Abstract
Microsatellites (or simple sequence repeats, SSR) are widely used markers in population genetics. Traditionally, genotyping was and still is carried out through recording fragment length. Now, next‐generation sequencing (NGS) makes it easy to obtain also sequence information for the loci of interest. This avoids misinterpretations that otherwise could arise due to size homoplasy. Here, an NGS strategy is described that allows to genotype hundreds of individuals at many custom‐designed SSR loci simultaneously, combining multiplex PCR, barcoding, and Illumina sequencing. We created three different datasets for which alleles were coded according to (a) length of the repetitive region, (b) total fragment length, and (c) sequence identity, in order to evaluate the eventual benefits from having sequence data at hand, not only fragment length data. For each dataset, genetic diversity statistics, as well as F ST and R ST values, were calculated. The number of alleles per locus, as well as observed and expected heterozygosity, was highest in the sequence identity dataset, because of single‐nucleotide polymorphisms and insertions/deletions in the flanking regions of the SSR motif. Size homoplasy was found to be very common, amounting to 44.7%–63.5% (mean over all loci) in the three study species. Thus, the information obtained by next‐generation sequencing offers a better resolution than the traditional way of SSR genotyping and allows for more accurate evolutionary interpretations.
Highlights
IntroductionMicrosatellites (short tandem repeats, STR, or simple sequence repeats, SSR) are widely used markers in population genetics due to their ubiquitous occurrence in the nuclear and organellar genomes, high levels of polymorphism, and codominant character
Microsatellites are widely used markers in population genetics due to their ubiquitous occurrence in the nuclear and organellar genomes, high levels of polymorphism, and codominant character
Our objectives were (a) to generate nucleotide sequence data of several non‐model plant spe‐ cies, for which prior genomic data did not exist, from both the SSR and the flanking regions, (b) to record the length of the repetitive region, as well as SNP and indel variation within the SSR and the FR, (3) to estimate the amount of molecularly accessible size homoplasy of each locus, and (4) to compare the degree of genetic variability between different datasets based on the number of repeat units, fragment length, and sequence identity
Summary
Microsatellites (short tandem repeats, STR, or simple sequence repeats, SSR) are widely used markers in population genetics due to their ubiquitous occurrence in the nuclear and organellar genomes, high levels of polymorphism, and codominant character. Single‐nucleotide polymorphisms aThese authors contributed to this work. (SNPs) or insertions/deletions (indel) polymorphisms in the nucleo‐ tide sequence of that fragment, either within the repetitive array or in the flanking regions (FR), remain undetected by length assessment alone. Using only length information, SSR alleles may appear identical in state (i.e., length/ size), but they are not necessarily identical by descent in case of convergent mutation(s) to the same size (“size homoplasy”, Estoup, Jarne, & Cornuet, 2002) or variability only in sequence but not in size. Still, sequencing cannot re‐ solve homoplasy that arises from the convergence of two alleles to the same sequence and length
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