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A crush on small fungi: An efficient and quick method for obtaining DNA from minute ascomycetes
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
Uppsala University, Music and Museums, Museum of Evolution.ORCID iD: 0000-0003-3021-1821
Uppsala University, Music and Museums, Museum of Evolution.
2018 (English)In: Methods in Ecology and Evolution, ISSN 2041-210X, E-ISSN 2041-210X, Vol. 9, no 1, p. 148-158Article in journal (Refereed) Published
Abstract [en]

We have developed a reliable technique for extracting DNA from single microscopic fungal thalli, including efficient cell disruption and transfer of cell content for subsequent polymerase chain reaction (PCR). The technique was primarily developed for members of the ascomycete order Laboulbeniales, which are minute fungi with tough cell walls that are exceedingly difficult to disrupt with standard extraction techniques. Our method makes routine amplification of DNA from single thalli possible, even from small species or poorly developed individuals. DNA release is accomplished in an entirely mechanical manner using an arbor press fitted with custom-made components. This approach has eliminated additional treatment such as laborious freeze-thaw cycles, enzymes, or lysing agents. The overall PCR success rate of 89% is comparable to or better than alternative protocols that make use of substantially larger amounts of fungal tissue. From 97% of the successful PCRs a total of 156 sequences from four gene regions were produced. Being able to restrict DNA extractions to a single thallus is critical to all genetic studies requiring data at the level of individual, e.g. population genetics. As all researchers working with minute uncultivable organisms in many respects face the same problems (effective handling of the material, small quantities of DNA etc.), the methodology described here has a potential to be widely applicable. Necessary custom-made components can be manufactured at fairly low cost by any precision-tool workshop using our detail drawings.

Place, publisher, year, edition, pages
2018. Vol. 9, no 1, p. 148-158
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-334516DOI: 10.1111/2041-210X.12850ISI: 000419821200015OAI: oai:DiVA.org:uu-334516DiVA, id: diva2:1159707
Funder
Helge Ax:son Johnsons stiftelse Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2018-08-16Bibliographically approved
In thesis
1. Contributions to the understanding of diversity and evolution in the genus Coreomyces
Open this publication in new window or tab >>Contributions to the understanding of diversity and evolution in the genus Coreomyces
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Laboulbeniales is an order of enigmatic ascomycete fungi that are obligate microscopic parasites of arthropods, which are usually non-detrimental. They typically display a high degree of host specificity, and the genus Coreomyces, which is the recurring theme of this thesis, is no exception. Coreomyces is a small aquatic genus parasitising water boatmen (Corixidae). Members in this genus present something that is unique for Laboulbeniales, position specificity. This means that a parasite is restricted to a specific position on the host. This thesis aims at elucidating the patterns of host and position specificity present in the genus Coreomyces by the use of molecular markers. A second aim is to describe new taxa encountered during the course of the project. Finally we wanted to reveal what mating system is present in a member of the Laboulbeniales, something that has never been done before. Understanding the mode of sexual reproduction and to get insights into mating type organisation can give important clues to how the species patterns we observe may have evolved. To achieve these goals we first had to develop a robust and simple molecular methodology that made use of as little material as possible, preferably a single thallus. Our results showed that position specificity indeed exists in this genus, but that it is not as strict as previously presumed, all species we analysed occupied more than one position. Further we were able to show that thalli found in the same position on different hosts constitute the same species, and also that two sister species utilise the same position. We conclude that, in most cases, growth position is more important than host species or host sex in species delimitation. We confirmed the presence of four discrete taxa, two of which were described as new species, Coreomyces confusus H. Sundb. et al. and C. dextrorsus H. Sundb. et al. Finally we were able to show that C. macropus and C. confusus are likely to display a homothallic breeding system.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 47
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1703
Keywords
ascomycetes, Coreomyces, Corixidae. DNA, DNA extraction, Fungi, ge-nome, host specificity, Laboulbeniales, mating types, MAT-loci, meth-odology, NGS, parasite, position specificity, systematics, taxonomy
National Category
Biological Systematics Evolutionary Biology
Research subject
Biology with specialization in Systematics; Biology with specialization in Evolutionary Organismal Biology
Identifiers
urn:nbn:se:uu:diva-357110 (URN)978-91-513-0405-2 (ISBN)
Public defence
2018-10-02, Lindahlssalen, Gamla Zoofysiologen (Hus 5), Norbyvägen 14, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2018-09-11 Created: 2018-08-16 Last updated: 2018-10-02

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Sundberg, HenrikEkman, StefanKruys, Åsa

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