Identification of Epichloë Endophytes In Planta by a Microsatellite-Based PCR Fingerprinting Assay with Automated Analysis

Abstract

Epichloë endophytes are a group of filamentous fungi that include both sexual (Epichloë) and asexual (Neotyphodium) species. As a group they are genetically diverse and form both antagonistic and mutualistic associations with temperate grasses. We report here on the development of a microsatellite-based PCR system for fingerprinting this group of fungi with template isolated from either culture or infected plant material. M13mp19 partial genomic libraries were constructed for size-fractionated genomic DNA from two endophyte strains. These libraries were screened with a mixture of DIG-labeled dinucleotide and trinucleotide repeat probes. Positive clones were sequenced, and nine unique microsatellite loci were identified. An additional microsatellite was serendipitously identified in the 3′ untranscribed region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from N. lolii Lp19. Primers were designed for each locus and a panel of endophytes, from different taxonomic groupings, was screened to determine the degree of polymorphism. On the basis of these results a multiplex assay was developed for strain identification with fluorescently labeled primers for five of these loci. Using this system the size of the products amplified can be precisely determined by automated analysis, and an allele profile for each strain can be readily generated. The assay was shown to resolve endophyte groupings to the level of known isozyme phenotype groupings. In a blind test the assay was used successfully to identify a set of endophytes in planta. A reference database of allele sizes has been established for the panel of endophytes examined, and this will be expanded as new strains are analyzed.

Global Genetic Diversity of the Perennial Ryegrass Fungal Endophyte Neotyphodium lolii

“…The markers have been shown to display high levels of cross-species transfer in in vitro-based studies, null alleles being less frequent than for genomic DNA-derived SSR markers (Moon et al, 2000(Moon et al, , 2004van Zijll de Jong et al, 2003). In contrast to some of the more commonly used methods, such as histological, serological and immunoblot detection methods (Belanger, 1996;Hiatt et al, 1999;Musgrave, 1984), SSR markers have been shown here, and in other studies, not only to detect endophyte but also to discriminate diff erent taxa and even strains of endophyte (Moon et al, 1999;van Zijll de Jong et al, 2003. This level of discrimination has not been reported for sequence tagged site markers (Dombrowski et al, 2006;Doss et al, 1998).…”

“…As for genomic DNA-derived SSR markers (Groppe and Boller, 1997;Moon et al, 1999), expressed sequence tag (EST)-derived SSR markers were highly eff ective for in planta endophyte detection. The effi ciency of marker conversion from in vitro to in planta-based detection was high (>80%), both for tillers and seeds.…”

“…These groupings correlated with alkaloid profiles of isolates. Later studies performed with simple sequence repeat (SSR) markers also detected low levels of genetic variation (Moon et al, 1999;van Zijll de Jong et al, 2003). To date, only a restricted number of isolates have been genetically characterized.…”

“…Simple sequence repeat markers have proven to be informative, in comparisons with isoenzyme and amplifi ed fragment length polymorphism (AFLP) markers, for assessment of endophyte genetic variability (Moon et al, 1999;van Zijll de Jong et al, 2003) and are suitable for in planta detection of endophytes. However, only a small number of SSR markers have so far been developed for this application (Groppe and Boller, 1997;Moon et al, 1999). Additional markers for sensitive in planta detection are described in this study.…”

Mutualistic fungal endophytes in theTriticeae- survey and description

“…All SSR primers were synthesised by Integrated DNA Technologies, Inc. (Coralville, IA). PCR using B10 and B11 was performed as described by Moon et al (1999), except that a 10 lL reaction volume was used. For all other SSRs, PCR amplifications were conducted in a 10 lL reaction volume as per Faville et al (2004) except that a final concentration of 2.5 mM magnesium chloride and 0.75 units of Platinum Taq DNA polymerase (Life Technologies, Carlsbad, CA) were used.…”