19.7 Random Amplified Polymorphic DNA (RAPD)
Analysis of Cannabis
Several early studies have demonstrated the applicability of RAPD’s as a method to
distinguish among strains ofC. sativaoriginating from different geographic loca-
tions (Gigliano et al. 1995 ; Gillan et al. 1995 ; Jagadish et al. 1996 ; Shirota et al.
1998 ). One of the utilities of RAPD markers in hemp was described by Faeti et al.
( 1996 ), in which the variability among 13 cultivars and accessions of hemp was
assessed using 10 primers of arbitrary sequence. They showed that groupings of the
cultivars was correlated with their geographic origin (Italy, Hungary, Korea), and a
high degree of polymorphism was present. In a subsequent study by Forapani et al.
( 2001 ), 5 arbitrary decamer primers (Operon Technologies, Alameda, CA) were
used to assess the extent of genetic diversity among 6 varieties of hemp. The study
included a dioecious landrace, a dioecious selection from it, a cross-bred cultivar, a
monoecious variety, a strain containing THC (‘Northern Lights’), and an inbred
female line. The genetic complexity of each variety was investigated by deter-
mining the number of bands produced by the primers used, the number offixed and
polymorphic loci, the average allele frequency, and the heterozygosity. A good
correlation was found between these parameters and the genetic origin and breeding
strategy of each variety. The average polymorphism over all varieties and loci was
found to be high, at 97.1%. Heterozygosity ranged from 0.05 (female inbred line) to
0.26 (the cross-bred cultivar). A test based on allele frequencies suggested that
complete differentiation among all hemp varieties was possible (Forapani et al.
2001 ). From 102 markers, 99 had a variant in at least one variety, and the com-
bination of all 5 primers allowed discrimination among all 6 varieties. The lowest
variation was seen in the female inbred line. The use of RAPD analysis to separate
samples based on geographic regions within Turkey was confirmed by Pinarkara
et al. ( 2009 ).
19.8 Inter Sequence Simple Repeat (ISSR)
Analysis of Cannabis
Thefirst application of ISSR markers was to distinguish between 3 strains of hemp
(from France, Czech Republic and Japan) as described by Kojoma et al. ( 2002 ).
Primers UBC 808, 811, 827, and 834 (Biotechnology Laboratory, University of
British Columbia, Vancouver, BC) produced clear and reproducible polymorphic
bands which could readily distinguish among the 3 samples. Hakki et al. ( 2003 ,
2007 ) used inter simple sequence repeats (ISSR) to distinguish hemp (9 samples)
from marijuana (23 samples) obtained from different locations in Turkey. A high
degree of polymorphism (93.7%) was observed using 18 primers, and hemp
samples could be distinguished from marijuana samples. When comparing the
RAPD method with ISSR with a similar group ofC. sativa plant samples,
19 Assessing Genetic Diversity inCannabis sativa... 403