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A protocol for targeted enrichment of intron-containing sequence markers for recent radiations: A phylogenomic example from Heuchera (Saxifragaceae).

Folk RA, Mandel JR, Freudenstein JV - Appl Plant Sci (2015)

Bottom Line: Organellar phylogenies were also well-supported and conflicted with the nuclear signal.Our approach shows promise for resolving a recent radiation.Enrichment for introns is highly successful with little or no sequencing dropout at low taxonomic levels despite higher substitution and indel frequencies, and should be exploited in studies of species complexes.

View Article: PubMed Central - PubMed

Affiliation: Herbarium, The Ohio State University, Columbus, Ohio 43212 USA.

ABSTRACT

Premise of the study: Phylogenetic inference is moving to large multilocus data sets, yet there remains uncertainty in the choice of marker and sequencing method at low taxonomic levels. To address this gap, we present a method for enriching long loci spanning intron-exon boundaries in the genus Heuchera.

Methods: Two hundred seventy-eight loci were designed using a splice-site prediction method combining transcriptomic and genomic data. Biotinylated probes were designed for enrichment of these loci. Reference-based assembly was performed using genomic references; additionally, chloroplast and mitochondrial genomes were used as references for off-target reads. The data were aligned and subjected to coalescent and concatenated phylogenetic analyses to demonstrate support for major relationships.

Results: Complete or nearly complete (>99%) sequences were assembled from essentially all loci from all taxa. Aligned introns showed a fourfold increase in divergence as opposed to exons. Concatenated analysis gave decisive support to all nodes, and support was also high and relationships mostly similar in the coalescent analysis. Organellar phylogenies were also well-supported and conflicted with the nuclear signal.

Discussion: Our approach shows promise for resolving a recent radiation. Enrichment for introns is highly successful with little or no sequencing dropout at low taxonomic levels despite higher substitution and indel frequencies, and should be exploited in studies of species complexes.

No MeSH data available.


Map of regions of interest in the mitochondrial genome of Heuchera parviflora var. saurensis, prepared using OrganellarGenomeDRAW (Lohse et al., 2013). The outer circle depicts protein-coding genes, rRNAs, and tRNAs; the inner circle depicts the large repeat regions. Genes annotated on the inner face of the circle indicate genes transcribed in a clockwise orientation, and genes on the outer face are transcribed counterclockwise. The orientation of the repeat regions is arbitrary; the green and blue regions are direct repeats, while the yellow and lavender regions exist both as direct repeats (where they overlap the green direct repeat) and inverted repeats (elsewhere). Annotated gene regions include both introns and exons. Plant mitochondrial genomes have several trans-spliced genes (e.g., nad5); only the cistronic portions are annotated to avoid overlap.
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fig5: Map of regions of interest in the mitochondrial genome of Heuchera parviflora var. saurensis, prepared using OrganellarGenomeDRAW (Lohse et al., 2013). The outer circle depicts protein-coding genes, rRNAs, and tRNAs; the inner circle depicts the large repeat regions. Genes annotated on the inner face of the circle indicate genes transcribed in a clockwise orientation, and genes on the outer face are transcribed counterclockwise. The orientation of the repeat regions is arbitrary; the green and blue regions are direct repeats, while the yellow and lavender regions exist both as direct repeats (where they overlap the green direct repeat) and inverted repeats (elsewhere). Annotated gene regions include both introns and exons. Plant mitochondrial genomes have several trans-spliced genes (e.g., nad5); only the cistronic portions are annotated to avoid overlap.

Mentions: The chloroplast genome of H. parviflora var. saurensis (154,696 bp; mean coverage 1939.8×; no map shown) is conventional among angiosperms in terms of structure and size, with complete synteny shared with other members of the order. The H. parviflora var. saurensis mitochondrial genome (541,954 bp; mean coverage 141.9×, draft map in Fig. 5) is much less conserved in terms of structure, as is typical for plant mitochondrial genomes (Sloan, 2013), with only a few very short regions of synteny and only short stretches of similar sequence preserved when compared to the closest reference, V. vinifera (Appendix S2). It has all functional mitochondrial protein-coding genes that are present in Vitis, as well as numerous interspersed sequences of chloroplast origin. BLAST searches against the Marchantia polymorpha L. mitochondrial genome showed that all conserved mitochondrial genes absent in Vitis were also absent in Heuchera. Processes of intramolecular recombination and multiple conformations of the plant mitochondrial genome are thought to be mediated by moderately long repeat regions (Alverson et al., 2011, and citations therein), of which we found four. These repeat regions were independently supported by locally higher read coverage. Unusually, the smaller three repeat regions (6428 bp, 1196 bp, 550 bp) that we found overlap with the largest repeat (32,849 bp), but with a single copy of each elsewhere in the genome. The issue of multiple genome conformations is not critical for assemblies intended for analysis with phylogenetic methods. Using these two organellar references, we were able to infer resolved phylogenies with high support values as well (chloroplast, Fig. 4; mitochondrion, Fig. 5).


A protocol for targeted enrichment of intron-containing sequence markers for recent radiations: A phylogenomic example from Heuchera (Saxifragaceae).

Folk RA, Mandel JR, Freudenstein JV - Appl Plant Sci (2015)

Map of regions of interest in the mitochondrial genome of Heuchera parviflora var. saurensis, prepared using OrganellarGenomeDRAW (Lohse et al., 2013). The outer circle depicts protein-coding genes, rRNAs, and tRNAs; the inner circle depicts the large repeat regions. Genes annotated on the inner face of the circle indicate genes transcribed in a clockwise orientation, and genes on the outer face are transcribed counterclockwise. The orientation of the repeat regions is arbitrary; the green and blue regions are direct repeats, while the yellow and lavender regions exist both as direct repeats (where they overlap the green direct repeat) and inverted repeats (elsewhere). Annotated gene regions include both introns and exons. Plant mitochondrial genomes have several trans-spliced genes (e.g., nad5); only the cistronic portions are annotated to avoid overlap.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542943&req=5

fig5: Map of regions of interest in the mitochondrial genome of Heuchera parviflora var. saurensis, prepared using OrganellarGenomeDRAW (Lohse et al., 2013). The outer circle depicts protein-coding genes, rRNAs, and tRNAs; the inner circle depicts the large repeat regions. Genes annotated on the inner face of the circle indicate genes transcribed in a clockwise orientation, and genes on the outer face are transcribed counterclockwise. The orientation of the repeat regions is arbitrary; the green and blue regions are direct repeats, while the yellow and lavender regions exist both as direct repeats (where they overlap the green direct repeat) and inverted repeats (elsewhere). Annotated gene regions include both introns and exons. Plant mitochondrial genomes have several trans-spliced genes (e.g., nad5); only the cistronic portions are annotated to avoid overlap.
Mentions: The chloroplast genome of H. parviflora var. saurensis (154,696 bp; mean coverage 1939.8×; no map shown) is conventional among angiosperms in terms of structure and size, with complete synteny shared with other members of the order. The H. parviflora var. saurensis mitochondrial genome (541,954 bp; mean coverage 141.9×, draft map in Fig. 5) is much less conserved in terms of structure, as is typical for plant mitochondrial genomes (Sloan, 2013), with only a few very short regions of synteny and only short stretches of similar sequence preserved when compared to the closest reference, V. vinifera (Appendix S2). It has all functional mitochondrial protein-coding genes that are present in Vitis, as well as numerous interspersed sequences of chloroplast origin. BLAST searches against the Marchantia polymorpha L. mitochondrial genome showed that all conserved mitochondrial genes absent in Vitis were also absent in Heuchera. Processes of intramolecular recombination and multiple conformations of the plant mitochondrial genome are thought to be mediated by moderately long repeat regions (Alverson et al., 2011, and citations therein), of which we found four. These repeat regions were independently supported by locally higher read coverage. Unusually, the smaller three repeat regions (6428 bp, 1196 bp, 550 bp) that we found overlap with the largest repeat (32,849 bp), but with a single copy of each elsewhere in the genome. The issue of multiple genome conformations is not critical for assemblies intended for analysis with phylogenetic methods. Using these two organellar references, we were able to infer resolved phylogenies with high support values as well (chloroplast, Fig. 4; mitochondrion, Fig. 5).

Bottom Line: Organellar phylogenies were also well-supported and conflicted with the nuclear signal.Our approach shows promise for resolving a recent radiation.Enrichment for introns is highly successful with little or no sequencing dropout at low taxonomic levels despite higher substitution and indel frequencies, and should be exploited in studies of species complexes.

View Article: PubMed Central - PubMed

Affiliation: Herbarium, The Ohio State University, Columbus, Ohio 43212 USA.

ABSTRACT

Premise of the study: Phylogenetic inference is moving to large multilocus data sets, yet there remains uncertainty in the choice of marker and sequencing method at low taxonomic levels. To address this gap, we present a method for enriching long loci spanning intron-exon boundaries in the genus Heuchera.

Methods: Two hundred seventy-eight loci were designed using a splice-site prediction method combining transcriptomic and genomic data. Biotinylated probes were designed for enrichment of these loci. Reference-based assembly was performed using genomic references; additionally, chloroplast and mitochondrial genomes were used as references for off-target reads. The data were aligned and subjected to coalescent and concatenated phylogenetic analyses to demonstrate support for major relationships.

Results: Complete or nearly complete (>99%) sequences were assembled from essentially all loci from all taxa. Aligned introns showed a fourfold increase in divergence as opposed to exons. Concatenated analysis gave decisive support to all nodes, and support was also high and relationships mostly similar in the coalescent analysis. Organellar phylogenies were also well-supported and conflicted with the nuclear signal.

Discussion: Our approach shows promise for resolving a recent radiation. Enrichment for introns is highly successful with little or no sequencing dropout at low taxonomic levels despite higher substitution and indel frequencies, and should be exploited in studies of species complexes.

No MeSH data available.