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.     

Development of primers to characterize the mitochondrial control region of Galápagos land and marine iguanas (Conolophus and Amblyrhynchus)

Primers were developed for the amplification and sequencing of the mitochondrial control region of Galápagos land (Conolophus) and marine (Amblyrhynchus) iguanas. Sequences were obtained for four land iguana samples from two islands and for 28 marine iguana samples from three islands. A series of 70–80 bp tandem repeats adjacent to the control region are described and preliminary quantification of intra‐ and interspecific sequence divergence is included.     

Microsatellite DNA loci in the rock bream Oplegnathus fasciatus

Nine novel microsatellite loci were isolated from Oplegnathus fasciatus by screening an enriched genomic library using nonradioactive PCR (polymerase chain reaction) techniques. All loci were found to be polymorphic with an average of 8.1 alleles per locus (range 3–15). The mean observed and expected heterozygosities were 0.71 (range 0.40–1.00) and 0.74 (range 0.50–0.90), respectively. Two loci showed significant Hardy–Weinberg disequilibrium at the P < 0.05 level. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for genetic variation monitoring of O. fasciatus.     

Molecular phylogeny of Pacific Island Colymbetinae: radiation of New Caledonian and Fijian species (Coleoptera, Dytiscidae)

We present a molecular phylogeny and taxonomic review of the Pacific island colymbetine diving beetles, focusing on the Fijian and New Caledonian faunas. Four new species are described: Rhantus monteithi and R. poellerbauerae from New Caledonia, and R. kini and R. bula from Fiji. We also describe the 3rd instar larvae of R. monteithi and R. poellerbauerae spp. nov., assigned to adults using mtDNA sequence data and discuss larval characters in the light of phylogeny. The phylogenetic hypotheses derived from both parsimony and Bayesian inference based on 3508 aligned nucleotides from a combination of mitochondrial (cox1, cob and rrnL‐tRNALeu‐Nad1) and nuclear genes (18S rRNA and H3) reveal a clade comprising R. novaecaledoniae, R. alutaceus, R. pseudopacificus, R. monteithi sp. nov. and R. poellerbauerae sp. nov., which agrees with the R. pacificus group sensu Balke (1993). Carabdytes upin was included within this clade, possibly indicating paraphyly of the genus Rhantus. Rhantus annectens, R. bacchusi, R. supranubicus, R. suturalis, R. simulans, and the Palearctic R. exsoletus, R. latitans and R. bistriatus formed a clade corresponding to the R. suturalis group sensu Balke (2001). Rhantus vitiensis, previously assigned to the R. pacificus group, was included in the R. suturalis clade. We find some support for a scenario where the Pacific was colonized out of the Northern hemisphere only during the past c. 12 million years, rejecting a Gondwanan origin of the morphologically isolated endemics. The new species are all characterized by mtDNA haplotype clusters, the degree of divergence between sister species pairs ranging from 1.3 to 7%, while R. novaecaledoniae individuals from all over New Caledonia apparently form one morphospecies, with moderate genetic diversity (up to 2.3% mtDNA divergence between populations). The sisters R. pollerbauerae sp. nov. + R. monteithi sp. nov. occur sympatrically on Mont Panié but appear ecologically segregated, while the sisters R. vitiensis + R. bula sp. nov. were encountered syntopically on Viti Levu. Comparing genetic and morphological data of Fijian Rhantus and Copelatus diving beetles, we here show that even in island radiations it is not per se possible to know if mitochondrial DNA barcoding would perform well (Rhantus: YES, Copelatus: NO). At the same time we show that fixed cutoff values, as sometimes used to discriminate between barcodes, thus species, might be meaningless. We underpin the importance of morphology for sustainable exploration of global diversity.     

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.     

Polymorphic tetranucleotide microsatellite loci in the Hwamei (Garrulax canorus canorus) (Timaliidae)

Eight polymorphic tetrarepeat (GATA)_n, microsatellite loci were isolated from a babbler, Hwamei (Garrulax canorus canorus). We evaluated the polymorphism of these microsatellite loci by genotyping 36–48 individuals from the Asian mainland. The number of alleles for each locus ranged from eight to 29. The heterozygosity was between 0.587 and 0.978. Except for one locus, genotype frequencies of these microsatellites did not significantly deviate from the Hardy–Weinberg expectation. These markers should be useful for monitoring potential hybridization between different Hwamei subspecies and provide new insights into the mating system and geographical differentiation of these birds.     

Isolation and characterization of eight polymorphic microsatellite markers in Aphanius fasciatus (Teleostei: Cyprinodontidae)

The first set of eight polymorphic microsatellites markers was successfully optimized from a partial genomic library enriched for an AC motif of the killifish Aphanius fasciatus Nardo, 1827 (Teleostei, Cyprinodontidae), a brackish‐water fish widely distributed along the coasts of the Mediterranean Sea. The developed loci proved to be polymorphic, displaying from two to six alleles per locus with expected heterozygosity ranging from 0.277 to 0.752. All loci were under Hardy–Weinberg equilibrium, except Af8 that showed significant heterozygotes deficiency, and there was no evidence of linkage associations between any pair of loci. Markers reported here will be potentially useful in monitoring of genetic population structure in this species.     

Characterization of 10 microsatellite loci in the spiny‐tailed iguana Ctenosaura hemilopha

Ten microsatellite loci were developed for the Baja California spiny‐tailed iguana Ctenosaura hemilopha, using an enriched genomic DNA library. In the Cerralvo Island population, seven loci were polymorphic and presented moderate levels of variation. Number of alleles ranged from two to eight (average 4.43), and observed heterozygosity from 0.150 to 0.857 (average 0.492). Polymorphism was detected at six loci on C. hemilopha individuals from the southern Baja California Peninsula. These markers will be useful to study familiar relationships and behaviour on the Cerralvo Island population.     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Isolation and characterization of RAPD-based markers linked to the beet cyst nematode resistance locus (Hs1 pat-1) on chromosome 1 of B. patellaris

A beet cyst nematode (BCN)-resistant telosomic addition of B. patellaris chromosome 1 in B. vulgaris was used to isolate 6 RAPD markers linked to the BCN resistance locus Hs1 ^pat-1. Southern analysis showed that the analyzed RAPD products contain either low-, middle or high-repetitive DNA. The relative positions of the random amplified polymorphic DNA (RAPD) markers and of the restriction fragment length polymorphism (RFLP) loci corresponding to the low-repetitive RAPD products were determined by deletion mapping using a panel of seven nematode-resistant B. patellaris chromosome-1 fragment additions. One RAPD marker, OPB11₈₀₀, was found to be present in two copies on the long arm telosome of B. patellaris chromosome 1. These copies are closely linked to the BCN resistance gene and flank the gene on both sides. On the basis of the nucleotide sequence of OPB11₈₀₀, sequence-tagged site (STS) primers were developed that amplify specific fragments derived from the two OPB11₈₀₀ loci. These STS markers can be used in the map-based cloning of the BCN gene, as they define start and finishing points of a chromosomal walk towards the Hs1 ^pat-1 locus. Two copies of the middle-repetitive OPX2₁₁₀₀ marker were mapped in the same interval of the deletion mapping panel as the resistance gene locus and thereby belong to the nearest markers as yet found for the BCN gene in B. patellaris.     

Polymorphic microsatellite loci isolated from the marine sponge Scopalina lophyropoda (Demospongiae: Halichondrida)

A total of seven microsatellites out of 88 isolated from a genomic library enriched for (CA)_n and (GA)_n repeats were characterized in the Mediterranean marine sponge Scopalina lophyropoda. The microsatellite motifs were large (34.81 ± 13.9 bp) and imperfect. The seven microsatellite loci were screened in 30 individuals collected from Blanes, northwestern Mediterranean. All of them were polymorphic (allele numbers and observed heterozygosities ranged from 3 to 6 and from 0.16 to 0.76, respectively). No significant linkage disequilibrium between pairs of loci and no departure from Hardy–Weinberg equilibrium were found. These markers are therefore promising for studies of the population structure of the species.     

Isolation and characterization of seven microsatellite loci in Chioglossa lusitanica (Urodela: Salamandridae)

Seven polymorphic microsatellite markers were isolated and characterized for golden‐striped salamander, Chioglossa lusitanica (Bocage 1864), a salamandrid endemic to the northwestern part of the Iberian Peninsula, from genomic libraries enriched for (GATA)₈ (GACA)₈ (ATG)₉ and (CA)₁₆. These loci were screened in 37–51 individuals from two populations. The number of alleles per locus ranged from five to 19. Heterozygosity ranged from 0.241 to 1.0. The high level of polymorphism revealed by these loci will be extremely useful for the study of population structure and evolutionary history of this species.     

Polymorphic microsatellite DNA markers for Banksia hookeriana (Proteaceae)

We developed 11 polymorphic microsatellite DNA markers for an Australian native shrub Banksia hookeriana (Proteaceae). The number of alleles per locus in 37 individuals varied from three to 17, observed and expected heterozygosities ranged from 0.297 to 0.838 and from 0.279 to 0.900, respectively. Two loci (BH‐B5 and BH‐B107) showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05), and null alleles may be present at these two loci. All loci showed independent inheritance.     

Highly polymorphic microsatellite loci from the giant mottled eel (Anguilla marmorata)

Eighteen polymorphic microsatellite loci from the giant mottled eel (Anguilla marmorata) were identified and their characteristics were described. These markers were tested in two wild populations from China (n = 20) and Australia (n = 20) respectively. The number of alleles ranged from 9 to 14 with an average of 11.28 per locus. The mean expected heterozygosity (H_E) was 0.8696 in the population from China, 0.8823 in the population from Australia. No locus deviates significantly from Hardy–Weinberg proportions (P < 0.05).     

A set of highly discriminating microsatellite loci for the Galápagos marine iguana Amblyrhynchus cristatus

We describe here the cloning of 12 (7 dinucleotide, 1 trinucleotide and 4 tetranucleotide) microsatellite loci for the Galápagos marine iguana Amblyrhynchus cristatus. When tested for individuals from five different island populations on the Galápagos archipelago, high genetic diversities (9–20 alleles per locus) and heterozygosities (0.200–0.944) were observed. All loci showed no obvious deviations from Hardy–Weinberg equilibrium. The new set of microsatellite loci was able to assign individuals reliably to their island of origin, thus being able to discriminate between residents and migrants between islands.     

Absence of phytosterol dealkylation and identification of the major ecdysteroid as makisterone A in Dysdercus fasciatus (Heteroptera,Pyrrhocoridae)

The absence of phytosterol dealkylation in the cotton stainer bug, Dysdercus fasciatus, has been established and the major ecdysteroid in the fifth-stage larvae identified. The demonstration that the free and esterified sterols in D fasciatus consisted of 95–96% sitosterol and 4–5% campesterol, a similar composition to the cottonseed diet, together with the lack of conversion of [¹⁴C]sitosterol into cholesterol, establishes that phytosterol dealkylation does not occur in this insect species. The ecdysteroid titer determined by radioimmunoassay in the fifth instar of D fasciatus shows a distinct peak at day 6, the instar lasting for 7 days. Makisterone A was purified by HPLC from insects at a time of high ecdysteroid titer and identified as a major component by both fast atom bombardment and electron impact mass spectrometry. Gas-liquid chromatography/mass spectrometry (selected ion monitoring) confirmed the occurrence of makisterone A and revealed the presence of two unidentified compounds. One of these occurs in a similar amount to makisterone A and may be 26-hydroxymakisterone A, whereas only a minute amount of the other compound, which may be 20-deoxymakisterone A, was present; further identification of the latter compounds is necessary. C₂₇ ecdysteroids (eg, ecdysone and 20-hydroxyecdysone) and C₂₉ ecdysteroids (eg, podecdysone A) were undetectable. The specificity of the enzymes of ecdysteroid biosynthesis is discussed.     

Microsatellites for tree of heaven (Ailanthus altissima)

Nine (CT)_n microsatellites were developed for tree of heaven, Ailanthus altissima, from invasive populations on the Mediterranean islands. These loci had seven to 12 alleles in 96 trees from five islands. Two loci had significant deficits of heterozygotes within islands while the other loci were in Hardy–Weinberg equilibrium, and four pairs of loci had significant linkage disequilibrium within a single island. These loci were also polymorphic in one to three individuals of the tree of heaven varieties, Ailanthus altissima erythrocarpa, Ailanthus altissima sutchuenensis and Ailanthus altissima tanakai, and the related species Ailanthus giraldii and Ailanthus vilmariniana.     

Isolation of microsatellite markers in Cannabis sativa L. (marijuana)

We have identified 15 variable microsatellite loci in Cannabis sativa. In 48 samples from five fibre crop seed accessions, we detected an average of 10 alleles per locus (range 2–28) with mean heterozygosity of 0.68 (range 0.28–0.94). Significant genetic differentiation was found between accessions (F_ST = 0.12, P < 0.001). These markers have utility for characterizing genetic diversity in cultivated and naturalized Cannabis populations.     

Characterization of four tetranucleotide and six dinucleotide microsatellite markers for use in the tropical freshwater fish Telmatherina antoniae and related species

Ten primer pairs were designed from two genomic libraries enriched for (GACA)₄ and (GACA)₇ in the sailfin silverside Telmatherina antoniae. Characterization with 57 T. antoniae individuals revealed between three and 30 alleles, with observed and expected heterozygosity values ranging from 0.47 to 0.98 and from 0.46 to 0.93, respectively. Eight of the 10 loci conformed to Hardy–Weinberg equilibrium, with no significant linkage detected between loci pairs. These microsatellite markers are intended for use in population genetic studies of T. antoniae and related fishes of the family Telmatherinidae.     

Characterization of microsatellite loci in Brachyderes rugatus,the Canary Islands pine weevil (Coleoptera: Curculionidae)

The Canary Island pine weevil Brachyderes rugatus (Wollaston) consists of four allopatric subspecies that are thought to have arisen from several historic colonization events within the archipelago. We have isolated and optimized seven microsatellite loci from Brachyderes rugatus calvus from Gran Canaria. Six of these loci are polymorphic within B. rugatus (11–22 alleles per locus; heterozygosity between 0.43 and 0.84). There is no evidence for heterozygote deficit within populations or for linkage disequilibrium between pairs of loci. These molecular markers are likely to prove useful tools for quantifying the genetic variability of bottlenecked island populations.