozygosity ranged from 0.191 to 0.851. ? Conclusions: We showed that all of the 12 microsatellite markers developed for H. chrysotricha are polymorphic within populations, which should provide a powerful tool for assessing population structure and genetic diversity across fragmented and continuous populations, and for studying the genetic effects of habitat fragmentation on this species.     

Isolation and characterization of 17 polymorphic microsatellite loci for the three-toed woodpecker (Picoides tridactylus)

We describe primers and polymerase chain reaction conditions to amplify 17 di‐, tri‐ and tetranucleotide microsatellite loci from the three‐toed woodpecker (Picoides tridactylus). The primers were tested on 26 to 30 individuals from a single population breeding in southern Finland. The developed primer pairs yielded an average of 7.6 alleles per locus (range two to 15), an average observed heterozygosity of 0.69 (range 0.07 to 0.97), and an average polymorphic information content of 0.68 (range 0.06 to 0.90).     

Cloning and characterization of 29 tetranucleotide and two dinucleotide polymorphic microsatellite loci from the endangered marbled murrelet (Brachyramphus marmoratus)

We developed 31 novel, polymorphic microsatellite loci in the marbled murrelet (Brachyramphus marmoratus), a critically endangered seabird. Variability was tested on 15 individuals from the Santa Cruz, California population, with each locus characterized by two to 12 alleles. Observed levels of heterozygosity ranged from 0.13 to 0.93. These loci provide a valuable means of assessing the population structure and demographic parameters of this species, which may be critical to its conservation across a fragmented habitat.     

Isolation and characterization of microsatellite loci from the invasive ant Pheidole megacephala

We report the characterization of eight microsatellite markers in the big-headed ant Pheidole megacephala, a pest ant registered in the list of '100 of the world's worst invasive alien species'. An enrichment protocol was used to isolate microsatellite loci, and polymorphism was explored with 36 individuals collected in an invasive population from Australia and 20 individuals collected in a population from the native mainland location in South Africa. These primers showed a number of alleles per locus ranging from two to 10, and expected heterozygosities ranging from 0.083 to 0.826. Moreover, results of cross-species amplification are reported in five other Pheidole species and in seven other ants of the subfamily Myrmicinae.     

Isolation and characterization of microsatellite loci in Merluccius australis and cross-species amplification

Eight novel and two heterologous microsatellite pairs of primers are presented for the Austral hake (Merluccius australis), representing the first microsatellite markers available for this species. Loci were characterized for 50 individuals from two populations in South America (Argentinean and Chilean coasts). All loci were polymorphic within M. australis (5 to 30 alleles per locus; observed heterozygosity between 0.320 and 0.840), and therefore useful for population genetic studies within the species. Cross-species transferability was tested for 100 individuals from four additional species within the Merluccius genus (M. albidus, M. bilinearis, M. gayi and M. hubbsi), and results indicate that most of these primers pairs will likely be useful for population genetic studies on Merluccius species.     

Microsatellite markers from Ceanothus roderickii (Rhamnaceae) using next-generation sequencing technology

? Premise of the study: Ceanothus roderickii is an endangered shrub endemic to California. To investigate the population genetics of this species, including the genetic consequences of population fragmentation and hybridization, 10 microsatellite markers were developed. ? Methods and Results: Using next-generation sequencing (454) data from a single C. roderickii individual, 10 microsatellite markers were developed. A group of 12 individuals representing all of the major C. roderickii populations were analyzed. All loci were found to be polymorphic, with a range from two to 12 alleles per locus. Observed heterozygosity ranged from 0.08 to 0.83 across loci. All 10 loci were also amplifiable in at least one other Ceanothus species. ? Conclusions: Results presented here indicate the utility of our new microsatellite primers in ongoing and future studies concerning population genetics and gene flow in C. roderickii, as well as the potential applicability of these primers in similar studies on other Ceanothus species.     

Isolation and characterization of microsatellite loci in a marine fish species,the tusk (Brosme brosme)

We developed polymerase chain reaction primers for nine dinucleotide microsatellite loci in the marine deep‐sea fish, the tusk (Brosme brosme). All markers were obtained from a partial genomic DNA library, and characterized in 100 unrelated individuals from one putative population. The number of alleles ranged from two to 60 with an average of 15/locus. The observed heterozygosity ranged from 0.020 to 0.826 (average 0.438). Several of the markers amplified multiple alleles from the two other gadoid species tested.     

Mark–recapture cloning: a straightforward and cost‐effective cloning method for population genetics of single‐copy nuclear DNA sequences in diploids

We describe a simple protocol to reduce the number of cloning reactions of nuclear DNA sequences in population genetic studies of diploid organisms. Cloning is a necessary step to obtain correct haplotypes in such organisms, and, while traditional methods are efficient at cloning together many genes of a single individual, population geneticists rather need to clone the same locus in many individuals. Our method consists of marking individual sequences during the polymerase chain reaction (PCR) using 5′‐tailed primers with small polynucleotide tags. PCR products are mixed together before the cloning reaction and clones are sequenced with universal plasmid primers. The individual from which a sequence comes from is identified by the tag sequences upstream of each initial primer. We called our protocol mark–recapture (MR) cloning. We present results from 57 experiments of MR cloning conducted in four distinct laboratories using nuclear loci of various lengths in different invertebrate species. Rate of capture (proportion of individuals for which one or more sequences were retrieved) and multiple capture (proportion of individuals for which two or more sequences were retrieved) empirically obtained are described. We estimated that MR cloning allowed reducing costs by up to 70% when compared to conventional individual‐based cloning. However, we recommend to adjust the mark:recapture ratio in order to obtain multiple sequences from the same individual and circumvent inherent technical artefacts of PCR, cloning and sequencing. We argue that MR cloning is a valid and reliable high‐throughput method, providing the number of sequences exceeds the number of individuals initially amplified.     

Isolation of 10 tetranucleotide microsatellite loci in the blackcap (Sylvia atricapilla)

We isolated 10 polymorphic microsatellite DNA loci from the blackcap (Sylvia atricapilla) and optimized them for future studies of population differentiation in populations with different migration strategies in southwestern Germany. The loci were screened for polymorphism using 178 individuals from two populations in Germany and Spain. The primers amplified highly variable loci characterized by two to 19 alleles per locus and their observed and expected heterozygosities range from 0.47 to 0.81 and from 0.50 to 0.91, respectively.     

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.     

Isolation and characterization of polymorphic microsatellite loci from Tetratheca ericifolia (Elaeocarpaceae)

We identified 11 informative microsatellite loci in Tetratheca ericifolia from an (AG)_n‐enriched library. Using these loci on 32 individuals from two populations (16 individuals from each), we detected an average of 11.3 alleles per locus (range of five to 21, average expected heterozygosity of 0.728), of which 56% were unique to one population or the other. All loci were amplifiable in seven to 12 of a further 12 species of Tetratheca under the same reaction conditions. The markers will be useful tools for evolutionary studies of this Australian endemic group.     

Cross-species amplification, non-invasive genotyping, and non-Mendelian inheritance of human STRPs in Savannah baboons

Twenty-nine human microsatellite primer pairs were screened for their utility in the cross-species amplification of baboon DNA derived from both blood and feces as part of a larger study to identify paternal half sisters in a population of wild baboons (Papio cynocephalus). Forty-one percent (12/29) of the human primers successfully amplified baboon DNA. Of these 12 primers, six amplified fragments that were both polymorphic and heterozygous (mean number of alleles = 6, mean heterozygosity = 87%) and yielded repeatable results. However, only five of these six simple tandem repeat polymorphisms (STRPs) showed patterns of Mendelian inheritance (i.e., mothers and offspring shared at least one allele at each locus), and were therefore useful for determining relatedness between individuals. Analysis of the sixth primer revealed non-Mendelian inheritance, i.e., three of the six known mother-daughter pairs had no shared alleles. This failure was probably due to non-specific fragment amplification, and may have resulted from a different STRP locus being amplified in mother and daughter. This finding highlights the importance of sampling DNA from known parent-offspring pairs when screening microsatellite primers for genetic studies. Multiple, independent replications of genotypes and Mendelian checks are both particularly important when using cross-species amplification or when using a low-quality source of DNA.     

Characterization of microsatellite markers for the tanoak tree,Lithocarpus densiflorus

We describe the development of 19 primers for amplification of microsatellite loci for tanoak, Lithocarpus densiflorus (Hook. & Arn.). A population from Soquel State Demonstration Forest, Santa Cruz County, California (n = 20), and eight individuals from four additional coastal populations and one interior (Sierra Nevada) population were used to investigate polymorphism. The total number of alleles per locus ranged from two to 10. Observed heterozygosity in the Soquel population ranged from 0.05 to 0.70, with two loci being fixed.