Isolation of six microsatellite markers from the pea aphid,Acyrthosiphon pisum (Homoptera,Aphididae)

Using the magnetic particles method, we isolated six polymorphic microsatellite loci from the pea aphid, Acyrthosiphon pisum (Harris), which feeds on a wide range of legume species. The isolated loci were polymorphic, with three to six alleles in 40 aphids. Expected heterozygosities ranged from 0.12 to 0.65. These loci can be used to quantify clonal diversity and compare genetic population structure between sexual and asexual populations.     

Microsatellite markers for Sclerotinia subarctica nom. prov., a new vegetable pathogen of the High North

Eight polymorphic microsatellite loci were isolated from the ascomycete fungus Sclerotinia subarctica nom. prov. In Alaska, this pathogen causes white mold vegetable diseases sympatrically with the cosmopolitan and closely related Sclerotinia sclerotiorum. Eighteen alleles were observed across the 41 isolates tested and ranged from two to three alleles per locus. Together, the alleles from the eight polymorphic loci yielded only four haploid multilocus genotypes and exhibited significant linkage disequilibrium, reflecting extreme selfing and clonal vegetative reproduction.     

Development and characterization of polymorphic microsatellite DNA markers for Sasa senanensis (Poaceae: Bambuseae)

Sasa senanensis is a dwarf bamboo species distributed on the floors of cool temperate forests in Japan and adjacent regions. We isolated eight polymorphic microsatellite loci from this species. The number of alleles ranged from two to eight and the observed heterozygosity per locus from 0.13 to 0.74. Seven of the eight loci were also polymorphic in Sasa nipponica. Most of these markers were successfully amplified in other dwarf bamboo species. These markers will be useful for investigating clonal structure and population genetics in some dwarf bamboo species.     

Development of microsatellite markers for the dwarf bamboo species Sasa cernua and Sasa kurilensis (Poaceae) in northern Japan

Ten polymorphic microsatellite markers were isolated from the dwarf bamboo species Sasa cernua and Sasa kurilensis. The applicability of these markers was confirmed by genotyping of open‐pollinated seeds and leaf samples from natural populations. Genotypes of seeds from each culm shared at least one allele from the maternal parent without contradiction. All 10 loci were polymorphic in S. cernua with 2–15 alleles (average H_E = 0.532). Eight loci were polymorphic in S. kurilensis with 2–10 alleles (average H_E = 0.532). These markers will be useful in detailing the extent of clonal and sexual reproduction in these species.     

Isolation of microsatellites in a parthenogenetic lizard Menetia greyii (Scincidae) and their utility in sexual species of the Menetia greyii complex

Tetra and tri‐nucleotide microsatellite loci were isolated from a parthenogenetic form of the Australian lizard Menetia greyii, using an enrichment method with polymerase chain reaction (PCR)‐based colony screening. Primers for 23 loci were designed and 11 of these loci amplified well in a panel of 10 parthenogenetic individuals from a single population. Seven loci were further characterized and six successfully amplified and were polymorphic in all five sexual species of the Menetia greyii complex they were tested in. These loci will be used to investigate the parental ancestry and clonal diversity of various parthenogenetic forms within the Menetia greyii species complex.     

Genetic analysis of salt-marsh sedge Carex scabrifolia Steud. populations using newly developed microsatellite markers

Nine microsatellite loci were isolated and characterized from the clonal salt-marsh sedge Carex scabrifolia, and genetic diversities within four populations were analyzed. The number of alleles per locus ranged from 2 to 7, with an average of 4.7. The observed and expected heterozygosities ranged from 0.000 to 1.000 and 0.000 to 0.679, respectively. In two populations, almost all polymorphic loci showed a significant excess of heterozygotes due to their high clonal reproduction and low number of genotypes (genets) composing the populations. Thus, the number of identical genets varied greatly among populations and ranged from 1 to 28, irrespective of population size (i.e., population area and ramet number). When attempting to conserve and restore clonal plants such as C. scabrifolia, it is important to preserve sufficient genetic diversity within a population, which can be assessed using genetic markers such as the simple sequence repeat markers described here.     

Genotyping the clonal structure of a gorgonian coral, Junceella juncea (Anthozoa: Octocorallia), using microsatellite loci

The identification of different clones is fundamental to the study of population structure among organisms with mixed reproductive modes such as cnidarians. However, due to the low genetic variation of coral mtDNA and contamination by zooxanthellate DNA, very few molecular markers are available for studying the clonal structure of cnidarians. Herein we used four polymorphic loci of microsatellite DNA isolated from a zooxanthellae-free octocoral, Junceella juncea, to study its clonal structure in seven populations collected from three localities in Taiwan. In total, 40 multilocus genotypes were found among 152 colonies, and the number of genotypes (clones) identified in the seven populations ranged from 2 to 16. Each of the 40 multilocus genotypes was restricted to a single population, even where adjacent populations were only 100 m distant. The ratio of observed to expected genotypic diversity (G_o:G_e) ranged from 0.217 to 0.650, and G_o showed a significant departure from G_e (p<0.05) at each site indicating that asexual fragmentation may play a major role in the maintenance of established populations. Mean relatedness (R) values showed that genotypes within reefs were more closely related than those between regions. The results indicate that microsatellites are useful for discerning the clonal structures among and within populations at different spatial scales. Electronic supplement: Unique multilocus genotypes (clones) revealed by 4 polymorphic loci for Junceella juncea colonies collected from Xiashuijui (Reefs A, B, C, Transplant, Transect), Nanwan and Shicheng     

Isolation and characterization of polymorphic microsatellite loci in Asiatic sand sedge,Carex kobomugi Ohwi (Cyperaceae)

Fourteen polymorphic microsatellite loci were isolated from Asiatic sand sedge, Carex kobomugi Ohwi (Cyperaceae), a clonal perennial herb predominant in sandy coast vegetation in Japan. Polymorphism was surveyed for 24 ramets within a single population and for eight distinct individuals from six populations. Within a population, between two and eight alleles were found per locus, and expected heterozygosity ranged from 0.041 to 0.720. Among populations, a higher level of polymorphism was observed. The microsatellite loci were shown to be useful for the study of spatial genetic structure at fine scale and genetic differentiation among populations.     

Cloning and Characterization of Microsatellite Loci in a Gorgonian Coral, Junceella juncea (Anthozoa; Octocorallia; Ellisellidae) and Its Application in Clonal Genotyping

We attempted to isolate microsatellites from a Symbiodinium-free gorgonian octocoral, Junceella juncea, using two methods, partial genomic library screening and enrichment. Among the 3856 clones screened by the partial library method, 10 possibly positive signals were found, and 3 of them could be used to design primers and amplified consistently. In contrast, only one locus isolated by the enrichment method gave reliable amplification and was useful. The results indicate that microsatellites are rare in Junceella juncea, as reported for other cnidarians. Overall, we obtained 4 polymorphic loci to test the feasibility in investigating clonal structure of J. juncea. A total of 40 multilocus genotypes were found among 152 colonies, and the number of genotypes (clones) identified at 7 reefs ranged from 2 to 16. The results of a nonmetric multidimensional scaling analysis indicated the recruitment of J. juncea populations mainly comes from self-retention. These novel microsatellite loci will provide a useful tool to study clonal structure and population genetics for J. juncea in the future.     

Characterization of microsatellite loci in the house fly,Musca domestica L. (Diptera: Muscidae)

The house fly, Musca domestica L., is a cosmopolitan species with a capacity to transmit human pathogens. Here, we report on the development of polymorphic microsatellite loci for house flies and present preliminary results from four house fly subpopulations from Manhattan, Kansas. Twenty‐four microsatellite loci were isolated and characterized using DNA enriched for repeat sequences. Forty individuals from four locations in Kansas were assayed to identify for polymorphic loci. Eight loci were polymorphic with the number of alleles ranging from three to six.     

Isolation and characterisation of polymorphic microsatellite loci in the vulnerable spectacled flying fox, Pteropus conspicillatus

The spectacled flying fox, Pteropus conspicillatus, is listed as vulnerable in Australia and is under threat from numerous impacts. Primers to amplify eight co-dominant microsatellite loci were designed for Pteropus conspicillatus, based on an enriched genomic library. Four loci were monomorphic in this species while the remaining four loci were highly polymorphic with 16–23 alleles. Two of the four monomorphic loci were found to be polymorphic in Pteropus alecto, a closely related congener. All but one of the six polymorphic loci were in Hardy Weinberg equilibrium. Additionally, six microsatellite loci isolated for Pteropus rodricensis were tested against individuals of P. conspicillatus with all loci amplifying reliably. These loci will be used to investigate population genetic structure in the vulnerable spectacled flying fox.     

Isolation and characterization of polymorphic microsatellite DNA markers in the rock bream (Oplegnathus fasciatus)

From a (GT)₁₃-enriched genomic library of Oplegnathus fasciatus, 14 polymorphic microsatellite were isolated and characterized in a test population with alleles ranging from two to nine, the observed and expected heterozygosities from 0.0000 to 1.0000, and from 0.1726 to 0.8507, respectively. Five loci deviated from the Hardy–Weinberg equilibrium, and linkage disequilibrium between two loci was significant. Two loci were also polymorphic in Pagrosomus major assessed for cross-species amplification. These polymorphic microsatellites will be useful for genetic diversity analysis and molecule-assisted breeding for O. fasciatus. Changwei Shao contributed equally.     

Highly polymorphic tetranucleotide microsatellite loci for the eastern Canary Island lizard,Gallotia atlantica

We describe 10 polymorphic tetranucleotide microsatellite loci from the eastern Canary Island lacertid lizard, Gallotia atlantica. Loci were isolated from a partial genomic library that had been enriched for AAAG repeat sequence. All loci were highly polymorphic (eight alleles or more) with observed heterozygosities from 0.75 to 1.00. At least four loci were successfully amplified and polymorphic in the Gran Canarian lacertid, Gallotia stehlini. These loci will be used to examine correlations between patterns of gene flow and recent volcanism on the island of Lanzarote.     

Genetic structure of Halotydeus destructor and Penthaleus major populations in Victoria (Acari: Penthaleidae)

The redlegged earth mite (Halotydeus destructor) and the blue oat mite (Penthaleus major) are major pests of pastures and crops in southern Australia. Reproductive modes, migration rates and levels of differentiation between populations were investigated using allozyme electrophoresis. Collections were made throughout Victoria and a sample was also obtained from Western Australia. Three enzyme loci were polymorphic in H. destructor (Mdh-1, Mdh-2 and Idh). Genotype frequencies of these loci did not differ between phenotypic males and females, providing no evidence for haplodiploidy. Allele frequencies were in Hardy-Weinberg equilibrium, indicating that H. destructor is diploid and sexual. This was confirmed via crosses between males and females. Allele frequencies differed between Victorian sites, although F statistics indicated little differentiation over all loci. A sample from Western Australia did not differ in allele frequencies from the Victorian sites. Four polymorphic loci were found in P. major (Mdh-1, Mdh-2, Idh and Gpi). Only a few multilocus genotypes occurred in a sample, indicating that P. major is parthenogenic. No male P. major were found in this study. A number of colour morphs were also identified and a genetic association between genital plate colour and clonal type was found in one population of P. major. Two different body colour morphs were associated with different clonal types.     

Characterization,multiplex conditions,and cross‐species utility of tetranucleotide microsatellite loci for Blanchard's cricket frog (Acris crepitans blanchardi)

We have isolated and characterized 17 tetranucleotide microsatellite loci for Blanchard's cricket frog (Acris crepitans blanchardi), an anuran common in the central USA. Sixteen loci were organized into four multiplex amplification reactions. These loci were highly polymorphic when screened in 55 individuals from two distant populations, with 11–48 alleles per locus (average = 24.8). Observed and expected heterozygosities ranged from 0.18 to 0.97 and from 0.17 to 0.96, respectively. Nine loci were also polymorphic in Acris crepitans crepitans, with seven polymorphic in Acris gryllus. Five loci amplified in all three taxa. These loci will be useful for population‐ and species‐level investigations of this widespread group.     

Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks

We isolated and characterized 16 microsatellite loci from the blacktip shark, Carcharhinus limbatus, and tested cross‐species amplification in 11 Carcharhinus species and five additional shark genera. Thirty‐six (1.6%) and 180 (48%) colonies were positive for dinucleotide repeat motifs from unenriched and enriched libraries, respectively. Heterozygosities of polymorphic loci ranged from 0.04 to 0.96 with two to 22 alleles per locus. Amplification products were observed at nine to 13 loci (five to 11 of which where polymorphic) in 10 Carcharhinus species. Several loci were also polymorphic in each of the additional genera examined.     

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.     

Microsatellite loci in the Japanese subterranean termite,Reticulitermes speratus

Reticulitermes termites have such a cryptic life style and complex colony structure that polymorphic microsatellite markers are desired to investigate their population and colony structures. We successfully isolated seven microsatellite loci in R. speratus , the Japanese subterranean termite, five of which were polymorphic. These polymorphic loci had 2–8 alleles per locus and their observed heterozygosities ranged from 0.059 to 0.438. These five loci were also polymorphic in R . kanmonensis , distributed sympatrically with R. speratus in the Kanmon Region, western Japan; the number of alleles per locus was 2–5, and observed heterozygosity was 0.176–0.625.     

Isolation of six microsatellite markers from apterous grasshopper Podisma kanoi (Orthoptera: Podisminae)

Using the magnetic particles method, we isolated six polymorphic microsatellite loci from an apterous grasshopper, Podisma kanoi Strozhenko, which shows conspicuous structural variation in chromosomes. The isolated loci were polymorphic, with three to 10 alleles among 20 individuals. Expected heterozygosities ranged from 0.23 to 0.90. These loci can be used to examine the history of population fragmentation and the establishment of chromosomal variation.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of seven-band grouper (Epinephelus septemfasciatus) and cross-species amplification

Seven-band grouper (Epinephelus septemfasciatus) is a commercially important fishery species. Sixty-six microsatellite loci were isolated from a dinucleotide-enriched genomic library of E. septemfasciatus. Twelve of these loci were polymorphic in a test population with alleles per locus ranging from two to five, and observed and expected heterozygosities per locus from 0.04 to 1.00 and from 0.28 to 0.76, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci after Bonferroni correction. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of E. septemfasciatus and other related species. Lili Zhao and Changwei Shao have contributed equally.