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Intragenomic Polymorphisms Among High-Copy Loci: A Genus-Wide Study of Nuclear Ribosomal DNA in Asclepias (Apocynaceae)

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Intragenomic Polymorphisms Among High-Copy Loci: A Genus-Wide Study of Nuclear Ribosomal DNA in Asclepias (Apocynaceae)

Kevin Weitemier et al. PeerJ.

Abstract

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual's consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the "noncoding" ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming).

Keywords: 2ISP; Asclepias; Concerted evolution; Genome skimming; High-copy; ITS; Intra-individual site polymorphism; Intragenomic polymorphism; Nuclear ribosomal DNA (nrDNA); Partial SNP (pSNP).

Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Polymorphic site frequency among species of Asclepias.
Histogram of polymorphic site frequency among species of Asclepias. Individuals contained from 0.4% to 15.1% polymorphic sites.
Figure 2
Figure 2. Polymorphic sites across the nrDNA cistron of Asclepias.
Number of individuals that are (A) polymorphic and (B) highly polymorphic at each position. Polymorphic positions are those with ≥2% of reads differing from the consensus; highly polymorphic positions are those with ≥10% differing reads. Subunit regions, white background; spacer regions, shaded background. Numbers in each region are the percentage of sites polymorphic or highly polymorphic in at least one individual.
Figure 3
Figure 3. Polymorphism probability by region and structure.
Probability that at least one individual is (A) polymorphic or (B) highly polymorphic at a position that is either within a spacer (ITS1, ITS2) or subunit region (18S, 5.8S, 26S), and either paired (stems) or unpaired (loops). Error bars indicate 95% confidence intervals. Values derived from two-factor multiple logistic regressions (Table 3).
Figure 4
Figure 4. Ancestral state reconstruction of polymorphic site abundance.
Ancestral state reconstruction of the number of polymorphic positions in nrDNA in Asclepias obtained with squared-change parsimony. The tree topology is that pruned from Fig. 2 of Fishbein et al. (2011) with clades indicated by letters, following that study.
Figure 5
Figure 5. Ancestral state reconstruction of highly polymorphic site abundance.
Ancestral state reconstruction of the number of highly polymorphic positions in nrDNA in Asclepias obtained with squared-change parsimony. The tree topology is that pruned from Fig. 2 of Fishbein et al. (2011) with clades indicated by letters, following that study.

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Grant support

Funding for this work is supported by the United States National Science Foundation Systematic Biology program DEB 0919583 and DEB 0919389. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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