Neurospora crassa is a model pyrenomycete fungus for which there are MAT-2 (= mat a) HMG box primers available, hence it is an ideal positive control. Isolates of both mating types (mt a and mt A) were obtained from the Fungal Genetics Stock Center (FGSC), genomic DNA was extracted and used in PCR with the primers NcHMG1 & NcHMG2. PCR conditions were optimised to yield a product of the expected size (300 bp) with the mt a strain. DNA sequence analysis of the 300 bp product confirmed its identity as part of the MAT (mt a) gene. In Southern blots (580C) the 300 bp fragment hybridised to N. crassa mt a genomic DNA but not to N. crassa mt A or E. lata DNA. This confirmed our expectation that the overall nucleotide sequence identity between the N. crassa and E. lata MAT-2 HMG boxes would not be sufficiently high to allow identification of the E. lata MAT-2 by hybridisation. However, the PCR conditions developed for N. crassa provided a starting point for PCR using E. lata as a template.
Four NZ isolates of E. lata were initially available in our laboratory. It was not known whether these were mating type 1 or 2 (or heterothallic). However in order to increase the probability that at least one of them carried a MAT-2 gene it was important to dismiss the possibility that they were genetically identical to one another (clonal), as found with other fungal plant pathogens (e.g. Dothistroma pini) in New Zealand (Hirst et al 1999; NZ J Forest Sci. 29:459). Molecular profiles were generated with microsatellite-based primers (anchored PCR; Ganley 2000, Massey University Masterate thesis). The profiles showed extensive polymorphisms between each of the isolates.
The NcHMG1&2 primers were tested with the four isolates of E. lata. It was not known what size intron (if any) would be present in the E. lata MAT-2 HMG box, hence the PCR products were expected to be = 215 bp. Despite extensive testing with different PCR conditions, no abundant distinctive PCR products of the expected size were seen. Two limitations of this work were that (a) the E. lata sequences might differ from those of N. crassa and (b) the isolates tested so far could all be MAT-1 rather than MAT-2. Further strategies were therefore developed to address these limitations.
MAT-2 HMG box amino acid sequences of pyrenomycete fungi were obtained from the GenBank database and aligned using Gene Jockey 2. The positions of the Neurospora crassa primers designed by Arie et al 1997 (Fungal Genetics and Biology 21:118) are indicated. The upstream (5?) region (primer NcHMG1) is highly conserved between all the pyrenomycetes for which sequence information is available. However, the downstream (3?) region (primer NcHMG2) is variable in amino acid sequence between the different species. Although Eutypa species are most closely related to Sordaria and Neurospora species, new primers were designed for use with E. lata based on the sequences of two species of Gibberella (primer GfHMG2) and Podospora anserina (primer PaHMG2) to maximise the probability of successful PCR amplification.
Use of the new primer combinations (NcHMG1 with GfHMG2 or PaHMG2) with N. crassa genomic template gave an amplified product of the expected size as well as additional products. Several of the E. lata isolates gave abundant products in the expected size range with the NcHMG1 + GfHMG2 combination. Amplification with single primers (e.g. GfHMG2 only) was also carried out to identify which products were only produced in the presence of both primers (and hence were more likely to be genuine MAT gene products). Direct sequencing of one of these products yielded a mixed sequence, hence this and other PCR products ranging in size from 215 ? 300 bp are currently being cloned in E. coli prior to further sequence analysis.