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Hairul Azman ROSLAN et al.
Table 2. PCR amplification parameters using the M13 primers and OPD-10
Separation of DNA fragments by gel
electrophoresis
Parameters Temperature and Reaction time
Initial denaturation 94ºC for 3 minutes
Denaturation 84ºC for 30 seconds
Annealing 46ºC / 30ºC for 1 minute (M13 /
OPD10)
Extension 72ºC for 2 minutes
Number of cycles 35
Final extension 72ºC for 7 minutes
The amplicons were separated using 1.3 % (w/v)
agarose gel electrophoresis in 1X TAE (Tris-Acetic
acid-EDTA) buffer. The electrophoresis was
performed at 100V for 90 minutes. The gel was
visualised using ethidium bromide under UV
transilluminator and documented using Gel
Documentation System (BioRad).
Construction of phylogenetic relationships
Each individual RAPD band was considered as
equivalent independent characters and all the bands
were scored as present or absent for each isolate.
Banding patterns were converted into binary tables.
The data was analyzed using genetic data analysis
software, Numerical Taxonomy and Multivariate
Analysis System (NTSYSpc) version 2.2. The data
was quantified by similarity index, Jij = Cij / (ni +
nj – Cij), where Jij= the number of individuals i and
j, ni= the number of bands in individual i, nj= the
number of bands in individual j. A dendogram was
generated using Unweighted Pair-Group Method
with Arimethrical Averages (UPGMA) as
described by Sneath and Sokal (1973).
Results and Discussion
Morphological groupings of Penicillium isolates
All the isolates were initially identified based on
the cultural characteristics and structure of
conidiophores using stereo and compound
microscopes. Among the 20 isolates, 4 isolates
were grouped as Clade 1, 2 isolates were grouped
as Clade 2, 3 isolates were grouped as Clade 3, 2
isolates were grouped as Clade 4, 4 isolates were
grouped as Clade 5, 2 isolates were grouped as
Clade 6, and one isolate each for Clade 7, Clade 8
and Clade 9 respectively. Table 3 shows the clades
based on morphological characters, isolate name,
substrate it was found and origin of the isolates.
The detailed morphological classifications of
selected isolates are presented in Figures 2 to 7
below representing Clade 1 to Clade 6.