Detection and Identification of Neotyphodium Species from DNA Extracted from Single Seeds Stored in Ethanol.
2013
Walid Naffaa | Koya Sugawara
Seed samples of three Lolium species from Syria, as well as two Lolium, four Festuca and one of Melica sp. from France were submerged in ethanol and shipped to Japan for analysis. Living seeds of three Lolium and one Festuca cultivar from Japan and USA were also used for comparison. Microscopic observations showed the presence of typical mycelium of Neotyphodium endophytes in 90 - 100 % of the seeds. Genomic DNA from individual seeds in ethanol, and from living seeds were extracted with the same protocol. PCR amplification of all DNA samples , using universal primers for rDNA-ITS region and specific primers for Neotyphodium species (Noc1 and / or Noc2), have successfully generated amplicons from the genome of endophytes in most cases, regardless of storage conditions of the seeds. Among SSR markers for Neotyphodium species, primers for loci B9 and B11 generated amplicons of expected size. This study demonstrated the possibility to detect and identify the Neotyphodium endophytes from single seeds stored in ethanol which can make shipping living samples between countries easy.
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