Amplification of cox2 (~620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens, Comparing 19 Extraction Methods and 15 Polymerases

Abstract

During the past years an increasing number of studies have focussed on the use of herbarium specimens for molecular phylogenetic investigations and several comparative studies have been published. However, in the studies reported so far usually rather large amounts of material (typically around 100 mg) were sampled for DNA extraction. This equals an amount roughly equivalent to 8 cm2 of a medium thick leaf. For investigating the phylogeny of plant pathogens, such large amounts of tissue are usually not available or would irretrievably damage the specimens. Through systematic comparison of 19 DNA extraction protocols applied to only 2 mg of infected leaf tissue and testing 15 different DNA polymerases, we could successfully amplify a mitochondrial DNA region (cox2; ∼620 bp) from herbarium specimens well over a hundred years old. We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens. Through a combination of suitable DNA extraction protocols and DNA polymerases, only a fraction of the preserved plant material commonly used is necessary for successful PCR amplification. This facilitates the potential use of a far larger number of preserved specimens for molecular phylogenetic investigation and provides access to a wealth of genetic information in preserved in specimens deposited in herbaria around the world without reducing their scientific or historical value.

Muyocopronales, ord. nov., (Dothideomycetes, Ascomycota) and a reappraisal of Muyocopron species from northern Thailand

“…Methods for sample preparation, DNA extraction and PCR reaction were carried out according to Telle and Thines (2008). Fungal colonies were grown on MEA at 25 °C for two weeks.…”

Phylogenetic investigations in the genus Pseudoperonospora reveal overlooked species and cryptic diversity in the P. cubensis species cluster

“…DNA-extraction, PCR and sequencing DNA was extracted using the method best suited for herbarium samples as described in Telle and Thines (2008). For amplification of the mitochondrial cox2 gene, primers designed by Hudspeth et al (2000) were employed as described previously (Choi et al 2007), but using the MangoTaq Polymerase (Bioline, Luckenwalde, Germany), adding BSA to a final concentration of 8 μg/ml in the PCR reaction mix.…”

Phylogenetic investigations in the downy mildew genus Bremia reveal several distinct lineages and a species with a presumably exceptional wide host range

“…DNA-extraction, PCR and sequencing DNA was extracted using the Innupure Plant DNA extraction kit, with the modifications for herbarised material described in Telle and Thines (2008). For amplification of the mitochondrial cox2 gene, the primers described by Hudspeth et al (2000) were employed as described previously (Choi et al 2007b), but using the MangoTaq Polymerase (Bioline, Luckenwalde, Germany), and with the addition of BSA to a final concentration of 8 μg/μl in the PCR reaction mix.…”