APPLICATION ARTICLE INVITED SPECIAL ARTICLE For the Special Issue: Conducting Botanical Research with Limited Resources: Low-Cost Methods in the Plant Sciences A basic ddRADseq two-enzyme protocol performs well with herbarium and silica-dried tissues across four genera Ingrid E. Jordon-Thaden1,8 , James B. Beck2,3,11 , Catherine A. Rushworth4,5, Michael D. Windham6, Nicolas Diaz7,9, Jason T. Cantley7,10, Christopher T. Martine7 , and Carl J. Rothfels1 Manuscript received 1 September 2019; revision accepted PREMISE: The ability to sequence genome-scale data from herbarium specimens would allow 28 January 2020. for the economical development of data sets with broad taxonomic and geographic sampling 1 University Herbaria and Department of Integrative that would otherwise not be possible. Here, we evaluate the utility of a basic double-digest Biology, University of California Berkeley, 3040 Valley Life restriction site–associated DNA sequencing (ddRADseq) protocol using DNAs from four Sciences Building, Berkeley, California 94720, USA genera extracted from both silica-dried and herbarium tissue. 2 Department of Biological Sciences, Wichita State University, 1845 Fairmount, Wichita, Kansas 67260, USA METHODS: DNAs from Draba, Boechera, Solidago, and Ilex were processed with a ddRADseq 3 Botanical Research Institute of Texas, 1700 University Drive, protocol. The effects of DNA degradation, taxon, and specimen age were assessed. Fort Worth, Texas 76107, USA RESULTS: Although taxon, preservation method, and specimen age affected data recovery, 4 Department of Evolution and Ecology and Center for Population Biology, University of California Davis, One Shields large phylogenetically informative data sets were obtained from the majority of samples. Avenue, Davis, California 95616, USA DISCUSSION: These results suggest that herbarium samples can be incorporated into 5 Department of Plant and Microbial Biology, University of ddRADseq project designs, and that specimen age can be used as a rapid on-site guide Minnesota, 1500 Gortner Avenue, St. Paul, Minnesota 55108, USA for sample choice. The detailed protocol we provide will allow users to pursue herbarium- 6 Department of Biology, Duke University, 130 Science Drive, Durham, North Carolina 27708, USA based ddRADseq projects that minimize the expenses associated with fieldwork and sample 7 Department of Biology, Bucknell University, 1 Dent Drive, evaluation. Lewisburg, Pennsylvania 17837, USA KEY WORDS Boechera; double-digest restriction site–associated DNA sequencing (ddRAD- 8 Current address: Department of Botany, University of Wisconsin, seq); Draba; herbarium specimens; Ilex; Solidago. 430 Lincoln Drive, Madison, Wisconsin 53706, USA 9 Current address: Biology Department, Portland State University, 1719 SW 10th Avenue, Portland, Oregon 97201, USA 10 Current address: Department of Biology, San Francisco State University, 1600 Holloway Avenue, San Francisco, California 94132, USA 11 Author for correspondence: [email protected] Citation: Jordon-Thaden, I. E., J. B. Beck, C. A. Rushworth, M. D. Windham, N. Diaz, J. T. Cantley, C. T. Martine, and C. J. Rothfels. 2020. A basic ddRADseq two-enzyme protocol performs well with herbarium and silica-dried tissues across four genera. Applications in Plant Sciences 8(4): e11344. doi:10.1002/aps3.11344 Genomic tools that best combine data quality, ease, and cost- categories of genomic tools outlined by McKain et al. (2018), re- effectiveness become standard in the empirical studies that ad- striction site–associated DNA sequencing (RADseq) and target vance our knowledge of diversity and phylogeny. Of the six broad enrichment approaches most effectively combine the potential for Applications in Plant Sciences 2020 8(4): e11344; http://www.wileyonlinelibrary.com/journal/AppsPlantSci © 2020 Jordon-Thaden et al. Applications in Plant Sciences is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 of 7 Applications in Plant Sciences 2020 8(4): e11344 Jordon-Thaden et al.—ddRADseq flexibility • 2 of 7 generating large data sets with applicability to samples of varying with 100–200 μg of RNase A (QIAGEN). All extracts were desalted DNA quality. Although herbarium-derived DNAs (“herbarium (see Appendix S1). DNA”) are now being included in both target enrichment (Hart DNA degradation was quantified as the DNA integrity num- et al., 2016; Brewer et al., 2019) and RADseq (see references below) ber (DIN) for the Draba, Boechera, and Solidago samples, which studies, concerns remain regarding the link between data recov- was determined using a Genomic DNA ScreenTape Assay on a ery and herbarium DNA degradation. Here, we define herbarium TapeStation 2200 (Agilent Technologies, Santa Clara, California, DNA degradation as comprising any type of alteration that occurs USA). Ilex DIN values were not determined for logistical reasons. during collection, processing, and museum storage that negatively DIN values range from 1–10, with lower numbers indicating more affects DNA extract quality. The most recognized form of DNA deg- degradation. Genome sizes estimated from C-values (C-value da- radation are double-strand breaks, which we will refer to as DNA tabase; Leitch et al., 2019) included 0.23 Gbp (Boechera), 0.39 Gbp shearing. DNA shearing is viewed as particularly problematic for (Draba), and 1.1 Gbp (Solidago and Ilex). While the Draba data set RADseq, because highly sheared DNAs could include relatively few included both diploids and polyploids, all Boechera, Solidago, and intact fragments flanked by appropriate cut sites (Graham et al., Ilex individuals were diploid. 2015). Graham et al. (2015) found a negative relationship between DNA shearing and RADseq data recovery in freshly collected fish Enzyme choice, library preparation, sequencing, and single- tissue, and data loss was significantly more severe at the highest nucleotide polymorphism (SNP) calling level of shearing. Beck and Semple (2015) similarly reported a strong relationship between data loss and herbarium specimen age Prior success with EcoRI/SphI in Draba (Jordon-Thaden, University when using genotyping-by-sequencing, but relied on only a coarse, of California Berkeley, unpublished data) suggested that this en- agarose gel–based visual assessment of shearing. Other studies re- zyme pair would also be suitable for the confamilial Boechera. porting RADseq success with herbarium specimens did not include In silico digests of the Daucus carota L. genomic sequence in a formal evaluation of specimen age or DNA degradation on the Geneious version 10.2 (Biomatters Ltd., Auckland, New Zealand; levels of RADseq data recovery (Massatti et al., 2016; Wessinger see Appendix S1) suggested that the EcoRI/SphI enzyme pair would et al., 2016; Gilman and Tank, 2018). produce the target number of fragments in the Solidago and Ilex A broader evaluation of the feasibility of RADseq with her- genomes as well. Library preparation involved a modified version barium DNA is needed. In this study, we assess the relationships of the protocol from Peterson et al. (2012), and followed the de- between preservation method, specimen age, DNA shearing, and tailed protocol presented in Appendix S1. A significant cost-saving data recovery both within and across four sample sets representing measure of this protocol is the “freeze and squeeze” size selection three angiosperm families, four genera, and both silica-dried and approach during library preparation (Appendix S1, section I). This herbarium tissues. A double-digest RADseq (ddRADseq) protocol technique eliminates the need for a digital size selection apparatus was used to process all samples. The success of a single, streamlined (BluePippin; Sage Science, Beverly, Massachusetts, USA) in the user protocol incorporating a single restriction enzyme pair would re- lab. Rather, fragment analysis (TapeStation) and size selection of fi- duce upfront enzyme costs and allow for the simultaneous prepara- nal libraries (if needed) can be performed at the chosen sequencing tion of ddRADseq libraries from diverse sample sets. This flexibility facility for a modest fee. would allow a researcher to quickly assemble geographically and The Boechera/Draba and Solidago/Ilex pools were combined, taxonomically broad sample sets by utilizing herbarium DNAs, thus and each pool pair was sequenced with 150-bp paired-end sequenc- reducing the need for costly fieldwork and allowing for sampling ing on separate HiSeq 2500 lanes (Illumina, San Diego, California, regimes that would otherwise be unattainable. USA) at the University of Kansas Genome Sequencing Core. Demultiplexing, clustering, and SNP calling were conducted for each 48-sample pool using PyRAD version 3.0 (Eaton, 2014). The METHODS PyRAD settings for each of the data runs were as follows. In step 1, the restriction overhangs CATG and AATT (EcoRI and SphI) were Sampling and DNA extraction, desalting, and assessment specified, with data type “pairddrad.” Runs were performed on the SAVIO server of the University of California Berkeley Computing A detailed bench protocol is presented in Appendix S1, and de- Facility (one node with 20 tasks per node running in parallel), al- scriptive data for the 192 samples are included in Appendix S2. lowing zero barcode mismatches during demultiplexing. Before The 48-sample,