1. Gene expression during fruit body initiation modulated by environmental parameters in Agaricus bisporus Bram HERMAN, D.C. Eastwood, S. Sreenivasaprasad, A. Dobrovin-Pennington, R. Noble, K.S. Burton Warwick HRI, University of Warwick, Warwick, CV35 9EF, U.K. Mushroom initiation - the phase change of the homobasidiomycete Agaricus bisporus from vegetative mycelium to reproductive growth is induced by manipulating a series of environmental parameters during cultivation (‘airing’). Little is known on the molecular mechanisms controlling this process or the function of each environmental parameter (‘black box’). Probe and microarray design Established a database containing ~450 differentially expressed sequences identified from mushroom initiation stages using cDNA suppression subtractive hybridisation and 1160 publicly available sequences from A. bisporus Designed up to three 50-60 oligomer probes for each of the 1110 unique sequences in the database Developed a custom oligonucleotide microarray comprising 9,654 probes including replicates (Agilent Technologies) This information will assist in understanding the molecular basis of the phase change during mushroom initiation and the effect of each environmental trigger on A. bisporus fruit body development. Microarray validation RNA was extracted from different stages of mushroom development, including colonised casing and mature fruit bodies. This RNA was used in a series of 18 two-colour microarray hybridisation experiments to validate the various processes. Future work Design of a modified microarray comprising up to 2000 probes – 8 arrays/ slide using one reproducible probe for each sequence Expression profiling of A. bisporus genes during mushroom initiation to assess the effect of each environmental trigger at airing Identification of key initiation genes and their functional analysis (e.g. comparative bioinformatics and RNAi) Array hybridisations showed strong and reproducible signals (e.g. figure b, control probes). Analysis of the data identified reliable probes for ~90% of the A. bisporus unique sequences, including the majority of genes identified by SSH. Only ~8% of the probes and replicates hybridised inconsistently in self versus self hybridisations of RNA extracted from colonised casing (red and green probes in figure c). These results enabled the selection of reliable probes for future initiation experiments. pin stage colonised casing (a) Result of a self vs. self hybridisation of RNA from the pin stage (left) and a self vs. self hybridisation of RNA from colonised casing 24 h after airing (right). (b) Quality report of a self vs. self hybridisation with RNA from colonised casing 24 h after airing, illustrating the expected log ratios versus observed log ratios performance of spike-in controls. (c) The quality report showing the log ratios of non-control probes vs. the average of their signals, illustrating their degree of expression Custom microarray technology will be used to investigate the expression profile of A. bisporus genes under normal and modified growth conditions during the phase change. Comparative bioinformatics and functional analysis will be used to identify key initiation genes. a b c 2 mm diam. 5 mm diam.