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The PCR products were labeled with SybrGold
fluorescent dye (Invitrogen, Canada) and were loaded
on 5% polyacrylamide gels in GS2000 (Corbette
Australia). Electrophoresis was performed at 1 200
volt constant power in TBE (1×) buffer as a running
buffer, and stopped depending on the real-time
dimension of digital image and expected product size
of each primer set. Molecular sizes of the amplified
fragments were estimated using a 100 bp ladder.
Digitally captured image subjected to future analysis.
3.4 Data analysis
Data were scored as presence (1) or absence (0) of a
band. Only distinct and well-separated bands were
included in the analysis. The polymorphic information
content (PIC) was calculated according to Anderson et
al. (1993). The Observed and effective number of
alleles, Shannon information index (Lewontin, 1972)
and Nei's genetic diversity (Nei and Li, 1979) were
estimated in POPGEN program (Yeh et al., 1997).
Relationships among individuals were estimated
by unweight pair-group mean analysis (UPGMA) by
using NTSYS pc 2.02e program (Rohlf, 1998).
Cophenetic correlation was used to choose the best
clustering method and similarity coefficient. According
to cophenetic correlation results, a similarity matrix
was generated using Simple Mathing. Principal
Coordinate Analysis (PCo) and Genetic distances (Nei,
1972) were conducted by using GenAlex v.6.2. This
multivariate approach was chosen to complement the
cluster analysis information, because cluster analysis
is more sensitive to closely related individuals,
whereas PCo is more informative regarding distances
among major groups (Hauser and Crovello, 1982).
Acknowledgements
Special thanks to the all foresters in the provinces throughout
Iran who helped us to collect the samples.
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