DNA extraction from old herbarium material of Veronica subgen. Pseudolysimachium (Plantaginaceae)

Abstract

Herbarium specimens have become a major source of information in molecular biodiversity research, framing the term "herbarium genomics". However, obtaining good DNA from old herbarium specimens is still a challenge. Currently, DNA extraction methods from old herbarium material often yield highly degraded and fragmented DNA. A number of studies have discussed such methods, especially how to avoid further DNA fragmentation. This study aims to compare different DNA extraction methods applied to old herbarium material from Veronica subg. Pseudolysimachium. One such method is a CTABbased DNA extraction followed by a clean-up with paramagnetic beads that is used in the Jodrell Laboratory, Royal Botanic Gardens Kew, UK. This method was compared to a modified NucleoSpin Plant II protocol, based on silica columns, as used at the Technical University Munich-Freising, which was already successfully used for extracting DNA from a Linnean type specimen. Further tests were conducted on the influence of incubation time on the CTAB DNA extraction protocol with a subsample of specimens. Our preliminary results suggest that CTAB DNA extraction might have some advantages in specific cases but also that silica column-based methods have fewer problems with contamination by polysaccharides and polyphenolic compounds. Regarding the incubation time, we did not observe a clear pattern, but we developed several ideas on how to proceed with tests to find an optimal DNA extraction protocol to deal with highly fragmented DNA. Taking practical considerations into account, the column-based method proves to be preferable, especially when trying to reduce the amount of leaf tissue used, but further modifications of both methods should be explored.

Using herbarium samples for NGS methods – a methodological comparison

“…Older samples had in general lower yield, especially when using the commercial extraction kit. Our results contrast those of other studies (19,39), where no correlation was found between age of the specimens and DNA yield. The reason of this discrepancy might be explained by sampling peculiarities.…”

“…The first studies on ancient or archival DNA (aDNA) from plants were published in the early 90s of the last century and dealt with plant remains in archaeological sites (e.g., (15,16) among others). Studies dealing with DNA extraction from old herbarium samples have used one of the following approaches: i) Early studies on herbarium material aiming at sequencing single markers, e.g., ITS, simply used standard CTAB protocols for extraction (17) or a modified version of it (12,(18)(19)(20)(21); ii) commercial kits were used with few adaptations, e.g., increasing incubation times (18,19,22,23); or, iii) more specific protocols for aDNA extraction were applied (e.g., optimized to obtain short sequences and to increase the proportion of endogenous DNA in the extracts; (10,12,13,24). Since then, more and more studies included historical plant material in phylogenomic studies (7,23,25,26).…”

“…Although a few recent studies focused on comparing the efficiency of CTAB-based extraction protocols with commercial kits (19), or comparing CTAB extractions with protocols specific for aDNA (10), no studies yet have investigated the circumstances in which aDNA methods (e.g., the PTB-DTT protocol described in (28)) should be preferred to commercial kits when extracting DNA from old and damaged herbarium material. For systematists (systematic botanists) extraction kits represent still the easiest and most convenient solution for DNA extraction.…”

Strategies for reducing per‐sample costs in target capture sequencing for phylogenomics and population genomics in plants

“…Extra modifications (recommended when polysaccharides and secondary metabolites are abundant) include sorbitol and high‐salt (4 M NaCl) CTAB (e.g., for mucilage‐rich tissue, Tel‐Zur et al., ; Štorchová et al., ) or 2% polyvinylpyrrolidone (PVP) (e.g., for latex‐rich tissue, Michiels et al., ). When phenol use is restricted (e.g., in light of health and safety concerns), additional clean‐up steps are recommended, such as column (e.g., CsCl‐EtBr density gradient centrifugation, Albach and Chase, ; Ahmed et al., ; Höpke et al., ) or bead cleaning (e.g., solid‐phase reversible immobilization [e.g., SPRIselect beads, Beckman Coulter, Indianapolis, Indiana, USA]; Shee et al., ), and even additional modified CTAB protocols optimized for plant lineages with high levels of polysaccharides and secondary metabolites (Štorchová et al., ; Sahu et al., ).…”

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“…In recent years, assessing techniques of DNA isolation and sequencing from herbarium plant material has gained broad interest (e.g. Rogers & Bendich, 1985;Goff & Moon, 1993;Savolainen et al, 1995;Porebski, Bailey & Baum, 1997;Blattner, 1999;Drábková, Kirschner & Vlĉek, 2002;DeCastro & Menale, 2004;Jankowiak, Buczkowska & Szweykowska-Kulinska, 2005;Cota-Sánchez, Remarchuk & Ubayasena, 2006;Telle & Thines, 2008;Soni & Kumar, 2009;Lehtonen & Christenhusz, 2010;Staats et al, 2011;Särkinen et al, 2012;Staats et al, 2013;Shepherd & Perrie, 2014;Shepherd, 2017;Do & Drábková, 2018;Höpke et al, 2019). The primary aim of such technical investigations is to establish appropriate methods of efficient DNA analysis from preserved plant specimens, taking into account the specificity of such source material.…”