A MICROSATELLITE MARKER FOR STUDYING THE ECOLOGY AND DIVERSITY OF FUNGAL ENDOPHYTES (EPICHLOE SPP) IN GRASSES

Randomly amplified polymorphic DNA fingerprinting, which is based on PCR with arbitrary 10-nucleotide primers, were used to analyze genetic diversity among isolates of the endophytic ascomycete Epichloe typhina, which were collected at a single field site from a population of one of its hosts, the grass Bromus erectus. One of the polymorphic randomly amplified polymorphic DNA PCR products occurred in all isolates as single bands with different but closely related sizes. Two of the size variants of this product were cloned and sequenced, and they were found to represent the same DNA sequence, except for a stretch of tandem repeats of the trinucleotide AAG . TTC, which differed in size, consisting of 8 and 18 repeats, respectively. Tandem repeats of this type are called microsatellites. Oligonucleotides were synthesized corresponding to portions of the sequence flanking the microsatellite and were used for PCR amplification of the loci from the genomic DNAs of different Epichloe isolates. A single PCR product was found for most isolates, indicating that the sequence represented a single genetic locus. Five alleles that could clearly be distinguished in size were found in a population of 91 field isolates. PCR with (AAC)(8) and (AAG)(8) as primers yielded a number of amplified bands from genomic DNA of Epichloe isolates, indicating that these types of microsatellites occur frequently in the genome of this fungus. A survey of all fungal DNA sequences currently deposited in the DNA sequence databases of EMBL and GenBank revealed that microsatellites of different repeating units are widespread in fungi. Our findings indicate that microsatellite-containing loci may be used as molecular markers for population studies of Epichloe species and many other unrelated fungi.