The researchers used Suppression Subtraction Hybridization PCR (SSHP) to create two gene libraries for a peanut-infecting Sclerotium rolfsii strain using a CLONTECH PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, Calif). One library was enriched for genes more highly expressed in sclerotia than in hyphae, while the other was enriched in the reverse manner. Individual clones from these libraries were then selected, sequenced, and compared to sequences available in the National Center for Biotechnology Information (NCBI) database.

The team identified a method using cellophane filters to cleanly isolate sclerotia at each of the early, mid and late stages of sclerotium development, and used this method to collect more than a gram of tissue from each stage, and over a gram of mycelial tissue. They then isolated and purified RNA from the tissues and used them in the SSHP gene library kit. Ms. Takach generated two libraries of approximately 1,000 clones apiece and showed that they contained appropriately sized inserts, suggesting that the libraries were of good quality.

At the time of this report (10/10/06), sequence analysis of the libraries was underway and bioinformatic analysis of the sequences was soon to begin, which was expected to increase understanding of the expression of genes vital to the critical resting structures of this important peanut pathogen. Ms. Takach was planning to conduct expression blot analysis at a future date and publish a report detailing her findings. She presented her results from this project at the Annual Phytopathology Society Conference, held in Quebec in the summer of 2006.