Kayis et al. ( 2010 ) demonstrated that both methods provided similar results with
respect to identifying genetic diversity and polymorphisms. However, the ISSR
method was shown to provide a slightly higher resolution of samples compared to
RAPD’s. Patzak ( 2001 ) reported a similarfinding while comparing several molec-
ular methods used for assessment of genetic diversity in hops (Humulus lupulusL.).
In our research, we have studied the extent of genetic diversity among marijuana
strains that can be characterized as landraces, as well as cultivated strains from a
broad range of geographic locations and sources. In total, the DNA from over 75
such samples were analyzed with a set of 7 ISSR primers (Table19.1), selected on
the basis of production of multiple and reproducible bands in repeated experiments.
The methods used for polymerase chain reaction (PCR) and the analysis of
results were conducted as described by Punja et al. ( 2015 ). Briefly, PCR amplifi-
cations were performed in a volume of 25lL. Each PCR reaction contained
0.1lM of primer, 1 unit of Taq DNA Polymerase, 200lM of dNTP’s, 1.5 mM
MgCl 2 , 20 ng template DNA, and 1PCR buffer. Amplifications were carried
out in an M&J Research PTC-225 gradient cycler with a heated lid programmed at
94 °C for 3 min for initial denaturation, followed by 94 °C for 30 s, 50 °C for 30 s,
and 72 °C for 3 min for 45 cycles and then afinal extension step at 72 °C for
7 min. After amplification, each PCR reaction was subjected to electrophoresis on a
2% TAE agarose gel and visualized under UV light. Gels were scanned with a
Bio-Rad Gel Imaging System and analyzed with Quantity One analysis software
version 4.3.0 (Bio-Rad Laboratories, Inc.). The sizes of the PCR products were
compared with the molecular size standard (1 kb plus) DNA ladder. Only
well-separated bands of 0.1–4.0 kb size with high intensity were scored as present
or absent for ISSR markers. Data were scored as 1 for the presence and 0 for the
absence of DNA bands in each sample. Each set of experiments was repeated three
times to ensure consistency of results. Based on the scorings of consistent and
reproducible bands, a composite group of data was subjected to phylogenetic
analysis to establish relationships and degree of genetic variation among various
strains of marijuana. Data from all 7 discriminating primers were used in the
analysis to reveal inter-relationships amongst the lines selected as well as the extent
of genetic diversity. The FAMD (Fingerprint Analysis with Missing Data 1.31)
software was applied to generate the Neighbor-Joining trees (Schlüter and Harris
2006 ).
Table 19.1 ISSR primers
used to assess genetic
diversity among strains of
marijuana in this study
UBC 807 5’-AGAGAGAGAGAGAGAGT‐ 3 ’
UBC 808 5’-AGAGAGAGAGAGAGAGC‐ 3 ’
UBC 817 5’-CACACACACACACACAA‐ 3 ’
UBC 825 5’-ACACACACACACACACC‐ 3 ’
UBC 834 5’-AGAGAGAGAGAGAGAGYT‐ 3 ’
UBC 842 5’-GAGAGAGAGAGAGAGAYG‐ 3 ’
UBC 845 5’-CTCTCTCTCTCTCTCTRG‐ 3 ’
Primers originally described by Lata et al. ( 2010 , 2011 ) were
used. Primers were obtained from the Biotechnology Laboratory,
University of British Columbia (UBC), Vancouver, B.C404 Z.K. Punja et al.