Authors: Schnittler, Martin & Dagamac, Nikki H.A. & Leontyev, Dmitry & Shchepin, Oleg & Novozhilov, Yuri K. & Klahr, Anja
Journal: Karstenia, Volume 58 (2020), Issue 2, pages 385-392.
Full text: PDF
Key words: DNA barcoding, DNA extraction, elongation factor 1 alpha, direct PCR, spore collection, small ribosomal subunit, spore
Abstract: We present a workflow for efficient barcoding of myxomycete fructifications, which (i) requires less than 1000 spores, (ii) allows to collect spores with only a needle, (iii) works without any commercial kits, and (iv) is optimized for the use of 96-well PCR plates throughout the process. Specimens of 291 dark-spored nivicolous myxomycetes and 121 bright-spored members of the Trichiaceae were sequenced for the barcode marker 18S rDNA (SSU) with a low rate of failure and no detectable cross-contamination. Crude DNA extracts can be stored for further analyses: the elongation factor 1 alpha gene (EF1A), a single-copy marker, was successfully amplified after four weeks of storage.As such our procedure will allow a time- and cost-efficient barcoding of large series of specimens.
Supplement 1: Collecting procedure for spores
(Video at https://youtu.be/0A4kTLNt9L8).
Supplement 2: Documentation of preparation
steps, necessary equipment and time for
barcoding a 96 well plate of myxomycetes
Supplement 3A–C: List of specimens
sequenced and BLAST statistics