Erratum to: Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae) (original) (raw)
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Genetica, 2012
The Pilosocereus aurisetus group contains eight cactus species restricted to xeric habitats in eastern and central Brazil that have an archipelago-like distribution. In this study, 5–11 microsatellite markers previously designed for Pilosocereus machrisii were evaluated for cross-amplification and polymorphisms in ten populations from six species of the P. aurisetus group. The genotypic information was subsequently used to investigate the genetic relationships between the individuals, populations, and species analyzed. Only the Pmac101 locus failed to amplify in all of the six analyzed species, resulting in an 88 % success rate. The number of alleles per polymorphic locus ranged from 2 to 12, and the most successfully amplified loci showed at least one population with a larger number of alleles than were reported in the source species. The population relationships revealed clear genetic clustering in a neighbor-joining tree that was partially incongruent with the taxonomic limits between the P. aurisetus and P. machrisii species, a fact which parallels the problematic taxonomy of the P. aurisetus group. A Bayesian clustering analysis of the individual genotypes confirmed the observed taxonomic incongruence. These microsatellite markers provide a valuable resource for facilitating large-scale genetic studies on population structures, systematics and evolutionary history in this group.
American Journal of …, 2011
Chamaecyparis nootkatensis is an ecologically and economically important conifer of the north Pacific coastal forests. To aid in studies of clonal structure and genetic differentiation of this and related species, we isolated and characterized microsatellites from C. nootkatensis. A microsatellite-enriched library yielded 75 repeat-containing sequences for which primer pairs were designed. Only five showed reliable amplification and polymorphism, with an average of 13.7 alleles/locus and a mean expected heterozygosity of 0.592. In progeny tests with four families, few null alleles were directly detected and loci segregated according to Mendelian expectations. However, in one primer pair, high heterozygote deficiency was observed, suggesting the presence of a null allele. The ability of primer pairs to cross amplify was tested on 18 species of the Cupressaceae sensu lato; three primer pairs yielded polymorphic loci in Cupressus and Juniperus species, but not in other Chamaecyparis species. This also supports recent findings of a closer affinity of C. nootkatensis with Cupressus over other Chamaecyparis species.
Microsatellite transferability was used as a method to examine the genetic diversity and structure of populations in Pilosocereus gounellei seedling samples that have potential to implement effective restoration strategies for degraded and disturbed areas of the Caatinga biome. Genomic DNA was extracted from 85 seedlings obtained from fruit collected from plants growing in native areas in the Brazilian states of Piaui (PI), Rio Grande do Norte (RN), and Bahia (BA). Six microsatellite primers were polymorphic. AMOVA showed higher genetic variation within (72%) than among (28%) the samples from the three states. The high level of similarity between the seedlings from PI, BA, and RN indicated that samples collected at any of the three sites can be used to represent the genetic diversity of the species. Seeds of plants from the three States are recommended as samples for germplasm banks and/or the production of plantlets to i) plant in areas of strategic reserves for forage, ii) deploy new cultivation areas, iii) restore degraded areas in the semi-arid Northeast, and iv) maintain ecological reserve banks and fodder with genetically divergent plants.
Molecular Ecology Resources, 2009
The genus Conospermum Sm. (Proteaceae) represents an important component of the heathlands and woodlands of Western Australian sandplains. The genus has 53 species endemic to Australia, with its center of distribution in southwestern Western Australia (Bennett, 1995). Within the South West Australian Floristic Region, a global biodiversity hotspot (Myers et al., 2000; Hopper and Gioia, 2004), many Conospermum species are of increasing conservation concern, with four taxa already declared rare by the Western Australia government (Government Gazette, 2018). Moreover, as for many proteaceous species, various Conospermum species are widely utilized in floriculture (Bennett, 1995; Stone et al., 2006). Conospermum undulatum Lindl. is a diploid shrub with its range restricted to ca. 55 km 2 in a rapidly expanding urban zone in the metropolitan area of Perth (Close et al., 2006; Wardell-Johnson et al., 2016). This species is listed as Vulnerable under the Environment Protection and Biodiversity Conservation Act 1999. Habitat fragmentation and hybridization with sympatric Conospermum species are likely to pose a risk to the future persistence of C. undulatum. In Conospermum, studies of population genetics and reproductive biology have been undertaken using amplified fragment length polymorphism (AFLP) and random-amplified polymorphic DNA (RAPD) markers for only a few species (Stone et al., 2006; Sinclair et al., 2008). To our knowledge, no microsatellite resources have been developed for this genus to date. Considering the growing concern about this endemic genus and the number of species within it, we expect that microsatellite markers will have broad applicability for conservation and population genetic analyses. Here, we report the development and characterization of 20 microsatellite markers for C. undulatum that will be useful for the study of its genetic structure, spatial patterns of genetic diversity, and dispersal dynamics. Additionally, we tested for cross-amplification of these loci in three related Conospermum species to evaluate the utility of the marker set more broadly and specifically to allow assessment of hybridization between C. undulatum and neighboring species. METHODS AND RESULTS Genomic DNA was extracted from freeze-dried leaf material (ca. 50 mg) using a modified 2% cetyltrimethylammonium bromide (CTAB) method, with 1% polyvinylpyrrolidone and 0.1% sodium
PLOS ONE, 2015
Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as diand trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in nonmodel organisms.
2009
Chamaecyparis nootkatensis is an ecologically and economically important conifer of the north Pacific coastal forests. To aid in studies of clonal structure and genetic differentiation of this and related species, we isolated and characterized microsatellites from C. nootkatensis. A microsatellite-enriched library yielded 75 repeat-containing sequences for which primer pairs were designed. Only five showed reliable amplification and polymorphism, with an average of 13.7 alleles/locus and a mean expected heterozygosity of 0.592. In progeny tests with four families, few null alleles were directly detected and loci segregated according to Mendelian expectations. However, in one primer pair, high heterozygote deficiency was observed, suggesting the presence of a null allele. The ability of primer pairs to cross amplify was tested on 18 species of the Cupressaceae sensu lato; three primer pairs yielded polymorphic loci in Cupressus and Juniperus species, but not in other Chamaecyparis species. This also supports recent findings of a closer affinity of C. nootkatensis with Cupressus over other Chamaecyparis species.
Applications in Plant Sciences, 2016
Next-generation sequencing (NGS)-based methods have allowed the quick development of microsatellite primers specific to nonmodel organisms (e.g., Duwe et al., 2015; González et al., 2015). Here, microsatellite markers are presented for the grass genus Anthoxanthum L., comprising around 20 species often affected by reticulation (Pimentel et al., 2010, 2013). The phylogeny of Anthoxanthum defines a Euro-Siberian (as well as Macaronesian and Afroalpine) polyploid complex of species (Pimentel et al., 2013). It includes four diploid taxa: (i) the Mediterranean A. aristatum Boiss.-A. ovatum Lag. complex (Pimentel et al., 2010), (ii) the Macaronesian A. maderense Teppner, and (iii) the Arctic-alpine A. alpinum Á. Löve & D. Löve (Pimentel et al., 2013). The clade also includes at least three polyploid lineages (Chumová et al., 2015): the Iberian endemic A. amarum Brot. (16x-18x); the East African A. nivale K. Schum. (4x, 6x), and the Eurasian A. odoratum L. (4x). Fifteen microsatellite markers that can be applied to the Euro-Siberian complex of Anthoxanthum are presented here. These markers will be used to determine the geographic patterns of gene flow within and among the different diploid lineages in the complex, as well as to unravel the origin of its polyploid groups. METHODS AND RESULTS Microsatellite development-A microsatellite-enriched genomic library (motifs AC, AG, ACC, AGG, and ACG) was constructed at AllGenetics & Biology SL (A Coruña, Spain) from an equimolar mix of DNA extracts from the diploid A. aristatum-A. ovatum (two individuals) and the tetraploid A. odoratum (one individual; Appendix 1) using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, California, USA). Given the difficulty in morphologically distinguishing Anthoxanthum cytotypes (Chumová et al., 2015), between one and five individuals per population were assessed using flow cytometry following Galbraith et al. (1983). DNA was extracted from silica-dried leaves using the DNAeasy Plant Mini Kit (QIAGEN, Hilden, Germany). The enriched genomic library was sequenced in a fraction of an Illumina MiSeq PE300 run (Illumina), and the reads were processed using the software Geneious 7.1.5 (Biomatters, Auckland, New Zealand). Five hundred loci were detected containing a microsatellite and flanked by regions adequate to design PCR primers using Primer3 (Untergasser et al., 2012). Primer pairs were multiplexed with Multiplex Manager 1.0 (Holleley and Geerts, 2009). Forty microsatellite loci were combined so that differences in annealing temperatures were minimized and spacing between markers was maximized. Primers were tested for polymorphism on six diploid and two tetraploid samples (Appendix 1) that belonged to the different Anthoxanthum lineages and came from geographically distant populations. Each PCR reaction was performed following Schuelke (2000) with three primers (one of them fluorescently labeled using FAM or HEX; Table 1). PCR reactions were conducted in a final volume of 12.5 μL, containing 1 μL of DNA (10 ng/μL), 6.25 μL Type-it Microsatellite PCR Kit (QIAGEN), 4 μL PCR-grade water, and 1.25 μL of the primer mix (Schuelke, 2000). The optimal PCR protocol consisted of an initial denaturation step at 95°C for 5 min; followed by 30 cycles of 95°C for 30 s, 56°C for 90 s, and 72°C for 30 s; eight cycles of 95°C for 30 s, 52°C for 90 s, and 72°C for 30 s; and a final extension step at 68°C for 30 min. Labeled PCR products were then subjected to fragment analysis by Macrogen (Seoul, Republic of Korea). The resulting .fsa files were manually analyzed using Geneious 7.1.5 (Biomatters). Fifteen primers were selected based on amplification success and the number of alleles generated (Table 1).