Feasibility of using dried plant specimens for DNA barcoding. A case study of the Juncaceae Danka Do 1 , Lenka Záveská Drábková Corresp. 1, 2 1 Department of taxonomy, Institute of Botany Academy of Sciences of the Czech Republic, Průhonice, Czech Republic 2 Laboratory of pollen biology, Institute of Experimental Botany Academy of Sciences of the Czech Republic, Prague, Czech Republic Corresponding Author: Lenka Záveská Drábková Email address: [email protected] Background. Phylogenetic and barcoding studies usually employ fresh parts of plants as the source of DNA. Successful DNA amplification has been achieved in such investigations for different regions. However, there is need for the utilization of dried samples, due to frequent inaccessibility of fresh precious plants or their parts for genetic analyses or barcoding studies. Difficulties in obtaining amplifiable DNA have appeared as one of the major pitfalls that resulted in slowdown of the use of herbarium specimens for DNA analyses. Methods. Recent study highlights the crucial issues that are being faced by comparison of herbarium and fresh plants for barcoding purposes. We analyzed the performance of samples from herbarium specimens of different age and fresh plants in PCR reaction and sequencing of seven regions (cpDNA: rbcL, rpoC1, trnL-F intergenic spacer, trnL intron, psbA-trnH, mtDNA: atp1 and nrDNA: ITS1-5.8S-ITS2) with a combination of twenty-eight primers. Conclusions. We show that herbarium specimens may be successfully applied both for phylogenetic as well as for barcoding purposes. In comparison with fresh samples, working with dried herbarium specimens is more complicated, but may lead to amplification and sequencing success in almost all cases when appropriate internal primers are designed or optimization methods are used. Both attempts are useful for this aim: using the set of universal primers recommended by CBOL and design specific primers for a particular group of interest. We found limited detrimental effect of specimen age and length of the amplicon on the amplification success in most of the tested regions in the Juncaceae. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2293v1 | CC BY 4.0 Open Access | rec: 15 Jul 2016, publ: 15 Jul 2016 1 Feasibility of using dried plant specimens for DNA barcoding. A case study of the 2 Juncaceae 3 4 DANKA DO1 and LENKA ZÁVESKÁ DRÁBKOVÁ1,2 5 6 1 Department of Taxonomy, Institute of Botany, Academy of Sciences of the Czech Republic, 7 Zámek 1, CZ-252 43 Průhonice and 2 Laboratory of Pollen Biology, Institute of Experimental 8 Botany Academy of Sciences of the Czech Republic, Rozvojová 263, 165 02 Prague 6 - 9 Lysolaje, Czech Republic, [email protected] 10 11 Running title: Are dried samples appropriate for DNA barcoding? 12 13 ABSTRACT 14 Background. Phylogenetic and barcoding studies usually employ fresh parts of plants as the 15 source of DNA. Successful DNA amplification has been achieved in such investigations for 16 different regions. However, there is need for the utilization of dried samples, due to frequent 17 inaccessibility of fresh precious plants or their parts for genetic analyses or barcoding studies. 18 Difficulties in obtaining amplifiable DNA have appeared as one of the major pitfalls that resulted 19 in slowdown of the use of herbarium specimens for DNA analyses. 20 Methods. Recent study highlights the crucial issues that are being faced by comparison of 21 herbarium and fresh plants for barcoding purposes. We analyzed the performance of samples 22 from herbarium specimens of different age and fresh plants in PCR reaction and sequencing of 23 seven regions (cpDNA: rbcL, rpoC1, trnL-F intergenic spacer, trnL intron, psbA-trnH, mtDNA: 24 atp1 and nrDNA: ITS1-5.8S-ITS2) with a combination of twenty-eight primers. 25 Conclusions. We show that herbarium specimens may be successfully applied both for 26 phylogenetic as well as for barcoding purposes. In comparison with fresh samples, working with 27 dried herbarium specimens is more complicated, but may lead to amplification and sequencing 28 success in almost all cases when appropriate internal primers are designed or optimization 29 methods are used. Both attempts are useful for this aim: using the set of universal primers 30 recommended by CBOL and design specific primers for a particular group of interest. We found PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2293v1 | CC BY 4.0 Open Access | rec: 15 Jul 2016, publ: 15 Jul 2016 31 limited detrimental effect of specimen age and length of the amplicon on the amplification 32 success in most of the tested regions in the Juncaceae. 33 34 Keywords: DNA barcoding, dry plants, herbarium specimens, Juncaceae 35 36 INTRODUCTION 37 A traditional way of conserving specimens of higher plants is a preparation of herbarium sheets, 38 traditionally stored in the botanical collections. There are approximately 3,400 herbarium 39 collections through the world (Thiers, continuously updated). They provide the comparative 40 material essential for studies in taxonomy, phylogenetics, systematics, anatomy, morphology, 41 conservation biology, biodiversity, ecology and many other fields. Moreover, they represent a 42 veritable gold mine of information for comparative DNA studies. 43 Barcoding studies, potentially applicable in many areas of research and practice, may 44 allow rapid identification of the unidentifiable plant material. This may be useful not only in 45 specific cases of basic plant identification for research purposes and in forensic applications, 46 including the verification of composition of food supplements is required. In all these cases, only 47 dried plant material is often available for genetic analyses. Therefore, reliable techniques 48 enabling employment of dried plant material for such analyses of DNA are desirable. 49 There exist several prerequisites necessary for establishing an investigated DNA region 50 as a barcode. Among the most important issues, it must simultaneously contain unique identifier, 51 be short enough to be sequenced in one reaction and it should also contain invariant regions for 52 developing primers (e. g., Chase et al., 2007; Sass et al., 2007). The requirement for a short 53 length of DNA sequences may favor dried plant tissues as their DNA is fractioned into low 54 molecular weight fragments. However, there may be several pitfalls using this type of DNA. For 55 example, DNA may be broken in invariant primer potential region. In this case, the region has 56 lower potential and PCR reaction conditions should be optimized. The factors affecting the 57 quality and usefulness of DNA from herbarium samples and consequently the efficiency of DNA 58 analyses are known insufficiently. As the analyses of dried plant material are relatively scarce in 59 comparison with fresh material, the systematic studies focused on elucidation of these factors are 60 needed for DNA barcoding. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2293v1 | CC BY 4.0 Open Access | rec: 15 Jul 2016, publ: 15 Jul 2016 61 Many previous studies have been concentrated on testing different extraction/isolation 62 techniques to obtain DNA with high yield and quality from herbarium samples (e.g., Rogers, 63 1994; Erkens et al., 2008; Andreasen et al., 2009; reviewed Záveská Drábková, 2014; Choi et al., 64 2015). However, many pre-analytical factors may affect the quality and quantity of isolated 65 DNA, and subsequently the results of further applications. The way of drying the plant material 66 may be a considerable parameter as it has been suggested to be as rapid as possible to obtain 67 high-quality DNA (e.g., Záveská Drábková, 2014). Other factors may be associated with 68 material conservation after drying, for example application of fungicides and insecticides. 69 Herbarium material is usually very old. Protocols and guidelines for DNA extraction and 70 sequencing from plant herbarium specimens can be traced back to eighties of the 20th century. 71 However, obtaining DNA sequences from herbarium specimens can be far from routine even in 72 recent years. As the sufficient quality of DNA is essential for the success of the whole molecular 73 study, optimization steps in extraction methods, PCR and sequencing protocols are often needed. 74 Unfortunately, these required modifications may differ in relation to a particular taxonomic 75 group (Hollingsworth et al., 2011). 76 Elucidation of above-mentioned factors is necessary for establishing the DNA barcoding 77 techniques based on dried conserved plant material. This will have a considerable importance for 78 interpretations of how realistic is DNA barcoding on conserved plant material. In the present 79 study, we provide a systematic investigation of PCR amplification, optimization and sequencing 80 efficiency from herbarium specimens of different age, and in fresh samples across the model 81 monocot family Juncaceae. We tested twenty-eight primers for seven regions (rbcL, rpoC1, 82 trnL-F intergenic spacer, trnL intron, psbA-trnH, atp1 and ITS1-5.8S-ITS2), most of them being 83 universal within the plant kingdom. We also applied different PCR optimization tests based on 84 differential temperature and cycling protocols including PCR additives. The main effort of the 85 study was to address and answer these questions: (1) Are DNA samples of old herbarium 86 specimens with usually degraded DNA (checked on the gel) useful for DNA barcoding in spite 87 of problematic methodology? (2) Is a successful DNA amplification correlated with age of the 88 specimen and differs between fresh and dried samples? (3) Is the amplification success 89 dependent on length of the amplicon? (4) Is it possible to use universal primers recommended by 90 CBOL PWG for herbarium specimens? 91 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2293v1 | CC BY 4.0 Open Access | rec: 15 Jul 2016, publ: 15 Jul 2016 92 MATERIAL AND METHODS 93 Plant sampling 94 The Juncaceae family comprise eight genera and about 450 species.