An efficient field and laboratory workflow for plant phylotranscriptomic projects1

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-NC-SA 4.0), which permits unrestricted noncommercial use and redistribution provided that the original author and source are credited and the new work is distributed under the same license as the original. Phylotranscriptomics, or using transcriptome sequences to investigate phylogenetic relationships and gene family evolution in nonmodel plants, has gained popularity in recent years due to decreases in cost and improvements in analysis pipe-It is often possible to recover at least 15,000 genes from the target species using de novo–assembled transcriptome data (Yang and Smith, 2013). Among these, approximately 5000 are shared among most species within an order (Yang et al., 2015), with the rest being tissue-and/or taxon-specific. Together they provide enormously rich data both for phylogenetic reconstruction and for investigating gene family evolution that underlies lineage-specific adaptations. Generating plant phylotranscriptomic data has become much easier over the past few years due to improvements in sequenc-ing and extraction protocols but may still be challenging for a variety of reasons. Previous literature on phylotranscriptomic methods has focused on RNA extraction and fragment analyses of those extracted RNA samples (Johnson et al., 2012; Yockteng et al., 2013; Jordon-Thaden et al., 2015) and sequence data analyses (Yang and Smith, 2013, 2014). However, as phylotrans-criptomic studies expand to nonmodel systems that often require field sampling, the logistics of obtaining fresh tissues becomes a limiting factor. Likewise, some taxa such as cacti pose special challenges due to high levels of mucilage (Jordon-Thaden et al., 2015). Moving forward, the issues of long-term preservation and curation of cryogenic genetic materials will also be of the utmost importance for laboratories seeking to pursue these studies.Net BiodivERsA project " WhoIsNext, " with the national funders Agence Nationale de la Recherche (ANR; ANR-13-EBID-0004), Deutsche Forschungsgemeinschaft (DFG), and Fonds zur Förderung der wissen-schaftlichen Forschung (FWF); the Joseph-Fourier Alpine Station provided lodging and logistic support. • Premise of the study: We describe a field and laboratory workflow developed for plant phylotranscriptomic projects that involves cryogenic tissue collection in the field, RNA extraction and quality control, and library preparation. We also make recommendations for sample curation. • Methods and Results: A total of 216 frozen tissue samples of Caryophyllales and other angiosperm taxa were collected from the field or botanical gardens. RNA was extracted, stranded mRNA libraries were prepared, and libraries were sequenced on Illu-mina HiSeq platforms. These …