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

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

Herbarium vouchers investigated.

1<p>BR: National Botanic Garden of Belgium, BP: Hungarian Natural History Museum, HOH: Herbarium of the University of Hohenheim.</p

Comparison of the performance of various DNA polymerases.

<p>Square fields indicate amplification of the ~350 bp fragment with different intensity. Black fields: amplicon amount >90 ng, dark grey: amplicon amount 30–90 ng, grey: amplicon amount 10–30 ng, light grey: amplicon amount <10 ng, white: no amplicon detectable, asterisks: very faint band visible (≪10 ng); white dots indicate additional amplification of the ~620 bp fragment. +: positive control, −: negative (water) control.</p

List of protocols used for DNA extraction.

<p>List of protocols used for DNA extraction.</p

Molecular phylogenetic analysis of Peronosclerospora (Oomycetes) reveals cryptic species and genetically distinct species parasitic to maize

Downy mildews are amongst the most widespread and economically important pathogens of cultivated grasses in the tropics and subtropics. Despite their importance, molecular methods, particularly DNA sequence analysis, have rarely been applied to either species identification or to the determination of phylogenetic relationships between species. Here we report the presence of several cryptic species in the genus Peronosclerospora. Further we confirm that maize can be parasitised by several species of Peronosclerospora, including P. eriochloae, which has not been reported previously as a pathogen of maize. The presence of 14 distinct phylogenetic lineages, including three that are parasitic to maize, highlights the current fragmentary knowledge on the diversity and classification of species within Peronosclerospora. Species identification in Peronosclerospora has been traditionally based on the host genus and a set of variable morphological characteristics, which has meant that the identification of species is often unreliable. This situation is primed for the application of molecular techniques for the identification of species. One of the lineages parasitic to maize in Australia has not yet been formally described and its distribution is not known. Future investigation including a broad sampling of downy mildews from maize and other cultivated and native grasses on a world-wide basis is a prerequisite to a re-evaluation of quarantine regulations aimed at restricting or limiting their spread