QDD version 3.1: a user‐friendly computer program for microsatellite selection and primer design revisited: experimental validation of variables determining genotyping success rate

Microsatellite marker development has been greatly simplified by the use of high‐throughput sequencing followed by in silico microsatellite detection and primer design. However, the selection of markers designed by the existing pipelines depends either on arbitrary criteria, or older studies on PCR success. Based on wet laboratory experiments, we have identified the following factors that are most likely to influence genotyping success rate: alignment score between the primers and the amplicon; the distance between primers and microsatellites; the length of the PCR product; target region complexity and the number of reads underlying the sequence. The QDD pipeline has been modified to include these most pertinent factors in the output to help the selection of markers. Furthermore, new features are also included in the present version: (i) not only raw sequencing reads are accepted as input, but also contigs, allowing the analysis of assembled high‐coverage data; (ii) input data can be both in fasta and fastq format to facilitate the use of Illumina and IonTorrent reads; (iii) A comparison to known transposable elements allows their detection; (iv) A contamination check can be carried out by BLASTing potential markers against the nucleotide (nt) database of NCBI; (v) QDD3 is now also available imbedded into a virtual machine making installation easier and operating system independent. It can be used both on command‐line version as well as integrated into a Galaxy server, providing a user‐friendly interface, as well as the possibility to utilize a large variety of NGS tools.     

Twelve microsatellite loci for lake trout (Salvelinus namaycush)

We describe 12 microsatellite loci isolated from lake trout (Salvelinus namaycush). The number of alleles at these loci ranged from two to 11 with an average of 5.3 alleles per locus. The expected heterozygosity ranged from 0.29 to 0.76, with an average of 0.68. Accidental (or illegal) introductions of lake trout into watersheds are decimating native trout populations in the northern Rocky Mountains, and these loci will be useful for identifying the source of these introductions and for estimating the number of founding individuals.     

Isolation and characterization of 13 polymorphic microsatellite loci from the clam shrimp Eulimnadia texana (Crustacea: Spinicaudata)

Thirteen polymorphic microsatellite loci were isolated and characterized from the clam shrimp Eulimnadia texana. In analyses of 20–50 individuals from two populations the number of alleles ranged from two to seven with observed heterozygosity ranging between 0.00 and 0.37. The low values for heterozygosity were not unexpected for a group characterized by its unusual androdioecious mating system, in which males compete with self‐compatible hermaphrodites for offspring production. These microsatellites are likely to be useful for further evolutionary investigations of this rare mating system in these crustaceans.     

Ten polymorphic microsatellite loci for the endangered Buena Vista Lake shrew (Sorex ornatus relictus)

The ornate shrew (Sorex ornatus) is restricted to the vanishing wetlands of California, USA and Baja California, Mexico. Several subspecies of ornate shrews are considered ‘mammal species of special concern’ in California by the Department of Fish and Game, and one (Sorex ornatus relictus) has recently been listed as endangered. Populations of shrews around Buena Vista Lake have been diminished or extirpated due to habitat deterioration and human development. In order to study the patterns of genetic variation in isolated populations of Buena Vista Lake shrews, we developed 10 polymorphic microsatellite loci. There were 6–27 alleles per locus, and the loci had heterozygosity values that ranged from 20 to 80%. In addition, we screened 20 different populations of S. ornatus, eight species within two subfamilies of shrews (Soricinae and Crocidurinae), as well as in a mole (Talpidae, Neurotrichus gibbsii), to determine if these loci could be informative in other species as well.     

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.     

Characterization of microsatellite loci in the wall lizard Podarcis muralis (Sauria: Lacertidae)

In natural populations the mating success of males depends on different factors. By enhancing the intensity of secondary sexual characters, testosterone can play a role in mate choice. However, paradoxically, testosterone can also decrease immunocompetence and thus potentially diminish attractiveness. To estimate the influence of testosterone on male mating success in the wall lizard, Podarcis muralis, we characterized nine polymorphic microsatellite loci. There were three to 12 alleles per locus in the five to 16 individuals screened. These microsatellites will also be useful in determining population structure in this species.     

Characterization of eight polymorphic microsatellite loci from the Bengalese finch (Lonchura striata var. domestica)

Eight polymorphic microsatellite loci were isolated and characterized from the Bengalese finch (Lonchura striata var. domestica). In analyses of 25 individuals, the number of alleles ranged from two to four, and observed heterozygosity ranged between 0.05 and 0.73. At four loci, the observed heterozygosity of the Bengalese finches was significantly different from the expected heterozygosity. Primer sets were also tested in Javan munia (Lonchura leucogastroides), and the same eight loci were successfully amplified. In analyses of 20 unrelated individuals, the number of alleles ranged from one to seven, and the observed heterozygosity ranged from 0 to 0.56. In Javan munia, the observed heterozygosity differed significantly from the expected heterozygosity in only one locus.     

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.     

Polymorphic microsatellite loci for studies of bronze‐cuckoo species (Genus Chalcites: Aves)

A set of 17 microsatellite loci was shown to provide at least seven that were polymorphic in each of three bronze‐cuckoo species (Chalcites basalis, C. lucidus and C. minutillus) representing the taxonomic range of this genus. This set includes nine newly isolated loci from genomic libraries constructed from C. basalis and C. lucidus. For these three species, each had seven or more polymorphic loci that showed no significant linkage or Hardy–Weinberg disequilibrium with more than five alleles (mean 7.6–12.1) and expected heterozygosity greater than 0.5 (mean 0.78–0.85).     

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).     

Isolation and characterization of eight microsatellite loci in the mangrove mud‐nesting ant,Polyrhachis sokolova

We isolated and characterized eight anonymous microsatellite loci in the mangrove mud‐nesting ant, Polyrhachis sokolova. Three to 18 alleles were detected in 20 female workers collected from individual colonies at one location and observed heterozygosities ranged from 0.45 to 0.85. These microsatellite loci will be useful in studies on mating strategy, colony and population structure in P. sokolova.     

Species‐specific microsatellite loci for the European squid (Loligo vulgaris)

A microsatellite dinucleotide‐enriched library was obtained from the European squid (Loligo vulgaris) and five species‐specific dinucleotide markers were optimized. These markers are highly polymorphic with average expected heterozygosity ranging from 0.706 to 0.927 and allele number ranging from 7 to 17. This set of primers is suitable for population genetic studies.     

Nine new tetranucleotide microsatellite markers for the fire‐bellied toad (Bombina bombina)

We describe nine new polymorphic tetranucleotide microsatellite loci isolated from the fire‐bellied toad (Bombina bombina). The relative yield of new loci was higher than described in previous studies in amphibians: out of 12 loci initially evaluated, nine were polymorphic and amplifying reliably. Number of alleles ranged from four to 10 and observed heterozygosities from 0.47 to 0.91. Seven loci were polymorphic also in Bombina variegata and five in Bombina orientalis. Enrichment protocols yielding long flanking regions potentially overcome difficulties (i.e, low yield of reliable loci relative to number of clones screened) which have been reported in microsatellite development in anurans.     

Isolation of dinucleotide microsatellite loci from red‐backed salamander (Plethodon cinereus)

We isolated 13 variable dinucleotide microsatellites from red‐backed salamanders (Plethodon cinereus). After generating fragments using degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR), AC repeats were captured using biotinylated probes and streptavidin‐coated magnetic particles. Captured fragments were cloned into plasmids, screened for microsatellites with a simple PCR reaction, and select plasmids then sequenced. PCR primers were designed and optimized for robust amplification, and nine primers have been further optimized for multiplex reactions with fluorescent primers. These nine loci are variable with an average of 6.11 alleles per locus and an average heterozygosity of 0.61.     

Characterization of microsatellite loci in Lychnis flos‐cuculi (Caryophyllaceae)

We cloned microsatellite repeats from ragged robin Lychnis flos‐cuculi (Caryophyllaceae) and developed 20 primer pairs for microsatellite marker analysis. We used 18 individuals of a large Swiss population to screen microsatellites. Seven loci were polymorphic. Between seven and 11 alleles were found per locus and the observed heterozygosity was between 0.308 and 0.813. Observed heterozygote deficits were significant in five of the seven loci, in line with the mixed mating system of L. flos‐cuculi.     

M‐AFLP‐based protocol for microsatellite loci isolation in Cynara cardunculus L. (Asteraceae)

Nine microsatellite markers for Cynara cardunculus L. were developed using a two‐step ‘primer extension’ procedure, based on microsatellite‐amplified fragment length polymorphism (M‐AFLP) technique. In the first step, highly enriched SSR gel profiles were produced and, from the derived sequences of selected bands, forward primers directed towards the microsatellite motif were designed. In the second step, the opposite microsatellite flanking sequence was isolated using a nested approach on a restricted‐ligated genomic fraction. Polymorphism was explored in 24 plants of wild cardoon (Cynara cardunculus L. var. sylvestris) as well as two accessions of both globe artichoke (Cynara cardunculus L. var. scolymus), and cultivated cardoon (Cynara cardunculus L. var. altilis).     

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.     

Development of microsatellite markers in the grey‐crowned babbler (Pomatostomus temporalis)

The grey‐crowned babbler (Pomatostomus temporalis) is a cooperative breeding bird species in which nonbreeding helpers of both sexes care for the young of breeding individuals. To measure the genetic relatedness between breeders and their offspring and helpers, we developed nine microsatellite markers. Most of the loci were highly polymorphic. These loci will be useful in understanding the evolution and maintenance of cooperative breeding and helping behaviour in this 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).     

Polymerase chain reaction primers for polymorphic microsatellite loci from the túngara frog Physalaemus pustulosus

We developed eight PCR?primer pairs of polymorphic microsatellite loci for the túngara frog Physalaemus pustulosus. Genomic libraries were enriched for one of four microsatellite repeat sequences (CA_n, GA_n, ATG_n and TAGA_n). Following characterization of microsatellite loci by sequencing, primers were designed and PCR conditions optimized. Microsatellite PCR‐amplification was tested in 37 frogs from 8 populations in Costa Rica and Panama. Primer sequences, PCR conditions, allelelic diversities and observed as well as expected heterozygosities in the screened populations are described.