Objective 1 – To isolate the mating type gene from E. lataFurther attempts to clone fragments of mating type genes (idiomorphs MAT1-1 or MAT1-2) have so far not been successful. Primers designed to all conserved regions of the three MAT1-1 genes and the MAT1-2 gene have been tested in all possible combinations, under a range of conditions, without success. PCR amplification using primers designed to one of the yeast mating type genes gave a product of the expected size, but this was also obtained when using one of the primers alone, hence is unlikely to be a genuine mating type gene product.
Objective 2 – Production of the sexual stage in cultureThe sexual stage of our single ascospore isolates of a Diatrypaceous fungus has been achieved in culture and optimization experiments have reduced the time required from 47 weeks to 17-19 weeks. However, all perithecia produced have around 32-40 ascospores per ascus. Given the similarity to E. lata of culture morphology, anamorph and ITS product size, but not sequence, it would appear that the multispored condition is not a facet of the incubation conditions but an indication that we have either a very unusual strain, or, a closely related fungus. Progress has been made in developing molecular methods to distinguish parents from one another and to determine the parentage of progeny. RAPD-PCR has been used to identify polymorphic loci and specific SCAR (Sequence Characterised Amplified Region) primers have been designed to these. Analysis of progeny from a single ascus of a cross of mixed parentage suggests homothallism (i.e. selfing, not a cross between the different isolates. To further clarify conditions under which perithecia can be produced and to define conditions under which heterothallism can be achieved, we are currently investigating further crosses in which both parents are known.
Objective 3 – To use the tools developed in objectives 1 and 2 to determine the distribution of mating types in the natural environment. Work on this objective has not commenced since it was entirely dependent on identification of the MAT genes in Objective 1.