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In situ PCR: applications and prospects in the study of
arbuscular mycorrhizal fungi
Talk session 2: Ecology
BAGO, BERTA1, LUC SIMON2 and YVES
PICHÉ1.
1Centre de recherche en biologie forestière
2Recherche en sciences de la vie et de la
santé, Pavillon C.-E. Marchand, Université Laval,
Québec, G1K 7P4 Canada
ICOM1 Abstract
The polymerase chain reaction (PCR) has been successfully
applied to the study of arbuscular-mycorrhizal (AM)
symbiosis, especially after the design of specific primers
targeting ribosomal genes. However, DNA extraction prior to
solution-phase PCR makes it impossible to directly correlate
nucleic acid analysis with the histological features of the
sample. Until recently, in situ hybridization protocols had
to be developed to address this question. In situ PCR, a
relatively new technique, allows both more sensitive and more
specific detection of the target sequence within specimens
fixed on microscope slides, permitting to spatially correlate
PCR results with cell morphology. Although it has never been
applied before to AM-research, in situ PCR could prove an
interesting tool in the study of AM genetics (e.g. to confirm
the presence of different alleles in AM fungal (AMF) genes,
to show possible different nuclear populations within a
single AMF spore), cytology (e.g. nuclei behavior,
distribution of different endophytes colonizing the same
plant) and physiology (e.g. expression of symbiosis-related
genes along the different stages of mycorrhiza formation). We
have developed a fluorescent in situ PCR protocol which has
allowed us to obtain specific amplification signal in the
nuclei of two AMF. The sensitivity and specificity of this
technique and its suitability for AM research will be
discussed.