Abstract
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 di- and 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 non-model organisms.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cactaceae / genetics*
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Computational Biology
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Cost-Benefit Analysis
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DNA Primers
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DNA Restriction Enzymes / genetics
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DNA, Plant / genetics
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Genetic Markers / genetics
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Genetic Variation
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Genotype
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High-Throughput Nucleotide Sequencing / methods*
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Linkage Disequilibrium
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Microsatellite Repeats / genetics*
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Molecular Sequence Data
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Polymerase Chain Reaction
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Polymorphism, Genetic
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Sequence Analysis, DNA
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Species Specificity
Substances
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DNA Primers
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DNA, Plant
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Genetic Markers
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DNA Restriction Enzymes
Associated data
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Grants and funding
This work was supported through grants from the São Paulo Research Foundation (FAPESP) (2012/22857-8 to IASB and 2012/22477-0 to EMM) and CAPES (BEX: 5639/13-1 to IASB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.