DNA Damage in Plant Herbarium Tissue

Martijn Staats,Gitte Petersen,Ole Seberg,James E Richardson,Ria Vrielink-Van Ginkel,Argelia Cuenca,Freek T Bakker,Carles Lalueza-Fox

doi:10.1371/journal.pone.0028448

Abstract

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4–3.8% of fresh control DNA and 1.0–1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

Highlights

The world’s approximately 3400 herbaria contain an immense number of plant specimens covering virtually all known species, making herbaria invaluable assets for understanding plant biodiversity [1,2], and largely underutilised genomic treasure troves
The expansion of next-generation sequencing (NGS) capabilities will potentially open up possibilities for cost-effective sequencing of genomes from type specimens and rare or extinct species stored in herbaria [3]
We investigated whether polymerase misincorporation errors alone explain the levels of miscoding lesions observed or whether they represent true damage-derived lesions in herbarium DNA

Summary

Introduction

The world’s approximately 3400 herbaria (http://sciweb.nybg. org/science2/IndexHerbariorum.asp) contain an immense number of plant specimens covering virtually all known species, making herbaria invaluable assets for understanding plant biodiversity [1,2], and largely underutilised genomic treasure troves. Org/science2/IndexHerbariorum.asp) contain an immense number of plant specimens covering virtually all known species, making herbaria invaluable assets for understanding plant biodiversity [1,2], and largely underutilised genomic treasure troves. The expansion of next-generation sequencing (NGS) capabilities will potentially open up possibilities for cost-effective sequencing of genomes from type specimens and rare or extinct species stored in herbaria [3]. DNA extraction results in irreparable damage to specimens, which conflicts with their historic and scientific importance, typically only a few milligrams of herbarium material need to be sampled. Up until twenty years ago, herbarium specimen preparation techniques were not aimed at preserving DNA. Commonly used collection methods involved chemical treatments of specimens with formalin or ethanol, both of which severely affect DNA preservation in plants [7,10,11]

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