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Kazarina, Anna [1], Dea, Hannah I. [2], Castro-Ross, Ayanna [1], Ary, Evie [1], Loeke, Terry [3], Lee, Sonny [1], Platt, Tom [1], Jumpponen, Ari [1].

Performance comparison of direct DNA extraction and amplification kits: promising tool to analyze fungal communities.

Next-generation DNA sequencing (NGS) analyses of fungal communities that inhabit plant tissues have become increasingly commonplace. Although these NGS tools are expedient and cost-efficient, the nucleic acid isolation usually requires substantial monetary and time investment. A variety of tools exist to acquire environmental DNA and so-called direct extraction and amplification tools designed for plant tissues provide an alternative to more commonly used extraction systems. We sampled leaf and root tissues from a common prairie plant Schizachyrium scoparium (little bluestem) from four grassland plots at two different times for a total of eight samples and compared direct extraction and amplification kits (Phire Plant Direct PCR  - ThermoFisher Scientific, Wilmington, Delaware; E.Z.N.A. Plant Direct -  Omega Bio-tek, Narcoss, Georgia; Extract-N-Amp - Invitrogen, Carlsbad, California) with a conventional DNA isolation kit (PowerSoil DNA Isolation Kit - Qiagen, Hilden, Germany). Pilot experiments indicated that all four systems permitted PCR amplification of the ITS2 region using fITS7 and ITS4 primers. We selected one of the three direct kits (Phire Plant Direct PCR) for further comparisons and analyzed pairs of ITS2 MiSeq libraries generated from DNA isolated with either the Phire or MoBio system and amplified with identical reagents under identical reaction conditions. The two DNA isolation systems provided comparable community data for root samples but not for leaf samples. These data suggest that the direct plant PCR kits bear a promise as a tool for expedient and cost-efficient tool to generate fungal community data albeit differences in tissues may require further validation of these systems.

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1 - Kansas State University, Division of Biology
2 - Kansas State University, Biology
3 - University of Kansas, Ecology and Evolutionary Biology

DNA extraction
Amplification kit
Direct extraction

Presentation Type: Poster
Session: MYP3, Mycology Posters III
Location: /
Date: Wednesday, July 21st, 2021
Time: 5:00 PM(EDT)
Number: MYP3015
Abstract ID:996
Candidate for Awards:MSA Best Poster Presentation Award by a Graduate Student

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