Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae) (original) (raw)
Related papers
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.
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.
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.
Phylogenetic analyses ofPilosocereus(Cactaceae) inferred from plastid and nuclear sequences
Botanical Journal of the Linnean Society, 2016
Pilosocereus is a large genus of Cactaceae with 42 species of columnar cacti distributed in the Americas. In this work we investigate the phylogenetics and evolutionary history of Pilosocereus based in plastid and nuclear DNA sequences. We use phylogenetic trees obtained as a basis to analyse infrageneric relationships and to study the evolution of selected morphological characters and geographical distribution in the group. Thirty-three species of the genus were sampled and five molecular regions were selected, four non-coding intergenic spacers of plastid DNA (trnS-trnG, psbD-trnT, trnL-trnT, petL-psbE) and one nuclear low-copy gene (phytochrome C). The phylogenetic analyses obtained point to a paraphyletic Pilosocereus, with P. bohlei and P. gounellei emerging nested in a clade of outgroup species (i.e. other genera of Cereinae). However, the majority of species of the genus form one well supported clade (excluding P. bohlei and P. gounellei) corresponding mostly to Pilosocereus subgenus Pilosocereus. Evidence indicates that the ancestor of Pilosocereus subgenus Pilosocereus clade was a shrub with a straight floral tube occurring in Brazil and the ancestor of Pilosocereus subgenus Gounellea was a shrub with a curved floral tube also occurring in Brazil. The ancestral distribution in central and eastern Brazil resulted in the diversification of most lineages in the same area, whereas the P. leucocephalus clade was able to disperse through the Amazonian areas and diversify further north and reach Central and North America.