Development of EST-microsatellites from the cycad <Emphasis Type="Italic">Cycas rumphii</Emphasis>, and their use in the recently endangered <Emphasis Type="Italic">Cycas micronesica</Emphasis>

We developed a set of 12 EST-microsatellite markers (EST-STRs) from Cycas rumphii Miq. and tested them on an ex situ collection of the endangered Cycas micronesica K.D. Hill. The number of alleles per locus in both species ranged from 1 to 15. Observed and expected heterozygosities ranged from 0.000 to 0.842, and 0.000 to 0.883, respectively. All primers amplified in four additional Cycas species. These markers are the first genome-enabled tools for cycad population level studies, and are now available to inform conservation efforts and disentangle the biogeographic history of the genus.     

Isolation and characterisation of di and tri nucleotide microsatellite loci in Rosmarinus officinalis (Lamiaceae), using enriched genomic libraries

An enrichment protocol was used to isolate and characterise microsatellite loci in Rosmarinus officinalis, a Mediterranean chamephyte. Twelve microsatellite loci were characterised and amplified a total of 117 alleles in a sample of 30 individuals from one population, with an average of 9.75 alleles per locus. Observed heterozygosities ranged from 0.333 to 0.900. Cross-species transferability was also assayed in the two other species of the genus. The cumulated probabilities of exclusion for paternity and parentage of the 12 loci were of 0.999971 and 1, respectively, supporting the usefulness of these microsatellite loci for parentage analyses. Nine out of 12 microsatellite loci amplified in the two species and were polymorphic detecting a total of 49 and 45 in R. eriocalyx and R. tomentosus, respectively. Twenty-two alleles were exclusive of R. eriocalyx and 12 of R. tomentosus, additionally, three alleles were shared between these two species but were otherwise absent in the analysed individuals of R. officinalis. In total, this set of markers amplified 154 different microsatellite alleles, supporting their usefulness to conduct population genetic, reproductive biology and hybridisation studies in Rosmarinus.     

A set of microsatellite markers for use in the endangered sea urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis> developed from <Emphasis Type="Italic">S. purpuratus</Emphasis> ESTs

The first set of nine microsatellite markers for the endangered sea urchin Strongylocentrotus nudus was developed from EST databases of S. purpuratus. Number of alleles per locus ranged from two to thirteen. The observed and expected heterozygosities ranged from 0.000 to 0.645 and from 0.063 to 0.912, respectively. These informative microsatellite markers will be useful in studies of population genetic structure for this species.     

Microsatellite markers for assessing DNA polymorphism of Emericella nidulans in nature

Based on genome sequence of Emericella nidulans, primers for 25 microsatellites were designed, of which, seven were chosen as polymorphic markers (five to 12 alleles). Simple sequence repeat (SSR) polymorphism was tested in 44 isolates from extremely arid soils of the southern Negev desert, Israel and some related species of Aspergillus. The SSR marker set appears suitable for population studies in diverse ecological conditions.     

Development and characterization of microsatellite markers from the yellow passion fruit (Passiflora edulis f. flavicarpa)

Here we described the development of the first set of Passiflora microsatellite loci isolated from an enriched genomic library. A sample of 43 individuals from 12 accessions of the yellow passion fruit was used to characterize those loci, which revealed up to 20 alleles per locus. Two loci were monomorphic. The observed (H_O) and expected (H_E) heterozygosities were very similar, as expected for a self‐incompatible species. Allelic diversity (H_T) was 0.444. This set of markers will permit genetic structure analyses of cultivated and wild populations of Passiflora, and contribute for integrating genetic maps based on dominant markers, as they can provide bridge alleles.     

Characterization of 22 microsatellites loci from the endangered Houbara bustard (Chlamydotis undulata undulata)

Here we present a new set of 22 microsatellite loci isolated from Chlamydotis undulata undulata, an endangered Houbara bustard found across North Africa. The number of alleles per locus ranged from one to nine, and heterozygosities ranged from 0.167 to 0.944. Total exclusionary probabilities using these loci for the first and the second parent were 0.992932 and 0.999915, respectively. Successful cross‐amplification was observed in eight other Otididae species (12–22 of the 22 loci). These microsatellite markers are powerful tools for genetic identification, paternity assignment and population genetic studies.     

Novel microsatellite loci in the threatened European long-snouted seahorse (<Emphasis Type="Italic">Hippocampus guttulatus</Emphasis>) for genetic diversity and parentage analysis

The long-snouted seahorse Hippocampus guttulatus is one of the two European seahorse species. We describe the isolation of the first 12 microsatellite loci in this threatened species. These new markers were tested in non-invasive samples of 32 seahorses from NW Spain. The number of alleles ranged from 2 to 15 (mean: 6.3) and expected heterozygosity from 0.031 to 0.912 (mean: 0.500). All loci conformed to Hardy–Weinberg expectations and no genotypic disequilibrium was observed between any pair of loci. The theoretical exclusion probabilities for this set of loci, when no parental information exists or when one parent is known, were 0.973 and 0.998, respectively. This study indicates the usefulness of these novel loci for population analysis and kinship studies in Hippocampus guttulatus. Their potential application is extended to the other European seahorse species, since all loci were successfully cross-amplified in H. hippocampus.     

Characterization of five microsatellite loci in the Pine Processionary Moth Thaumetopoea pityocampa (Lepidoptera Notodontidae Thaumetopoeinae)

Five microsatellite markers were developed for the lepidopteran species Thaumetopoea pityocampa using an enrichment protocol. All loci could be amplified with no evidence of null alleles and will be useful for population genetic studies. The number of alleles ranged from three to 12 for a population of 30 individuals. Observed heterozygosities ranged from 0.53 to 0.80. No significant heterozygote deficiency was detected. Four markers might be of interest for Th. wilkinsoni.     

Characterization of seven polymorphic microsatellites for the study of two Ranunculaceae: Ranunculus nodiflorus L., a rare endangered species and Ranunculus flammula L., a common closely related species

Ranunculus nodiflorus and Ranunculus flammula are two closely related Ranunculaceae. In France, the first one is rare and endangered, whereas the second is common. To compare their population genetic structure, seven polymorphic microsatellite markers were characterized. We found no polymorphism in R. nodiflorus, whereas R. flammula was polymorphic at each locus. The total number of alleles per locus varied from three to eight. Observed heterozygosities ranged from 0 to 0.261. This set of microsatellites offers an efficient tool to study population genetic structure and to compare the results between closely related endangered and common species.     

Isolation and characterization of microsatellite loci in the threatened wild toad lily Tricyrtis flava (Liliaceae)

Twelve polymorphic microsatellite loci were isolated and characterized from the threatened toad lily Tricyrtis flava. The number of alleles ranged from three to 17, and the observed and expected heterozygosities ranged from 0.35 to 0.96 and from 0.50 to 0.93, respectively, in a population of T. flava. We also tested cross‐species amplifications of the markers in three other species of Trycyrtis sect. Flavae. Most of 12 markers developed for T. flava were successfully amplified in the species of sect. Flavae and will be useful for population genetic studies of these species.     

Isolation and characterization of microsatellite loci in the Japanese pipistrelle (Pipistrellus abramus)

We describe the first set of ten microsatellite markers isolated in Pipistrellus abramus. The number of alleles per locus ranged from 7 to 13. The observed and expected heterozygosities values ranged from 0.486 to 0.971 and from 0.752 to 0.876, respectively. Three loci revealed significant departure from Hardy–Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These informative microsatellite markers will be a powerful molecular tool for studying the population genetic structure of P. abramus, as well as other species of this genus.     

Twenty new microsatellite loci for population structure and parentage studies of Parnassius apollo nevadensis (Lepidoptera; Papilionidae)

We characterized 20 new microsatellite loci isolated from Parnassius apollo nevadensis. The 20 loci displayed 2–12 alleles and expected heterozygosities varied from 0.22 to 0.89; the markers were amplified in nine multiplex reactions. The combined non-exclusion probabilities of this set are 0.001084 and 0.000005 for the first and second parents, respectively. This set of markers will thus enable other studies on reproductive strategies and population genetics in this endangered and previously understudied species. We show, for the first time, evidence of multiple paternity and that females may successfully copulate with more than one male despite the presence of mating plugs. Future studies on this mating behavior and population genetics of the species using the 20 loci here presented will be very important to define its conservation status and management measures.     

Development of 12 novel microsatellite loci in a traditional Chinese medicinal plant, Epimedium brevicornum and cross-amplification in other related taxa

A total of 12 polymorphic microsatellite loci were developed and characterized for a Chinese medicinal plant, Epimedium brevicornum (Berberidaceae). A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 38 individuals from one natural population. The observed number of alleles ranged from 2–14. The ranges of observed and expected heterozygosity were 0.00–0.83 and 0.15–0.88, respectively. In addition, successful cross-species amplification of this set of microsatellite markers in other four medicinal Epimedium species suggested that they would provide a useful tool for the genetic and conservation studies of Epimedium species.     

Assessing the genetic diversity in Argopecten nucleus (Bivalvia: Pectinidae), a functional hermaphrodite species with extremely low population density and self‐fertilization: Effect of null alleles

Argopecten nucleus is a functional hermaphroditic pectinid species that exhibits self‐fertilization, whose natural populations have usually very low densities. In the present study, the genetic diversity of a wild population from Neguanje Bay, Santa Marta (Colombia), was estimated using microsatellite markers, and the effect of the presence of null alleles on this estimation was assessed. A total of 8 microsatellite markers were developed, the first described for this species, and their amplification conditions were standardized. They were used to determine the genotype of 48 wild individuals from Naguanje Bay, and 1,010 individuals derived from the offspring of 38 directed crosses. For each locus, the frequencies of the identified alleles, including null alleles, were estimated using the statistical package Micro‐Checker, and the parental genotypes were confirmed using segregation analysis. Three to 8 alleles per locus with frequencies from 0.001 to 0.632 were detected. The frequencies of null alleles ranged from 0.10 to 0.45, with Ho from 0.0 to 0.79, and He from 0.53 to 0.80. All loci were in H‐W disequilibrium. The null allele frequencies values were high, with lower estimations using segregation analysis than estimated using Micro‐Checker. The present results show high levels of population genetic diversity and indicate that null alleles were not the only cause of deviation from H‐W equilibrium in all loci, suggesting that the wild population under study presents signs of inbreeding and Wahlund effect.     

Development and diversity of microsatellite markers for endangered species Clinostigma savoryanum

A set of microsatellite markers was developed for Clinostigma savoryanum, an endemic palm species distributed in the Bonin Islands. We obtained 233 sequences that were unique, containing microsatellites from an enriched library. Twelve loci were screened for their feasibility to be used as high resolution genetic markers using each 30 individuals from two insular populations, Haha-jima and Mukou-jima. They showed polymorphisms of two to eight alleles per locus and expected heterozygosities of 0.124–0.789. There is no evidence for significant scoring error due to stuttering, large allele dropout and null alleles at 95% confidence interval except for the presence of null alleles in CLS00-77 of Mukou-jima population.     

Isolation and characterization of highly polymorphic microsatellite loci for the garden tiger moth Arctia caja (Lepidoptera: Arctiidae)

A set of microsatellite markers was isolated from Arctia caja, a species that has been declining in UK for several decades. As has been found with other Lepidoptera species, this proved to be a difficult task. Eight enriched libraries identified 103 positive clones, and these yielded only seven polymorphic microsatellites. Allelic diversity ranged from 10 to 23 alleles per locus in a population of 30 individuals. Significant heterozygosity deficits were shown by three of these loci, presumably due to null alleles. The remaining four loci were in Hardy–Weinberg equilibrium and will be used to investigate the population genetic structure of A. caja.     

Characterization of microsatellite loci in fulvous fruit bat Rousettus leschenaulti

Rousettus leschenaulti is an abundant species in many countries of South‐East Asia, including south China. We isolated seven microsatellite loci in R. leschenaulti from genomic DNA enriched for CA repeats with the enriched library method. A total of 56 samples from a population in the Guangxi Province of China were tested with these microsatellite markers. The polymorphism ranged from seven to 16 alleles, and the observed heterozygosity was 84–94%. It is the first time microsatellite markers were characterized from R. leschenaulti, and these markers can be an important tool for analysing population structure and genotypic diversity.     

Isolation and characterization of 13 microsatellite loci in the pen shell Atrina pectinata (Bivalvia: Pinnidae)

The pen shell Atrina pectinata is a commercially important and over-exploited bivalve. In this survey, the first set of 13 polymorphic microsatellite loci for the pen shell was developed and characterized. The number of alleles ranged from 6 to 28 per locus with the observed and expected heterozygosities ranging from 0.640 to 1.000 and from 0.731 to 0.970, respectively. These informative microsatellite markers will be useful in studies of population and conservation genetics for this species.     

Development and characterization of microsatellite markers for <Emphasis Type="Italic">Camellia nitidissima</Emphasis>

Camellia nitidissima Chi (Theaceae), a rare and famous plant with golden-yellow flowers, narrowly distributes in Southwest China and North Vienam. It is now endangered and its distribution is ongoing shrinked due to deforestation and collection of seeds for horticulture. From repetitive DNA enriched libraries, we reported here the development and characterization of ten microsatellite markers for this species. The number of alleles ranged from 5 to 12. Observed and expected heterozygosities ranged from 0.4583 to 0.9474, and 0.6976 to 0.9033, respectively. These ten polymorphic microsatellite loci is the first set of microsatellite markers for C. nitidissima, and they will be used for future study of conservation genetics of this species.     

Isolation and characterization of polymorphic microsatellite loci from the pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae)

Thirteen polymorphic microsatellite markers were developed from an enriched partial genomic library for the pink bollworm, Pectinophora gossypiella (Saunders), one of the most important cotton pests in the world. The total number of alleles ranged from two to 12 for a sample of 50 individuals and the expected heterozygosity at these loci ranged from 0.042 to 0.830. Although the presence of null alleles in some loci is suspected, these polymorphic markers are likely to provide useful tools for the population genetic studies of this species.