New microsatellite markers for Xerophyta dasylirioides (Velloziaceae), an endemic species on Malagasy inselbergs.

PREMISE: Microsatellite markers were developed for Xerophyta dasylirioides (Velloziaceae), a species endemic to the Malagasy inselbergs, to explore the impact of its island-like distribution on genetic diversity and gene flow. METHODS AND RESULTS: A total of 7110 perfect microsatellite loci were recovered by shotgun sequencing on an Illumina MiSeq platform. Primer pairs were designed for 40 arbitrarily selected loci. Fifteen primer pairs that generated distinct PCR products were used to genotype 80 individuals of X. dasylirioides from three inselberg populations. All markers were polymorphic, revealing two to 17 alleles in the overall sampling. Levels of observed and expected heterozygosity per locus ranged from zero to 1.000 and from zero to 0.850, respectively. Success rates of cross-amplification in 10 additional species of Xerophyta (X. croatii, X. decaryi, X. isaloensis, X. labatii, X. lewisiae, X. pinifolia, X. retinervis, X. setosa, X. spekei, X. tulearensis) ranged from zero to 70%. CONCLUSIONS: Fifteen newly developed microsatellite markers provide a toolkit for assessing population genetic parameters of X. dasylirioides in its unique island-like habitats.

Development of 15 nuclear microsatellite markers in Deuterocohnia (Pitcairnioideae; Bromeliaceae) using 454 pyrosequencing.

PREMISE OF THE STUDY: Microsatellite markers were developed in Deuterocohnia longipetala (Bromeliaceae) to investigate species and subspecies boundaries within the genus and the genetic diversity of natural populations. METHODS AND RESULTS: We used 454 pyrosequencing to isolate 835 microsatellite loci in D. longipetala. Of 64 loci selected for primer design, 15 were polymorphic among 23 individuals of D. longipetala and 76 individuals of the heterologous subspecies D. meziana subsp. meziana and D. meziana subsp. carmineo-viridiflora. Twelve and 13 of these loci were also polymorphic in one population each of D. brevispicata and D. seramisiana, respectively. Numbers of alleles per locus varied from two to 14 in D. longipetala, two to 12 in D. meziana, one to nine in D. brevispicata, and one to 10 in D. seramisiana. STRUCTURE analyses clearly separated the taxa from each other. CONCLUSIONS: The 15 new microsatellite markers are promising tools for studying population genetics in Deuterocohnia species.

Population genetic structure of the rock outcrop species Encholirium spectabile (Bromeliaceae): The role of pollination vs. seed dispersal and evolutionary implications.

PREMISE OF THE STUDY: Inselbergs are terrestrial, island-like rock outcrop environments that present a highly adapted flora. The epilithic bromeliad Encholirium spectabile is a dominant species on inselbergs in the Caatinga of northeastern Brazil. We conducted a population genetic analysis to test whether the substantial phenotypic diversity of E. spectabile could be explained by limited gene flow among populations and to assess the relative impact of pollen vs. seed dispersal on the genetic structure of the species. METHODS: Nuclear and chloroplast microsatellite markers were used to genotype E. spectabile individuals from 20 rock outcrop locations, representing four geographic regions: northern Espinhaço Range, Borborema Plateau, southwestern Caatinga and southeastern Caatinga. F-statistics, structure, and other tools were applied to evaluate the genetic makeup of populations. KEY RESULTS: Considerable levels of genetic diversity were revealed. Genetic structuring among populations was stronger on the plastid as compared with the nuclear level, indicating higher gene flow via bat pollination as compared with seed dispersal by wind. structure and AMOVA analyses of the nuclear data suggested a high genetic differentiation between two groups, one containing all populations from the southeastern Caatinga and the other one comprising all remaining samples. CONCLUSIONS: The strong genetic differentiation between southeastern Caatinga and the remaining regions may indicate the occurrence of a cryptic species in E. spectabile. The unique genetic composition of each inselberg population suggests in situ conservation as the most appropriate protection measure for this plant lineage.

Microsatellites from Fosterella christophii (Bromeliaceae) by de novo transcriptome sequencing on the Pacific Biosciences RS platform.

PREMISE OF THE STUDY: Microsatellite markers were developed in Fosterella christophii (Bromeliaceae) to investigate the genetic diversity and population structure within the F. micrantha group, comprising F. christophii, F. micrantha, and F. villosula. METHODS AND RESULTS: Full-length cDNAs were isolated from F. christophii and sequenced on a Pacific Biosciences RS platform. A total of 1590 high-quality consensus isoforms were assembled into 971 unigenes containing 421 perfect microsatellites. Thirty primer sets were designed, of which 13 revealed a high level of polymorphism in three populations of F. christophii, with four to nine alleles per locus. Each of these 13 loci cross-amplified in the closely related species F. micrantha and F. villosula, with one to six and one to 11 alleles per locus, respectively. CONCLUSIONS: The new markers are promising tools to study the population genetics of F. christophii and to discover species boundaries within the F. micrantha group.

Development of 18 polymorphic microsatellite markers for Vinca minor (Apocynaceae) via 454 pyrosequencing.

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed in Vinca minor (Apocynaceae) to evaluate the level of clonality, population structure, and genetic diversity of the species within its native and introduced range. METHODS AND RESULTS: A total of 1371 microsatellites were found in 43,565 reads from 454 pyrosequencing of genomic V. minor DNA. Additional microsatellite loci were mined from publicly available cDNA sequences. After several rounds of screening, 18 primer pairs flanking di-, tri-, or tetranucleotide repeats were identified that revealed high levels of genetic diversity in two native Italian populations, with two to 11 alleles per locus. Clonal growth predominated in two populations from the introduced range in Germany. Five loci successfully cross-amplified in three additional Vinca species. CONCLUSIONS: The novel polymorphic microsatellite markers are promising tools for studying clonality and population genetics of V. minor and for assessing the historical origin of Central European populations.

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.

Development of microsatellite markers in Fosterella rusbyi (Bromeliaceae) using 454 pyrosequencing.

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed for Fosterella rusbyi (Bromeliaceae) to evaluate the population genetic structure and genetic diversity of natural populations of F. rusbyi and other Fosterella species in Bolivia. METHODS AND RESULTS: 454 pyrosequencing technology was used to generate 73027 sequence reads from F. rusbyi DNA, which together contained 2796 perfect simple sequence repeats (SSRs). Primer pairs were designed for 30 loci, of which 15 were used to genotype 30 F. rusbyi plants from two geographical areas in Bolivia. All markers were polymorphic, with two to nine alleles in the overall sample. Cross-species amplification was tested in 10 additional Fosterella species. Seven loci showed consistent amplification in six or more species. CONCLUSIONS: The 15 SSR markers developed for F. rusbyi are promising candidates for population genetic analyses within F. rusbyi and other species of Fosterella.

In silico mining for simple sequence repeat loci in a pineapple expressed sequence tag database and cross-species amplification of EST-SSR markers across Bromeliaceae.

A collection of 5,659 expressed sequence tags (ESTs) from pineapple [Ananas comosus (L.) Merr.] was screened for simple sequence repeats (EST-SSRs) with motif lengths between 1 and 6 bp. Lower thresholds of 15, 7 and 5 repeat units were used to define microsatellites of the mono-, di-, and tri- to hexanucleotide repeat type, respectively. Based on these criteria, 696 SSRs were identified among 3,389 EST unigenes, together representing 2,840 kb. This corresponds to an average density of one SSR every 4.1 kb of non-redundant EST sequences. Dinucleotide repeats were most abundant (38.4% of all SSRs) followed by trinucleotide repeats (38.1%). Flanking primer pairs were designed for 537 EST-SSR loci, and 49 of these were screened for their functionality in 12 accessions of A. comosus, 14 accessions of 5 additional Ananas species and 1 species of Pseudananas. Distinct PCR products of the expected size range were obtained with 36 primer pairs. Eighteen loci analyzed in more detail were all polymorphic in pineapple, and primer pairs flanking these loci also generated PCR products from a wide range of genera and species from six subfamilies of the Bromeliaceae. The potential to reveal polymorphism in a heterologous target species was demonstrated in Deuterocohnia brevifolia (subfamily Pitcairnioideae).

Isolation and characterization of 11 new microsatellite markers for Macaranga (Euphorbiaceae).

We provide primer sequences for 11 new polymorphic microsatellite markers developed in the tropical ant-plant genus Macaranga (Euphorbiaceae), after enrichment cloning of Macaranga tanarius and Macaranga hypoleuca. Allele numbers per locus ranged from two to 16 among 20 accessions of M. tanarius, and from three to 10 among 22 accessions of M. hypoleuca. Observed and expected heterozygosities ranged from 0.150 to 0.900 and from 0.375 to 0.894 in M. tanarius, and from 0.545 to 1.000 and from 0.434 to 0.870 in M. hypoleuca, respectively. Six of the 11 primer pairs successfully cross-amplified polymorphic polymerase chain reaction products in Macaranga winkleri.