plant surface microbiology.pdf

– Primers for ITS amplification: FGPS1490 (5¢–TGCGGCTGGATCACCTC-
CTT–3¢) and FGPS132¢ (5¢–CCGGGTTTCCCCATTCGG–3¢), 10 mM stock
solution in ddH 2O. These primers are selected for Rhizobia and may apply
to other bacterial groups, however specific primers for particular genera
can be designed on known 16S and 23S sequences retrieved from GenBank
or Ribosomal Database (RDP)
– 10-base random primers for RAPD (series OP from Operon Technologies),
80 mM stock solution in ddH 2O. The choice of the primers is highly relevant
for the usefulness of the results obtained (see the RAPD principles section
for details)
– DNA size marker: good examples are a 100-bp ladder for agarose gel electrophoresis
and TAMRA 500 (Applied Biosysem, PE) for capillary electrophoresis
– Genomic DNA: for RAPD and ITS concentrations at 10 ng/ml in ddH 2 O. For
the AFLP, use concentrations at 50 ng/ml. For general extraction protocols,
see Bazzicalupo and Fancelli (1997).Alternatively, use BIO 101 DNA extraction
kit
Note: All the above reagents should be kept at –20 °C
– TAE buffer: 40 mM Tris/Acetate, 1 mM EDTA, pH 8. Prepare a 50x stock
solution
– ethidium bromide stock solution: 10 mg/ml; store in a dark bottle
– agarose
– 10x loading buffer: 70 % (w/v) glycerol, 0.5 % (w/v) bromophenol blue;
store at 4 °C
Caution: Ethidium bromide is a powerful mutagen: wear gloves when handling
this compound; wear mask when weighing it
3 RAPD
28 Analysis of Microbial Population Genetics 553
Principle
The RAPD assay (Welch and McClelland 1990; Williams et al. 1990) is a PCR
amplification performed on genomic DNA template using a single short, arbitrary
oligonucleotide primer and low annealing temperature, conditions that
ensure the generation of several discrete DNA products. Each of these fragments
is derived from a region of the genome that contains two primer binding
sites on opposite strands and at an amplifiable distance. Polymorphism
between strains results from sequence differences which inhibit or enhance
primer binding or otherwise affect amplification. Single base mutations,
insertions and deletions are molecular events that produce RAPD polymorphism.
The large number of bands amplified with a single arbitrary primer
generates a complex fingerprinting that can be utilised to detect relative differences
in the random amplified DNA sequences from two different
genomes. RAPDs have been applied to bacterial population genetics for sev-