DOI: 10.5061/dryad.k3j9kd58k
title: A new approach using targeted sequence capture for phylogenomic studies across Cactaceae

description: Relationships within the major clades of Cactaceae are relatively well
 known based on DNA sequence data mostly from the chloroplast
 genome. Nevertheless, some nodes along the backbone of the
 phylogeny, and especially generic and species-level relationships, remain
 poorly resolved and are in need of more informative genetic
 markers. In this study, we propose a new approach to solve
 the relationships within Cactaceae, applying a targeted sequence
 capture pipeline. We designed a custom probe set for Cactaceae
 using MarkerMiner and complemented it with the Angiosperms353
 probe set. We then tested both probe sets against 36 different
 transcriptomes using Hybpiper preferentially retaining
 phylogenetically informative loci and reconstructed the relationships
 using RAxML-NG and Astral. Finally, we tested each probe set
 through sequencing 96 accessions, representing 88 species across
 Cactaceae. Our preliminary analyses recovered a well-supported phylogeny
 across Cactaceae with a near identical topology among major clade
 relationships as that recovered with plastome data. As expected,
 however, we found incongruences in relationships when comparing our
 nuclear probe set results to plastome datasets, especially at the
 generic level. Our results reveal great potential for the
 combination of Cactaceae-specific and Angiosperm353 probe set application
 to improve phylogenetic resolution for Cactaceae and for other
 studies.
description: We used MarkerMiner (MM) 1.0
 as implemented in the University of Florida High-Performance Cluster. We,
 therefore, used 15 transcriptomes representing all the main clades in
 Cactaceae. In addition, we selected <em>Arabidopsis
 thaliana</em> as the annotated reference genome (TAIR10) due to this
 lineage being the closest to Cactaceae among the datasets implemented in
 MarkerMiner. We then inspected the results from MarkerMiner and focused
 exclusively on the strictly single-copy loci. We manually trimmed our
 locus sequences in Geneious Prime 2020.0.5 to include only single-copy
 loci that contained at least one suitable exon of &gt;120 bp size and
 intronic regions of 100 bp or more. To avoid the inclusion of any
 non-nuclear loci, we performed several BLASTx analyses between all the
 target sequences and: 1) <em>Beta vulgaris</em> and
 <em>Arabidopsis thaliana</em> mitochondrial genomes. 2)
 <em>A</em>.<em> thaliana</em> and
 <em>Cylindropuntia bigelovii</em> chloroplast genomes. In
 addition, we performed a BLASTx analysis with <em>A. thaliana
 </em>whole nuclear genome (Araport11) to confirm and update the MM
 annotation results. At the same time, we explored the single-copy loci
 potential annotations from GenBank and TAIR database. Finally, we reduced
 to one copy any identical loci after a reciprocal BLAST between the
 MarkerMiner, Angiosperms353, and within each probe set. We then added
 a subset of the sequences for Caryophyllales that were recovered for
 <em>Nepenthes mirabilis</em>. This subset included the 296
 genes reported for <em>Nephentes </em>plus 26 additional genes
 retrieved from other Caryophyllales accessions in Johnson et al. (2019)
 for a total of 322 genes.
description: The Cactaceae120 loci names start with the corresponding
 transcriptome short name followed by loci name starting with
 "AT". The Angiosperms353 follow Johnson et al. (2019)
 names.