Cross‐species amplification of microsatellite loci in Aquilegia and Semiaquilegia (Ranunculaceae)

We developed 16 microsatellite loci from an F₂ hybrid between Aquilegia formosa and Aquilegia pubescens. In samples of 28 individuals, we found an average of 14 alleles per locus from each parental species. We tested these loci for cross‐amplification in 10 additional species of Aquilegia and found that all 16 loci amplified in other North American species and 12 consistently amplified in European or Asian species. Nine loci amplified in the sister species to Aquilegia, Semiaquilegia adoxoides. The success of cross‐species amplification suggests that these microsatellites should prove useful for studies in a broad range of Aquilegia species.     

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     

Primers from the orders Osteoglossiform and Siluriform detect polymorphic microsatellite loci in sun-catfish, Horabagrus brachysoma (Teleostei: Bagridae)

Horabagrus brachysoma (sun‐catfish, Bagridae, Siluriformes) is a valuable ornamental and food fish. The stock structure of H. brachysoma, necessary to conserve its declining natural populations, is not known. Twenty‐five primers developed for four fish species belonging to the orders Siluriform (3) and Osteoglossiform (1) were tested and eight primers amplified microsatellite loci in H. brachysoma. The results demonstrate that cross‐priming between fish species belonging to different families and even to different orders can yield microsatellite loci. Five of eight primers each amplified two loci. However, the loci that had repeat motifs after sequencing were considered only for genotyping. Finally, eight loci were polymorphic with hree to seven alleles. Individual fish genotype data (n = 42; 21 each in two rivers) at each locus was analysed. Significant genetic heterogeneity was detected at six loci. The identified loci exhibited potential for use in population genetics application in H. brachysoma.     

Polymorphic microsatellite loci in Eurytoma brunniventris,a generalist parasitoid in oak cynipid galls

Eurytoma brunniventris is a parasitic wasp with a wide range of host species. We have developed primers for nine polymorphic microsatellite loci to allow examination of intraspecific population subdivision associated with two aspects of the biology of their hosts: host species (oak gallwasps) and the tree on which the gallwasp develops (different oak species). All nine loci amplified well across individuals collected from a range of gallwasp species and across two oak taxa. These microsatellite loci are potentially of value in the study of closely related economic pests such as seed predators of almonds (E. amygdali) and pistachios (E. plotnikovi).     

Isolation and characterization of polymorphic microsatellite DNA markers in the brown sole,Pleuronectes herzensteini

New microsatellite DNA markers from brown sole were developed and characterized. Fourteen primer sets were designed from 40 microsatellite regions. Eight of 14 loci exhibited variations comprising 8–31 alleles. Observed and expected heterozygosities ranged from 0.611 to 0.833 and from 0.647 to 0.968 among 36 individuals, respectively. Phz3, Phz8 and Phz12 significantly deviated from Hardy–Weinberg equilibrium, and there was a significant linkage disequilibrium between Phz2 and Phz12. Seven of eight loci conformed to the Mendelian manner of inheritance in a full‐sib family. Seven to four loci of three related species were cross‐amplified by primers for brown sole.     

Development of microsatellite markers for muskellunge (Esox masquinongy) and cross‐species amplification in two other esocids

We report the development of seven polymorphic microsatellite loci in muskellunge (Esox masquinongy) using an unenriched subgenomic library. Polymorphic loci exhibited 2–11 alleles with observed heterozygosities ranging from 0.190 to 0.917 (n = 24). All seven loci amplified by their respective primer pairs resulted in monomorphic products in northern pike (E. lucius) whereas three loci amplified monomorphic products in grass pickerel (E. americanus americanus); these results imply conservation of flanking sequence but loss of polymorphism between these closely related species. Only one of six microsatellite primers developed in a previous study in northern pike amplified polymorphic products in muskellunge.     

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 for <Emphasis Type="Italic">Viola websteri</Emphasis> (Violaceae) and Cross-Species Amplification within the Genus <Emphasis Type="Italic">Viola</Emphasis>

We isolated and characterized microsatellite loci in Viola websteri (Violaceae), an endangered species from Korea and endemic to Northeast Asia. A total of 27 microsatellite loci were developed and tested in Korean and Chinese populations. The number of alleles per locus varied from two to eight. The observed and expected heterozygosities within two populations were 0.000 to 1.000 and 0.080 to 0.816, respectively. Korean and Chinese populations were clearly distinguished by the private alleles from 16 loci. A total of 21 loci out of the 27 developed loci were successfully cross-amplified in 39 other Viola species. We believe that these microsatellite loci will be useful for future studies on genetic diversity and population structure of V. websteri, as well as other Viola species.     

Cross-species amplification of 44 microsatellite loci developed for Chaetodon trifascialis, C. lunulatus and C. vagabundus in 22 related butterflyfish species

Forty‐four microsatellite primers developed for three species of butterflyfish were cross‐tested against 22 related confamilial species. Amplification success and cross‐species transferability of these markers were moderately high. Between 24 and 37 loci were amplified successfully in each species, with a mean success rate per species of 71.7% (± 1.8 SE). Rates of amplification success were comparable among primers designed for the three source species, ranging from a mean success rate per species of 16.9 loci (± 0.8 SE) for Chaetodon trifascialis source loci to 13.7 loci (± 1.5 SE) for C. vagabundus source loci. Polymorphism rates were high (76.1%± 3.1 SE of all successfully amplified loci), and 10 loci were polymorphic in all successfully amplified species (Tri14, B11, C5, D3, D113, D6, D117, D120, D111, D118). The number of alleles per polymorphic locus ranged from 2 to 8, and the average number of alleles across all polymorphic loci and all species was 3.6 (± 0.07 SE). Polymorphism rates were higher overall in primers designed for C. vagabundus (89.9%± 3.9 SE). Overall cross‐testing success was lowest for Heniochus chrysostomus, the most phylogenetically divergent species. The significant cross‐testing reported here provides a valuable resource that will enable population genetics studies to be undertaken on a range of butterflyfishes without the need for expensive and time‐consuming de novo microsatellite development.     

Isolation and characterization of microsatellite markers for Viola mirabilis (Violaceae) using a next‐generation sequencing platform

We isolated and characterized microsatellite loci in Viola mirabilis (Violaceae), an endangered species from South Korea. Twenty‐three polymorphic microsatellite loci were developed and tested in Korean, Chinese and Japanese populations. The number of alleles per locus varied from two to eight. The observed and expected heterozygosities within the three populations were 0.000–0.625 and 0.469–0.695, respectively. A total of six loci in the Korean population, one locus in the Chinese population and seven loci in the Japanese population deviated from Hardy–Weinberg equilibrium. We expect that these newly developed microsatellite markers will contribute to understanding the phylogeography and population genetics of V. mirabilis, which will aid in developing conservation strategies for this species.     

Trinucleotide microsatellite loci for the zebra mussel Dreissena polymorpha,an invasive species in Europe and North America

The high mutation rate at microsatellite loci can supply important demographic information on founder events and range expansion in an invasive species such as the zebra mussel Dreissena polymorpha, following its initial introduction. In order to facilitate studies into the colonization patterns of this species in new habitats in Europe and North America, five trinucleotide microsatellite loci were isolated from a partial DNA library. Allelic diversity at all described loci was high, ranging from 20 to 35 alleles per locus. Homologous loci were not amplified in a second related invasive species, Dreisenna bugensis, using the primers developed here.     

Characterization of 12 polymorphic microsatellite loci in the Japanese bush warbler Cettia diphone

Japanese bush warblers, Cettia diphone, are a common species in Japan and have a polygynous breeding system. In the breeding ground some males have their own territories with more than one female. Male floaters are also not uncommon in the breeding ground. To understand breeding strategy in this species, exact parentage should be elucidated. In order to obtain a tool for this purpose, we isolated 34 microsatellite loci from a genomic library in this species and developed primers for 12 loci. These primers were tested in the Japanese bush warbler and successfully amplified. In analyses of 49 unrelated individuals, allelic numbers ranged from two to 22, and observed heterozygosity (H_O) ranged from 0.27 to 0.854 except for two loci (Cdi29 and Cdi35a) with H_O < 0.1.     

Development of highly conserved primers for 12 new polymorphic microsatellite loci for the genus Rorippa Scop. (Brassicaceae), yellow‐cress

We isolated 12 highly conserved polymorphic microsatellite loci for the yellow‐cress species Rorippa amphibia and Rorippa sylvestris. We used a partial genomic library enriched for several repeat motifs. Obtained sequences containing repetitive elements were blasted and aligned with the Arabidopsis thaliana sequence. We evaluated the cross‐species compatibility of primers designed from sequences either aligning strongly or weakly with A. thaliana. The former proved much more efficient in obtaining primers that worked in both species. The developed conserved primers for microsatellite loci provide excellent markers for studying segregation, gene flow and hybridization in the genus Rorippa.     

Reproductive ecology of Viola mirabilis var. subglabra representing intermediate flowering characteristics between stemless and stemmed Viola

Viola (Violaceae) is one of the largest genera in angiosperms. This genus is essentially classified into stemless and stemmed groups based on growth morphology. However, Viola mirabilis var. subglabra is an exception in having intermediate flowering characteristics; cleistogamous (CL) flowers are formed in the axils of stem leaves, whereas chasmogamous (CH) flowers arise from basal rosettes (radical CH (CH(r)) flowers) and also in the axils of the stem (axially CH (CH(a)) flowers). To understand why the pattern of flower production varies in this Viola species, flower production was investigated in 10 Japanese populations from Hokkaido to the western part of Honshu in 2014 and 2015. Furthermore, flower characteristics were also compared between CH(r) and CH(a) flowers in Hokkaido. In this species, the production of CH flowers varied among individuals, and they were categorized into three groups, individuals that produced (i) only CH(r) flowers, (ii) only CH(a) flowers and (iii) both CH flowers. The frequency of these groups differed among populations, but some individuals changed the category between 2014 and 2015. Thus, the production of CH(r) and CH(a) flowers plastically changes depending on individual conditions and/or environmental factors. On the other hand, CH(r) and CH(a) flowers differed in flower size and flowering phenology. These results suggest that two types of CH flowers may play different roles in reproduction in each population, but fruit sets and seed sets did not differ between two types of CH flowers.     

Isolation and characterization of microsatellite markers in Labiotermes labralis (Isoptera,Termitidae, Nasutitermitinae)

We report here on the development of six polymorphic microsatellite loci for Labiotermes labralis. This soil‐feeding species is restricted to the Neotropical rainforest and then could represent a major candidate for being a bio‐indicator of anthropic disturbance resulting in the forest fragmentation. The microsatellite loci were isolated and characterized using a microsatellite‐enriched genomic library. The primers were tested on a French Guyanese population of L. labralis, represented by the sampling of one soldier in 22 nests. The primers were also tested against nine species of the same Nasutitermitinae subfamily and were successfully amplified at five loci in Armitermes minutus.     

Characterization of microsatellite markers in Fagus sylvatica L. and Fagus orientalis Lipsky

Using an enrichment procedure, we cloned microsatellite repeats from European beech (Fagus sylvatica L.) and developed primers for the amplification of microsatellite markers. Six polymorphic loci were characterized which produced 3–21 alleles in 70 individuals from one Italian population, with an observed heterozygosity between 0.58 and 0.85. All six loci amplified fragments which were polymorphic in the closely related species, Fagus orientalis, also. Owing to their very high degree of variation, these markers should be very useful in gene flow studies of these species.     

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.     

Isolation and characterization of five microsatellite loci in the ponerine ant Pachycondyla inversa (Hymenoptera,Formicidae)

We characterized five polymorphic DNA microsatellite loci for the neotropical ponerine ant Pachycondyla inversa. The variability was initially tested in 19 workers from nine colonies from a Brazilian population. We found five to 12 alleles per locus, with observed heterozygosities between 0.72 and 0.95. The allele size ranged from 73 to 197 bp. The primers also successfully amplified DNA at all five loci in the closely related species P. villosa.     

Large scale development of selectively amplified microsatellite polymorphic loci (SAMPL) markers in spruce (Picea)

The spruce (Picea) species are ecologically and economically important in Canada. Highly informative markers with high multiplex ratios are needed to assist spruce genomics, genetics, and breeding programs. Selectively amplified microsatellite polymorphic loci (SAMPL) markers are highly suitable for these programs. We have developed, optimized, and characterized a set of 10 new SAMPL primers in combination with 16 MseI primers and resolved a large number of polymorphic SAMPL markers in spruce. The SAMPL primers were designed from the compound microsatellite repeats found in Norway spruce (Picea abies) and white spruce (Picea glauca). A total of 6313 polymorphic SAMPL makers were produced by 160 SAMPL–MseI primers combinations in eight progeny of a spruce mapping population.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Melicope quadrilocularis</Emphasis> (Rutaceae), an endemic plant species of the Bonin Islands,Japan, and cross-species amplification in closely related taxa

Twelve microsatellite loci were isolated and characterized for Melicope quadrilocularis, an insular endemic tree species of the Bonin Islands. The observed number of alleles at each locus ranged from 1 to 18. The range of expected heterozygosity was 0.0000–0.9445. The inter-specific applicability of these loci was evaluated by analyzing two other endemic species and one endemic variety of Melicope that are also distributed on the Bonin Islands. All primer pairs for the 12 loci tested successfully amplified the loci in all taxa, except for primers for four loci in M. nishimurae.