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. 2012;7(7):e40699.
doi: 10.1371/journal.pone.0040699. Epub 2012 Jul 13.

Always look on both sides: phylogenetic information conveyed by simple sequence repeat allele sequences

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Always look on both sides: phylogenetic information conveyed by simple sequence repeat allele sequences

Stéphanie Barthe et al. PLoS One. 2012.

Abstract

Simple sequence repeat (SSR) markers are widely used tools for inferences about genetic diversity, phylogeography and spatial genetic structure. Their applications assume that variation among alleles is essentially caused by an expansion or contraction of the number of repeats and that, accessorily, mutations in the target sequences follow the stepwise mutation model (SMM). Generally speaking, PCR amplicon sizes are used as direct indicators of the number of SSR repeats composing an allele with the data analysis either ignoring the extent of allele size differences or assuming that there is a direct correlation between differences in amplicon size and evolutionary distance. However, without precisely knowing the kind and distribution of polymorphism within an allele (SSR and the associated flanking region (FR) sequences), it is hard to say what kind of evolutionary message is conveyed by such a synthetic descriptor of polymorphism as DNA amplicon size. In this study, we sequenced several SSR alleles in multiple populations of three divergent tree genera and disentangled the types of polymorphisms contained in each portion of the DNA amplicon containing an SSR. The patterns of diversity provided by amplicon size variation, SSR variation itself, insertions/deletions (indels), and single nucleotide polymorphisms (SNPs) observed in the FRs were compared. Amplicon size variation largely reflected SSR repeat number. The amount of variation was as large in FRs as in the SSR itself. The former contributed significantly to the phylogenetic information and sometimes was the main source of differentiation among individuals and populations contained by FR and SSR regions of SSR markers. The presence of mutations occurring at different rates within a marker's sequence offers the opportunity to analyse evolutionary events occurring on various timescales, but at the same time calls for caution in the interpretation of SSR marker data when the distribution of within-locus polymorphism is not known.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Two alternative genealogies for simple sequence repeat (SSR) alleles containing the same number of repeats.
SSR alleles are indicated by their number of repeats (n, n+1). The A/C letters indicate a SNP in the flanking regions. Red bars correspond to mutational events in the flanking region sequence or in the number of SSR repeats. (A) No sequence information: deduced genealogy of observed data (third line) groups alleles together according to their number of repeats and involves a single SSR mutation. Genealogy is recent. (B) Consideration of sequence information: deduced genealogy involves a SNP mutation and two SSR mutations (alternative topology will involve two identical and independent substitutions at the same nucleotide site and a unique SSR mutation, which is less likely). Genealogy is ancient and SSR alleles do not group according to their numbers of repeats.
Figure 2
Figure 2. Alignment of a subset of DNA fragments at simple sequence repeat (SSR) loci Jc3H10 (Jacaranda copaia) (A) and QrZAG30 (Quercus robur) (B).
Numbers in the top line indicate positions relative to the consensus sequence of (A) 190 bp and (B) 247 bp. Bold nucleotides in brackets indicate SSR motifs and their number of repeats. Dashes indicate gaps, highlighted nucleotides in green (light grey*) indicate indels of one to three bases, highlighted nucleotides in red (dark grey*) mark mutations from one base to another, and yellow (light grey*) boxes indicate groups of insertion/deletions longer than three bases and considered as a single mutational event. * In shades-of-gray printouts.
Figure 3
Figure 3. Linkage Disequilibrium (LD) for pairs of polymorphisms in the flanking regions.
Dark cells contain LD values significantly different from zero. An asterisk next to a locus name indicates that the SSR repeat(s) is (are) located between that locus and the next. Colouring (shading) indicates the degree of significance of the test: green (light grey*), P>0.05; orange (grey*), 0.05<P>0.001; red (dark grey*), P<0.001. * In shades-of-gray printouts.
Figure 4
Figure 4. Phylogenetic trees of Jacaranda populations based on different components of simple sequence repeat (SSR) data.
SSR variation (A), amplicon size variation (B), flanking region (FR) sequence variation (C) and amplicon sequence variation (D). Each type of data was analysed according to a pair of suitable genetic distance estimators: FST, suitable for loci following the infinite allele model (IAM); RST, for loci following the stepwise mutation model (SMM); and NST, for loci following the infinite site model (ISM) model. There are four geographic populations: Counami (C), Paracou (P) and Saint-Laurent (S) in French Guiana; Tapajos (T) in Brazil. Note that scales are not the same among trees.

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