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     

Microsatellite primers for fungus‐growing ants

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus‐growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly developed primers and earlier published primers that were developed for fungus‐growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus‐growing ants, are now available for studying the population genetics and colony kin‐structure of these ants.     

Development of simple sequence repeat markers for Botryosphaeria spp. with Fusicoccum anamorphs

We report the development of eight sets of microsatellite markers for the ascomycete fungus and tree pathogen, Botryosphaeria parva. The primers were identified after cloning and sequencing of fragments amplified using simple sequence repeat (SSR) primers. Genome walking was used to determine unknown sequences on either side of new SSRs. The primers were tested and proved useful in nine other Botryosphaeria species that all have Fusicoccum anamorphs, similar to B. parva.     

Isolation and characterization of ten new polymorphic microsatellite loci in the mud crab, Scylla paramamosain

We developed polymerase chain primers for ten microsatellite loci in the mud crab, Scylla paramamosain. All markers were obtained from a (CA)₁₅ and (CT)₁₅-enrichment DNA library, and characterized in 30 individuals from one wild population. The number of alleles per locus varies between 8 and 18, and the observed and expected heterozygosities ranged from 0.6207 to 0.9333 and from 0.5886 to 0.9243, respectively. These polymorphic loci provide a valuable tool for population genetic analysis and parentage determination in this species.     

Development of micro satellite markers for a critically endangered species, Ceropegia fantastica from the Western Ghats, India

Ceropegia fantastica L. (Asclepiadaceae) is a highly endemic and endangered species in the Western Ghats of India. Fourteen microsatellite markers were developed for C. fantastica. Eight microsatellite primers screened had 2–5 alleles per locus and the observed and expected heterozygosity ranged from 0.48 to 0.83 and 0.48 to 0.62, respectively. The primers were also evaluated for their cross amplification against two related species Ceropegia hirsuta and Ceropegia oculata. The microsatellites developed for this species could be used for addressing population genetics of this endemic and critically endangered species.     

Microsatellite loci for the critically endangered Santa Cruz long-toed salamander (Ambystoma macrodactylum croceum) and other Ambystoma taxa

The imperiled Santa Cruz long-toed salamander (Ambystoma macrodactylum croceum) is a geographically isolated lineage of the long-toed salamander species complex. Recovery of this taxon requires understanding its population genetic structure, which is necessary to identify critical habitat. To assess the population structure of A. m. croceum, I developed primers for 17 microsatellite loci and evaluated their performance. Polymorphism ranged from 3 to 10 alleles per locus, with an average of 6.3 per locus. Mean observed and expected heterozygosity were similar (H _O = 0.678; H _E = 0.708), with one locus demonstrating heterozygote deficiency. The performance of these and previously identified microsatellite loci were also characterized for two other long-toed salamander lineages, as well as two other species of Ambystoma. The utility of these markers has similar and overlapping applications for conservation and population genetics studies of other Ambystoma taxa, and will provide a basis for revising the recovery plan for A. m. croceum, particularly for designating corridors among interacting breeding populations and upland areas, and identifying areas where gene flow should be restored.     

Microsatellites for three distantly related genera in the Brassicaceae

Microsatellites are important genetic markers both in population genetics and for delimitation of closely related species. However, to develop microsatellites for each target organism is expensive and time consuming. In this study, we have therefore developed 65 new microsatellite primers for the species Draba nivalis and tested cross-species and cross-genus transfer success of these primers for two other genera in the Brassicaceae; Cardamine and Smelowskia. Furthermore, 15 previously developed microsatellites were tested for amplification in these three genera. The microsatellite markers that amplify across these genera may be useful for other genera in the Brassicaceae as well.     

Microsatellite markers for the large blue butterflies Maculinea nausithous and Maculinea alcon (Lepidoptera: Lycaenidae) and their amplification in other Maculinea species

We developed microsatellite markers for Maculinea nausithous and Maculinea alcon, two of five species of endangered large blue butterflies found in Europe. Two separate microsatellite libraries were constructed. Eleven markers were developed for M. nausithous and one for M. alcon. The primers were tested on both species as well as on the three other European Maculinea species. The number of alleles per locus ranged from two to 14. These markers will be useful tools for population genetic studies of Maculinea species.     

Interspecific Utility of Microsatellites in Fish: A Case Study of (CT)n and (GT)n Markers in the Shanny Lipophrys pholis (Pisces: Blenniidae) and Their Use in Other Blennioidei

We report the development of new microsatellite markers that can be used for population analyses in the shanny Lipophrys pholis. The procedure involved the construction of a microsatellite-enriched genomic bank. Five (GT)_n and (CT)_n microsatellites have been characterized, four of which are polymorphic. The analysis of one population allowed us to verify their usefulness as markers in population studies. Moreover, interspecific amplifications have been performed using primers defined in other species to amplify Lipophrys pholis, or using the primers defined in Lipophrys pholis to amplify other species. We use these results to discuss the hypothesis that microsatellites are highly conserved in fish. Received May 13, 1999; accepted October 28, 1999.     

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.     

Ten microsatellite loci from Zamia integrifolia (Zamiaceae)

Ten microsatellite loci isolated from Zamia integrifolia are described. All 10 are polymorphic, with three to 10 alleles across 36 members of a single population from South Florida. Heterozygosities ranged from 0.139 to 0.889. Two loci depart significantly from Hardy–Weinberg equilibrium, and exhibit heterozygote deficiency. One locus pair exhibits significant linkage disequilibrium. The primers have also successfully amplified loci from Zamia portoricensis and Zamia ambliphyllidia. These loci will be utilized for population studies in the Caribbean Zamia pumila complex.     

Characterization of microsatellite markers in the mosquito Ochlerotatus caspius (Diptera: Culicidae)

Seven polymorphic microsatellite loci were developed for the mosquito species Ochlerotatus caspius, using an enriched genomic library. The number of alleles per locus varied between two and 11; the expected heterozygosity (H_E) ranged from 0.18 to 0.77. These microsatellite primers should prove useful for population genetic studies of this mosquito species.     

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.     

Tetranucleotide microsatellites for aquila and haliaeetus eagles

A unique community of four syntopic eagle species exists in north‐central Kazakhstan. Questions about behaviour and genetics in these four species would benefit from the development of microsatellite markers. We isolated eight polymorphic microsatellite repeats (AAAG)_n from the eastern imperial eagle (Aquila heliaca) genome using a hybridization enrichment technique. These loci revealed moderate diversity in a local population of eastern imperial eagles (observed heterozygosity 0.26–0.78), and were also polymorphic in steppe eagles (A. nipalensis) and white‐tailed sea eagles (Haliaeetus albicilla). These primers may be polymorphic in other species of Aquila and Haliaeetus eagles.     

Development and characterization of microsatellite loci in the endangered Tricyrtis ishiiana (Convallariaceae), a local endemic plant in Japan

Nine microsatellite loci were isolated and characterized for the endangered and tertiary relict perennial herb, Tricyrtis ishiiana. The number of alleles ranged from 2 to 33. The expected (H _E) and observed (H _O) heterozygosities were 0.207–0.944 and 0.215–0.813, respectively, from 96 individuals on one population. Five loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). These primers amplifying microsatellites in this species may provide a useful tool for population genetics to establish conservation strategy.     

Characterisation of microsatellites from Actinidia chinensis

We have identified a set of informative microsatellite markers for genome analysis in kiwifruit and related Actinidia species. A small-insert genomic library was constructed from Actinidia chinensis DNA, and screened for microsatellites. About 1.2% of the total colonies hybridised to a (GA)₈ probe, 0.4% to (GT)₈, and 0.1% to a mixture of three different trinucleotide repeat probes, (CAA)₅, (GAA)₅ and (CTA)₅. From the DNA sequences of 35 hybridising clones, 18 primer pairs were designed, and used to amplify genomic DNA from 38 individual plants, representing 30 different accessions of ten Actinidia species. The banding patterns for most of the dinucleotide repeats showed a high degree of polymorphism in the diploid and tetraploid A. chinensis, and in the hexaploid A. deliciosa (kiwifruit). Heterozygosity levels of up to 100% were found among eight diploid accessions of A. chinensis examined, and the number of different-sized bands among all the species varied from 3 to 36 for each microsatellite. One simple CT microsatellite gave 21 bands with sizes suggesting that the number of repeats ranged from 9 to 37. The highest number of bands (36) and the largest size variation (>100 bp) were observed with a complex microsatellite harbouring four different repeat motifs. The majority of primer pairs amplified bands from most of the ten Actinidia species tested. The most polymorphic primer pairs were used successfully to fingerprint a range of closely related varieties of kiwifruit (A. deliciosa).Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - VNTR variable number of tandem repeats     

Novel microsatellite loci identified from the Australian eastern small‐eyed snake (Elapidae: Rhinocephalus nigrescens) and cross species amplification in the related genus Suta

A total of 15 microsatellite primers pairs were developed for the Australian small‐eyed snake Rhinoplocephalus nigrescens. Five primers were used to screen 93 individuals of R. nigrescens and were also tested against eight species of the closely related genus Suta. Allelic diversity in R. nigrescens was high in three loci (12–27) and there was high heterozygosity (0.58–0.82). Observed heterozygosity did not deviate from Hardy–Weinberg expectations for the five loci tested. These primers will be useful in studies of population genetics and mating systems of small‐eyed snakes and related species.     

Twenty‐seven new microsatellites for the migratory Asian catfish family Pangasiidae

In this paper, we describe primers for 27 new microsatellite loci tested on five species of migratory Asian catfish: Pangasius krempfi, P. bocourti, P. conchophilus, P. pleurotaenia, and Helicophagus waandersii. These primers were developed from a (GATA)_n library created from the pooled DNA of three species of pangasiid catfish. All 27 loci are polymorphic in at least one species. Fifteen loci are polymorphic in at least three species. The primers described in this paper are thus shown to be useful in several species within the catfish family Pangasiidae, and may prove useful in additional species in future tests.     

Isolation and characterization of microsatellite markers in Koompassia malaccensis (Leguminosae), an important tropical timber species

This paper reports the isolation and characterization of 24 polymorphic microsatellite markers in an important tropical timber species, Koompassia malaccensis (Leguminosae). The primers were designed from a genomic library enriched for dinucleotide (CT) repeats and screened on 24 samples from a natural population. The number of alleles detected per locus ranged from two to 13 while the observed heterozygosity ranged from 0.042 to 1.000. Significant departure from Hardy–Weinberg equilibrium (P < 0.05) was detected in two loci. These microsatellite markers were tested across 13 timber species of the same family. The amplification success appeared to be associated with taxonomy classification at the genus but not subfamily levels.     

Twenty‐three microsatellite primer pairs for Betula pendula (Betulaceae)

This paper reports the development of microsatellite primers for a widespread birch species Betula pendula. By screening genomic libraries with (TC)₁₀ and (TG)₁₀ probes, 38 microsatellite sequences were obtained, of which 23 proved to be polymorphic. Thirty genotyped individuals had two to 20 alleles per loci and a heterozygosity range of 0.17–0.92. Cross‐species amplification tests of four primer pairs on diverse birch species showed positive results in all cases. Also, one of two loci tested on related genus Alnus gave positive result.