1.8.19
MOLECULAR CYTOGENETIC ANALYSIS BY TWO-COLOUR FLUORESCENCE IN SITU HYBRIDIZATION IN NECTRIA HAEMATOCOCCA AND MAGNAPORTHE GRISEA

M TAGA1, T KAWADA1 and M MURATA2

1Department of Biology, Faculty of Science, Okayama University, Okayama 700-0082, Japan; 2Research Institute for Bioresources, Okayama University, Kurasiki 710-0046, Japan

Background and objectives
In recent years, chromosome analyses of fungi have relied mainly on pulsed-field gel electrophoresis (PFGE). Although a powerful tool, PFGE has drawbacks such as the limitation of resolvable DNA size and the lack of morphological information. Therefore, cytogenetic approaches are desirable to obtain data complementary to those from PFGE. The objective of this study is to establish fluorescence in situ hybridization (FISH) as a molecular cytogenetic method for Nectria haematococca (Nh) MPVI and Magnaporthe grisea (Mg), and to analyse their chromosomes by FISH.

Materials and methods
Specimens for FISH were prepared by the germ-tube burst method [1, 2], and the procedures of FISH have been described previously [2]. Probes were plasmid clones of rDNA, a cosmid clone (2H6) specific to 1.6-Mb conditionally dispensable chromosome (CD) of Nh and DOP-PCR-amplified DNAs from single-chromosome bands in PFGE gels. Probe labelling was done with biotin-14-dATP or digoxigenin-11-dUTP (DIG) either by nick translation (for plasmid and cosmid clone) or PCR, and the detection made with avidin-FITC or anti-DIG-rhodamine.

Results and conclusions
In both fungi, the probes successfully hybridized to mitotic metaphase chromosomes and interphase nuclei, and were detected by different fluorescence colours depending on the detection agents. Simultaneous identification of two chromosomes was possible by combining two probes labelled with different agents. With the PCR-prepared probe the entire metaphase chromosome was hybridized, indicating that each DNA band in PFGE gels corresponds to a chromosome observed by microscopy. With rDNA probes the nucleolus-organizer region (NOR) was visualized. In Nh, PCR-prepared probes revealed that 1.6-Mb CD and 1.1-Mb chromosome are not dispersed through the nucleus but are located in compact forms. The results of this study indicate that FISH is a very promising technique for chromosome analyses in fungal pathogens.

References
1. Shirane N, Masuko M, Hayashi Y, 1988. Phytopathology 78, 1627-30.
2. Taga M, Murata M, 1994. Chromosoma 103, 408-13.