Isolation and characterization of microsatellites in a medicinal plant,Phyllanthus emblica (Euphorbiaceae)

• Premise of the study: Microsatellite markers were isolated and characterized in a medicinal plant, Phyllanthus emblica, to study population genetics for designing an effective in situ and ex situ conservation of genetic resources of the species. • Methods and Results: Six microsatellite markers were developed using an enrichment and magnetic separation protocol. They were characterized in two natural populations of P. emblica. Out of the six microsatellites, five showed polymorphism, with the number of alleles ranging from four to seven. Observed and expected heterozygosities ranged from 0.360 to 0.760 and 0.499 to 0.806, respectively. • Conclusions: The five polymorphic microsatellite markers will be useful for studying the genetic structure, reproductive biology, and for identification of clones and provenances of this important medicinal plant.     

Development of microsatellite markers for the endangered medicinal plant Launaea arborescens (Asteraceae)

• Premise of the study: Microsatellite loci were developed in Launaea arborescens, an endangered and medicinal Asteraceae species in North Africa, for further investigation of its conservation genetics. • Methods and Results: We isolated and characterized 10 polymorphic and nine monomorphic microsatellite loci from L. arborescens using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). For the 10 polymorphic loci, the number of alleles detected per locus varied from two to six, and the observed and expected heterozygosities ranged from 0.000 to 0.833 and 0.059 to 0.713, respectively. • Conclusions: The polymorphic markers provide a useful tool for conservation genetics studies of L. arborescens, including analysis of mating system, estimating gene flow, and identifying discrete genetic units within the species.     

Characterization of 42 polymorphic microsatellite loci in Mimulus ringens (Phrymaceae) using Illumina sequencing

• Premise of the study: Microsatellite markers were isolated and characterized in Mimulus ringens (Phrymaceae), a herbaceous wetland perennial, to facilitate studies of mating patterns and population genetic structure. • Methods and Results: A total of 42 polymorphic loci were identified from a sample of 24 individuals from a single population in Ohio, USA. The number of alleles per locus ranged from two to nine, and median observed heterozygosity was 0.435. • Conclusions: This large number of polymorphic loci will enable researchers to quantify male fitness, patterns of multiple paternity, selfing, and biparental inbreeding in large natural populations of this species. These markers will also permit detailed study of fine-scale patterns of genetic structure.     

Isolation and characterization of microsatellite loci in a polyploid alpine herb,Callianthemum miyabeanum (Ranunculaceae)

• Premise of the study: Nuclear microsatellite primers were developed to analyze the clonal diversity and population genetic structure of the endemic polyploid herb Callianthemum miyabeanum. • Methods and Results: Using a protocol for constructing microsatellite-enriched libraries, 15 primer sets were developed for use in C. miyabeanum. The number of alleles found ranged from five to 22. The estimated range of expected heterozygosities was 0.574 to 0.907, and the Shannon–Weiner diversity index ranged from 1.061 to 2.733. Cross-amplification of all loci was also successful in the closely related endemic species C. kirigishiense and C. hondoense. • Conclusions: The development of these microsatellite loci will facilitate a deeper understanding of the genetic diversity, mode of reproduction, and population structure of not only C. miyabeanum, but also the other Callianthemum species endemic to Japan.     

Identification and characterization of 43 novel polymorphic EST-SSR markers for arum lily,Zantedeschia aethiopica (Araceae)

• Premise of the study: A new set of microsatellite or simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) was developed for arum lily (Zantedeschia aethiopica), which is one of the most iconic and widely recognized ornamental plants in the world. • Methods and Results: Using 2175 unigenes derived from 4283 random ESTs in arum lily, 166 primer pairs were designed and tested for amplification in 24 accessions from Asia, Europe, and Africa. A total of 43 loci were polymorphic, with the number of alleles per locus ranging from two to 10. The observed heterozygosity, expected heterozygosity, and polymorphism information content ranged from 0.2313 to 0.8480, 0.3034 to 0.8648, and 0.1015 to 0.7364, respectively. • Conclusions: These novel polymorphic EST-SSR markers will facilitate future studies of genetic variation and molecular-assisted breeding systems in arum lily.     

Isolation and characterization of polymorphic microsatellites in Iris ensata (Iridaceae)

• Premise of the study: Microsatellite primers were developed in Iris ensata (Iridaceae) to provide polymorphic markers for further studies into population genetics. • Methods and Results: Thirteen polymorphic microsatellite loci were isolated from I. ensata. These loci were successfully amplified in two natural populations of I. ensata from eastern China (Longwangshan, Zhejiang Province) and northeastern China (Jinchuan, Jilin Province). There was no significant linkage disequilibrium found for any pair of loci. These loci contained between two and 12 alleles per locus across all 48 individuals of I. ensata. The number of alleles per locus varied from two to 10 at the population level and the observed and expected heterozygosities ranged from 0.167 to 0.958 and from 0.284 to 0.853, respectively. • Conclusions: These loci showed high levels of polymorphism and could be used to study the population genetic structure, genetic relationships, and phylogeography of I. ensata.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

Development of microsatellite markers in species of Erigeron (Asteraceae) endemic to the Juan Fernández Archipelago,Chile

• Premise of the study: Microsatellite markers were developed in Erigeron rupicola and tested by amplification in six Erigeron species endemic to the Juan Fernández Archipelago, Chile, to investigate genetic diversity and population structure. • Methods and Results: Using 454 pyrosequencing, 24 primer pairs were developed in E. rupicola, 12 of which amplified and presented polymorphism among endemic species of Erigeron in the Archipelago. Two populations from E. rupicola and E. fernandezianus were genotyped, and one to eight alleles per locus per population were detected. The expected heterozygosity ranged from 0.000 to 0.812. • Conclusions: These results indicate the utility of primers for cross-species populational studies in all endemic species of Erigeron in the Archipelago.     

Isolation of microsatellite DNA and analysis on genetic diversity of endangered fish,Hucho taimen (Pallas)

Hucho taimen (Pallas) is an endangered species in China. To evaluate the species’ genetic diversity and population genetic structure, we isolated six polymorphic microsatellite markers from its genomic libraries by (ACA)₁₆ enriched, and characterized using 61 wild individuals. The number of alleles per locus ranged from two to seven, expected heterozygosity ranged from 0.3200 to 0.7410, polymorphism information content ranged from 0.3047 to 0.6896. These markers will be useful for the genetic variation assessment of taimen.     

A set of plastid microsatellite loci for the genus Dyckia (Bromeliaceae) derived from 454 pyrosequencing

• Premise of the study: Phylogeographical analyses of Dyckia (Bromeliaceae) suffer from low levels of sequence variation. Plastid microsatellite markers were developed to achieve a better-resolved genus-wide plastid genealogy of Dyckia. • Methods and Results: Approximately 84% of the D. marnier-lapostollei plastome was sequenced using 454 technology. Flanking primer pairs were designed for 34 out of 36 chloroplast simple sequence repeats (cpSSRs) detected, and 12 loci were further characterized by genotyping Dyckia samples at the level of populations and species. Three, five, and six cpSSRs were polymorphic among four individuals of D. limae, 12 individuals of D. dissitiflora, and 12 of D. pernambucana, respectively, with two to three alleles per locus and species. All loci were polymorphic among 19 different Dyckia species, with three to 10 alleles per locus. Ten primer pairs cross-amplified with bromeliad genera from five subfamilies. • Conclusions: The set of 12 cpSSR markers provides a toolbox to analyze phylogeographical patterns of Dyckia species.     

Microsatellite markers for Kleinia neriifolia,an endemic Asteraceae species on the Canary Islands

• Premise of the study: Microsatellite makers were developed in Kleinia neriifolia, an endemic Asteraceae species on the Canary Islands, for investigation of the population genetic structure and colonization history of this insular species. • Methods and Results: Seventeen polymorphic and seven monomorphic microsatellite loci were isolated and characterized from K. neriifolia using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). At the population level, the number of alleles detected per locus varied from two to 16, and the observed and expected heterozygosities were 0.105–1.000 and 0.100–0.923, respectively. • Conclusions: These highly informative loci are potentially useful to obtain a deeper understanding of the evolutionary process of K. neriifolia.     

Plastome sequences of two New World bamboos—Arundinaria gigantea and Cryptochloa strictiflora (Poaceae)—extend phylogenomic understanding of Bambusoideae

• Premise of the study: Two New World species of Bambusoideae, Arundinaria gigantea and Crytpochloa strictiflora, were investigated in a phylogenomic context. Complete plastome sequences have been previously determined and analyzed for nine bambusoid species that exclusively represent Old World lineages. The addition of New World species provides more complete information on relationships within Bambusoideae. • Methods: Plastomes from A. gigantea and C. strictiflora were sequenced using Sanger methods. Phylogenomic and divergence estimate analyses were conducted on both species with 23 other Poaceae. • Key Results: Phylogenomic and divergence analyses suggested that A. gigantea diverged from within Arundinarieae between 1.94–3.92 mya and that C. strictiflora diverged as the sister to tropical woody species between 24.83 and 40.22 mya. These results are correlated with modern relative diversities in the two lineages. • Conclusions: The two New World bamboos show unique plastome features accumulated and maintained in biogeographic isolation from Old World taxa. The overall evidence for A. gigantea is consistent with recent dispersal, and that for C. strictiflora is consistent with vicariance.     

Characterisation of polymorphic microsatellite markers for skates (Elasmobranchii: Rajidae) from expressed sequence tags

Simple sequence repeat markers (SSRs, microsatellites) were characterised for skates (Elasmobranchii: Rajidae) from published expressed sequence tags (ESTs) of Leucoraja erinacea. These were tested in four European species (Raja clavata, Raja montagui, Dipturus batis, and Leucoraja naevus). Thirteen of the fourteen amplified loci were polymorphic in at least one species. Polymorphic loci possessed on average 4.5–5.9 alleles per species, and expected heterozygosity ranged from 0.05 to 0.88. Possible null alleles were detected at three loci, while one locus showed significant deviation from Hardy–Weinberg Equilibrium proportions. Three locus-pairs exhibited significant linkage disequilibrium in one or more species. This marker set will be valuable for population genetic analyses of the focal taxa, and may prove useful for studies of other skate species.     

Development of STMS markers from the medicinal plant Madagascar periwinkle [Catharanthus roseus (L.) G. Don.]

We report the isolation and characterization of the first set of sequence‐tagged microsatellites sites (STMS) markers in Catharanthus roseus, a plant with a vast range of medicinal uses. The microsatellite loci were cloned from an enriched library constructed using degenerate primers. Based on the microsatellite motifs, seven STMS primer pairs were designed. They were used to amplify 32 accessions of C. roseus and one accession of Catharanthus trichophyllus. The primers amplified an average of 3.86 alleles per locus. The observed heterozygosity ranged from 0.2903 to 0.9688 with an average of 0.7511. The STMS markers of C. roseus also amplified corresponding loci in a related species (C. trichophyllus) suggesting conservation of the loci across the genus. These markers will prove useful for genetic diversity analysis and linkage map construction in C. roseus.     

Homoploid hybrid speciation in a rare endemic Castilleja from Idaho (Castilleja christii,Orobanchaceae)

• Premise of the study: Hybridization is an important evolutionary force in the history of angiosperms; however, there are few examples of stabilized species derived through homoploid hybrid speciation. Homoploid hybrid species are generally detected via the presence of genetic additivity of parental markers, novel ecological and spatial distinctions, and novel morphological traits, all of which may aid in the successful establishment of hybrid species from parental types. Speciation and diversification within the genus Castilleja (Orobanchaceae) has been attributed to high levels of hybridization and polyploidy, though currently there are no examples of homoploid hybrid speciation within the genus. We employed multiple lines of evidence to examine a putative hybrid origin in C. christii, a rare endemic, known only from 80 hectares at the summit of Mt. Harrison (Cassia Co., Idaho). • Methods: We used granule-bound starch synthase II (waxy) sequences and 26 morphological characters to address hybridization between C. christii and widespread congeners C. miniata and/or C. linariifolia in an area of sympatry. Chromosomes of C. christii were also counted for the first time. • Key results: All 230 direct-sequenced C. christii individuals had the additive genomes of both C. miniata and C. linariifolia. Castilleja christii shares traits with both parents but also has floral characters that are unique and transgressive. Cytological counts indicated that all three taxa are diploid. • Conclusions: We conclude that C. christii is a stabilized homoploid hybrid derivative of C. linariifolia and C. miniata and is likely following an independent evolutionary trajectory from its progenitors.     

Isolation and characterization of microsatellite DNA loci from Russell's snapper (Lutjanus russellii)

A total of 37 microsatellite loci from the Russell's snapper, Lutjanus russellii, were successfully isolated and characterized. Thirty‐four loci were polymorphic in L. russellii samples. Twenty of the 37 markers did not differ significantly from Hardy–Weinberg expected genotype proportions. Significant linkage disequilibrium was detected in one pairwise comparison. The numbers of alleles and observed heterozygosities in polymorphic loci ranged from two to 16 and from 0.41 to 0.95, respectively. These markers will be useful for studying the population genetic structure of this species.     

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.     

Development and characterization of microsatellites in Vanda varieties

The orchid genus Vanda includes more than 70 monopodial species and numerous hybrids. The aim of this study was to develop microsatellite markers for this horticultural valuable genus. Thirteen polymorphic microsatellite markers were isolated from the variety Vanda Miss Joaquim and were characterized in four Vanda species, 11 Vanda hybrids, and one Aranda intergenus hybrid. Populations from three species were also analysed. Number of alleles ranged from two to 19. For the V. sumatrana population, the expected heterozygosity ranged from 0 to 0.76 (mean 0.31) and the observed heterozygosity ranged from 0 to 1 (mean 0.36). All the varieties tested were distinct from one another (similarity index < 0.8). These microsatellites could be used for studying genetic diversity and population structure of wild populations within the orchid genus Vanda, as well as for distinguishing cultured Vanda varieties.     

Development of 12 polymorphic microsatellite markers in <Emphasis Type="Italic">Coilia ectenes</Emphasis> Jordan and Seale, 1905 (Clupeiformes: Engraulidae) and cross-species amplification in <Emphasis Type="Italic">Coilia mystus</Emphasis> Linnaeus, 1758

In this study, twelve polymorphic microsatellite markers were developed for Coilia ectenes (synonym C. nasus). In a sample of 30 C. ectenes individuals in a Wuhan population from Yangtze River, a total of 78 alleles were detected, and the number of alleles per locus ranged from 3 to 12. The observed and expected heterozygosities ranged from 0.100 to 1.000 and from 0.098 to 0.899, respectively. Six loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.004), and no significant linkage disequilibrium between pairs of loci was found. The applicability of these markers in a closely related species, C. mystus, was evaluated by cross-species amplification. These microsatellite markers will be useful in studies on population genetics, conservation genetics, and fishery management and in the construction of genetic linkage maps of C. ectenes and C. mystus.     

Isolation and characterization of microsatellite loci in the highland papaya Vasconcellea × heilbornii V. Badillo (Caricaceae)

Nine polymorphic microsatellite loci were identified in the tropical plant Vasconcellea ×heilbornii and used to estimate allelic diversity in two populations of southern Ecuador. Allelic richness ranged from two to five alleles, observed heterozygosity ranged from 0.150 to 0.947 and expected heterozygosity ranged from 0.186 to 0.701. Most of these markers also amplified microsatellite loci in two other Vasconcellea species (Vasconcellea stipulata and Vasconcellea cundinamarcensis). Hence, these markers will be useful for population genetic analysis and the evaluation of genetic diversity and gene flow in these species.