Development of microsatellite markers in the common fig,Ficus carica L.

We present a new set of 15 polymorphic microsatellite primer sequences developed from Ficus carica L. The variability of specific microsatellite regions was assessed in wild population of figs from the northern Adriatic coast and all 15 primer pairs showed single‐locus amplification with a total of 65 alleles and an observed heterozygosity ranging from 0.285 to 0.863. The 15 new microsatellite loci represent a significant tool for population genetic structure studies and will be further used to investigate the origin and maintenance of genetic variation within and between populations of figs along the Adriatic coastal region.     

Isolation and characterization of microsatellite loci from the endangered highlands scrub hypericum (Hypericum cumulicola)

We report the isolation of 19 primer pairs for amplification of polymorphic microsatellite loci for Hypericum cumulicola. These markers were evaluated in 24 individuals from one population; two to four alleles were detected per locus, and observed heterozygosity ranged from 0 to 0.5. Two loci demonstrated significant heterozygote deficiencies, possibly due to null alleles, and significant linkage disequilibrium was found between six pairs of loci. The remaining microsatellite loci will help determine if genetic differentiation is responsible for life‐history differences between natural and anthropogenically disturbed populations of H. cumulicola.     

A search for null alleles at the microsatellite locus of chum salmon (<Emphasis Type="Italic">Oncorhynchus keta</Emphasis> Walbaum)

Population studies with the use of microsatellite markers face a problem of null alleles, i.e., the absence of a PCR product, caused by the mutations in the microsatellite flanking regions, which serve as the sites of primer hybridization. In this case, the microsatellite primer associated with such mutation is not amplified, leading to false homozygosity in heterozygous individuals. This, in turn, results in biased population genetic estimates, including the excess of homozygotes at microsatellite loci. Analysis of the population structure of a Pacific salmon species, chum salmon (Oncorhynchus keta Walbaum), revealed the presence of null alleles at the Oke3 microsatellite locus in the population samples, in which an excess of homozygotes was observed. The analysis was performed using different combinations of modified primers chosen to match the Oke3 locus. The use of these primers enabled identification of true heterozygotes among those individuals, which were previously diagnosed as homozygotes with the use of standard primers. Removal of null alleles eliminated the excess homozygotes in the chum salmon samples described. In addition to the exclusion of false homozygosity, the use of modified primers makes it possible to introduce polymorphic primer variants associated with certain microsatellite alleles into population studies.     

Development and characterization of polymorphic microsatellites from Prunus avium‘Napoleon’

Primers were developed for 21 microsatellite loci isolated by enrichment from Prunus avium‘Napoleon’. Twelve loci contained uninterrupted dinucleotide repeats and nine were more complex. Nineteen primer pairs (EMPA001–019) showed single locus polymorphisms in a cultivar survey of 14 sweet cherries, with two to seven alleles per locus. Three primer pairs in combination (EMPA014, 015 and 018) discriminated all cultivars. Two primer pairs for loci monomorphic in P. avium were included: EMPA020 revealed segregation in an interspecific progeny and EMPA021 revealed polymorphism in P. dulcis. Twelve primer pairs reliably amplified products in three peach cultivars of which seven revealed polymorphisms.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Isolation and characterization of microsatellite loci from the orchid Serapias vomeracea (Orchidaceae) and cross‐priming to other Serapias species

In this paper we describe the isolation and characterization of six polymorphic microsatellite loci from the orchid Serapias vomeracea. This species is widely distributed in the Mediterranean region. Microsatellite loci were isolated from an enriched library and primer pairs were designed for 18 loci. Primer pairs for six loci amplified well and were tested on samples from southern Italy. Levels of genetic variability detected at these six loci are high, with numbers of alleles per locus ranging from 3 to 6, and observed heterozygosity (H_O) ranging from 0.35 to 0.86. All primer pairs tested amplified DNA from four other Serapias species, indicating that the primers are useful for population genetic studies throughout the genus.     

Development of new microsatellite primers for green and white sturgeon

Sixty-eight primer sets for microsatellite loci were developed from microsatellite motif enriched genomic libraries of pooled DNA from the polyploid green and white sturgeon (Acipenser medirostris and A. transmontanus). Four individuals from each species were screened for polymorphism at these loci. Forty-eight loci amplified in both species, and some exhibited species-specific amplification for white or green sturgeon (8 and 12 loci, respectively). The number of alleles per locus ranged from one to 12. At least 68% of the green and 65% of the white sturgeon loci we developed are polysomic.     

Eleven polymorphic microsatellite loci in Lindera benzoin,Lauraceae

Microsatellite markers were developed for studies of the genetic diversity and population substructure of Lindera benzoin, Lauraceae (spicebush). Nuclear microsatellite sequences were obtained from DNA libraries that were enriched for (CA), (GA), (AAG) and (ATG) repeat motifs. From 69 microsatellite sequences, 20 primer sets were developed. Of these, 11 primer pairs resulted in amplified polymorphic loci. In 29 samples of eastern Pennsylvania spicebush plants, the number of microsatellite alleles ranged from two to 16 per locus with observed heterozygosity values ranging from 0.10 to 0.82.     

Isolation and characterization of Ligustrum micranthum (Oleaceae) microsatellite loci using paired‐end Illumina reads

Twenty‐six microsatellite loci were developed and characterized for Ligustrum micranthum, a species endemic to the Ogasawara Islands, Japan. The genetic structure of this species must be clarified in order to restore the island's ecosystem. A total of 8511 primer pairs were designed from de novo sequencing. Of the 48 primer pairs selected, amplification and polymorphisms were tested using one population each from the Chichijima and Hahajima Islands of the Ogasawara Islands. Twenty‐six microsatellite loci were successfully amplified and the number of alleles for these loci ranged from five to 31 per locus, and the mean expected heterozygosities were 0.858 and 0.849, respectively. No significant deviation from the Hardy–Weinberg equilibrium was observed in either population, and no significant linkage disequilibrium was detected between any locus pair. The microsatellite loci reported in this study can be used in future studies to evaluate the genetic structure and mating system of L. micranthum.     

Isolation and characterization of microsatellite loci from yellowtail Seriola quinqueradiata and cross‐species amplification within the genus Seriola

We developed five microsatellite primer pairs for the yellowtail Seriola quinqueradiata. The loci were highly polymorphic, with eight to 14 alleles per locus, and can be used to study kinship and/or population structure. Many of these primer pairs amplified polymorphic loci in cross‐species amplification tests for two other Seriola species (S. lalandi and S. dumerili).     

Isolation and characterization of microsatellite DNA loci from the golden cuttlefish,Sepia esculenta Hoyle (Cephalopoda)

The first microsatellite markers were isolated from the golden cuttlefish, Sepia esculenta Hoyle. Eleven primer sets were designed to amplify the marker sequences via polymerase chain reaction. The 45–50 individuals from one wild population in the coastal waters of Ehime Prefecture, Japan were used to screen polymorphism in the 11 microsatellite loci. All the microsatellite loci were polymorphic, with the range of alleles from seven to 27 per locus. The observed and expected heterozygosities ranged from 0.380 to 0.980 and from 0.654 to 0.940, respectively. These marker loci except for one locus showing significant deviation from Hardy–Weinberg equilibrium will be useful for the assessment of genetic variation and population structure of this species.     

New microsatellite loci for the European bushcricket,Ephippiger ephippiger (Orthoptera: Tettigoniidae)

Ephippiger ephippiger is a model organism for studies of sexual selection and phylogeography but little is known about fine‐scale population structure. Available microsatellite loci have null allele problems so we used an enrichment technique to isolate 21 new microsatellite loci for E. ephippiger. We present primer pairs for 10 polymorphic loci (3–11 alleles per locus). Observed heterozygosities at polymorphic loci ranged from 0.118 to 0.787, but several were significantly lower than expected.     

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.     

Characterization of 24 microsatellite loci in delta smelt, Hypomesus transpacificus, and their cross-species amplification in two other smelt species of the Osmeridae family

We characterized 24 polymorphic tetranucleotide microsatellite loci for delta smelt (Hypomesus transpacificus) endemic to the San Francisco Bay Estuary, CA, USA. Screening of samples (n = 30) yielded two to 26 alleles per locus with observed levels of heterozygosity ranging from 0.17 to 1.0. Only one locus deviated from Hardy–Weinberg equilibrium, suggesting these individuals originate from a single panmictic population. Linkage disequilibrium was found in two pairs of loci after excluding the locus out of Hardy–Weinberg equilibrium. Twenty‐two primer pairs cross‐amplified in wakasagi smelt (Hypomesus nipponensis), and 15 primer pairs cross‐amplified in longfin smelt (Spirinchus thaleichthys).     

Isolation and characterization of polymorphic microsatellite DNA makers for Euphrasia nankotaizanensis (Orobanchaceae) and cross amplification in another Euphrasia L.

Euphrasia nankotaizanensis is an endangered flowering plant distributed restrictedly on rocky slope of high mountain peaks in central and northern Taiwan. In order to undertake a conservation program, especially given impacts of the global warming, it is essential to evaluate its genetic diversity and population structure. We described nine novel microsatellite primer pairs in E. nankotaizanensis and also examined its relative E. transmorrisonensis. The number of alleles per locus ranged from 7 to 29. Expected (H _E) and observed (H _O) heterozygosities ranged from 0.83 to 0.98 and 0.00 to 0.95, respectively. Eight of the nine microsatellite loci displayed significant departures from Hardy–Weinberg expectations, likely due to the loss of habitats and the small population size. Significant linkage disequilibrium (LD) was detected in most loci. Cross-species amplification of microsatellites took place at five loci. These primers may provide a tool for understanding the demography and population structure of Euphrasia species in Taiwan. Kuo-Hsiung Wang, Ming-Jou Wu, and Tzen-Yuh Chiang contributed equally to this work.     

Isolation and characterization of 20 polymorphic microsatellite loci for Scaptodrosophila hibisci

Scaptodrosophila hibisci is an endemic Australian Drosophilidae that breeds in the flowers of native Hibiscus . Here we report the isolation and amplification of 20 polymorphic microsatellite loci . We cloned these microsatellites because loci developed for Drosophila melanogaster failed to amplify in S. hibisci . Null alleles were detected at six loci, and five were X‐linked. Two of the primer pairs amplified an unlinked ‘bonus’ locus. One locus containing juxtaposed microsatellite loci was suitable for designing an additional set of primers. Mean number of alleles per locus was 10, mean H _O and H _E per locus were 0.532 and 0.636, respectively.     

Isolation and characterization of microsatellite markers in Indian neem (Azadirachta indica var. indica A. Juss) and cross-amplification in Thai neem (A. indica var. siamensis Valenton)

We developed eight polymorphic microsatellite loci in Indian neem (Azadirachta indica var. indica) and cross-amplified in closely related species Thai neem (A. indica var. siamensis). The number of alleles per locus in Indian neem and Thai neem ranged from 3 to 9 and 3 to 9, respectively. Average observed and expected heterozygosities in Indian neem and Thai neem were 0.63 and 0.70 and 0.61 and 0.65, respectively. Two loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium. These microsatellites may provide a useful tool for population genetics to establish conservation and management strategy.     

Polymorphic microsatellite markers from the formicine ant Lasius (Dendrolasius) fuliginosus

Six polymorphic microsatellite loci were isolated and characterized from the temporary social parasitic ant Lasius fuliginosus by a highly efficient enrichment procedure. Observed allele numbers ranged from three to 20 per locus, whereby four out of the six tested loci had more than 10 alleles and showed both observed and expected heterozygosities greater than 70%. For each locus we present suitable primer sequences. With these microsatellite markers we will be able to reveal colony and population structure of L. fuliginosus.     

Polymorphic tetranucleotide microsatellite markers in the Caribbean spiny lobster,Panulirus argus

Ten tetranucleotide microsatellite loci are described for the Caribbean spiny lobster Panulirus argus. Loci were polymorphic (4–15 alleles per locus) and exhibited high levels of expected (0.553–0.921) and observed heterozygosity (0.469–0.906) from samples caught off Belize and Puerto Rico coasts. No significant departure from Hardy–Weinberg equilibrium conditions were observed for any locus. All microsatellite loci should be useful for assessing population discrimination for this valuable marine animal currently subjected to excessive fishing efforts.     

Identification and characterization of microsatellite loci in Betula maximowicziana Regel

The sequences of eight primer pairs of microsatellite loci screened from a genomic library of Betula maximowicziana are reported in this paper. Seven loci showed polymorphism in 44 individuals of the Mitomi population in the central area of Japan and the observed and expected heterozygosities ranged from 0.045 to 0.659 (mean = 0.412) and from 0.044 to 0.642 (mean = 0.397), respectively. Although the one remaining locus is not polymorphic there, it showed variation in other populations. Thus, these eight microsatellite markers might be useful tools for studying the population and ecological genetics of B. maximowicziana.