1.10.5
COMPARISON OF INHERITANCE OF MITOCHONDRIA IN HETEROKARYONS FORMED BY HYPHAL ANASTOMOSIS AND PROTOPLAST FUSION IN FUSARIUM OXYSPORUM F.SP. CUBENSE

DN KUHN, BR CORTES, N D'ALESSIO and R RAMEAU

Department of Biological Sciences, Florida International University, Miami, Florida 33199 USA

Background and objectives
Parasexuality requires the initial formation of heterokaryons followed by karyogamy and haploidization. Heterokaryons can be formed readily in Fusarium by hyphal anastomosis between vegetatively compatible strains and infrequently between strains in different vegetative compatibility groups (VCG). Such inter-VCG heterokaryons are morphologically indistinguishable from those formed within a VCG. Heterokaryons can theoretically be formed between any two strains by fusion of protoplasts. We have used molecular markers including single strand conformational polymorphisms (ssCP) and DNA sequencing to identify mitochondrial types (mitotypes) of the different Foc VCGs. We have investigated the inheritance of mitochondria in inter-VCG heterokaryons formed by both hyphal anastomosis and protoplast fusion. In addition, we have attempted to document the time course of the loss of one of the parental mitochondrial types.

Materials and methods
Foc strains were obtained from the worldwide collection of Dr Randy Ploetz at the IFAS Center, University of Florida, Homestead, Florida. Auxotrophic isolates used to force heterokaryon formation were isolated after UV mutagenesis. Putative diploids, referred to as hybrids, were identified in our lab from inter-VCG heterokaryons. Primers were designed to an intergenic region of the mitochondrial genome [1]. This 600-bp fragment was initially analyzed by SSCP as described in [2]. All SSCP variants were sequenced on the ABI 377 automated sequencer. SSCP was used to determine the mitochondrial composition of heterokaryons and hybrids.

Results and conclusions
Seven different mitochondrial types (mitotypes) for 14 VCGs in Foc were identified by SSCP and sequencing. As little as one VCG per mitotype and as high as 4 VCGs per mitotype were observed. We forced heterokaryon formation between strains assigned to different mitotypes to be able to detect the individual parental contribution of mitochondria in the formed heterokaryons. In addition, we analyzed 28 individual hybrids, the result of karyogamy in 28 individual heterokaryons. In heterokaryons formed by hyphal anastomosis, plasmogamy does not result in a mixing of mitochondria from both parents, but rather uniparental inheritance. One parental mitochondrial type is lost within the first ten days after heterokaryon formation by hyphal anastomosis. These same inter-mitotype heterokaryons have been formed by protoplast fusion and the mode and time course of mitochondrial inheritance is currently under investigation.

Funded by NIGMS/NIH grant number GM08205.

References
1. D'Alessio N, 1998. PhD dissertation, Florida International University.
2. Orita M, Susuki Y, Sekiya T, Hayashi K, 1989. Nucleic Acids Research 20, 6741-6742.