differences between drug andfiber accessions. This observation suggests once again
that the boundaries between drug andfiber accessions are relatively artificial, which
strengthens the opinion that describesC. sativaas a monospecific, highly variable
genus (Small and Cronquist 1976 ; de Meijer 2014 ; see for a different view Hillig
2005 ), especially when no stringent selection is applied, as in the cases of
monoeciousfibre cultivars, or offinely selected drug strains. Indeed, Forapani et al.
( 2001 ) found the lowest level of polymorphism and of variability within a highly
inbred breeding line, developed specifically for pharmaceutical exploitation. It has
been observed that such inbred lines, obtained indoor upon repeated selfing fol-
lowing partial sex reversion (de Meijer et al. 2003 ) show low variability even when
compared to drug strains (Forapani et al. 2001 ).
The development of SSR-based assays, employing a single-reaction six-plex
microsatellite tool (Mendoza et al. 2009 ) was applied to the analysis ofCannabis
samples from materials of unknown origin, including both marijuana and hemp
samples. Not only was this assay able to differentiate each sample, but it also
revealed mixtures, when present: the presence of more than two alleles at the same
locus, along with a peak imbalance higher than 30% for heterozygous alleles, was
regarded as proof of mixed samples. A total of 29 alleles across the six loci were
identified accounting for an average observed heterozygosity of 0.47; four alleles
were found to be unique to marijuana samples, and two alleles unique to hemp
samples; nevertheless, the 10% genetic variance between the two types of samples
revealed by AMOVA made them not genetically distinguishable.
Inter-Simple Sequence Repeats (ISSRs) differ from SSRs (microsatellites) since
they represent regions amplified by primers binding directly to SSRs and therefore
have the advantage that no information on the sequence is required to analyze this
kind of molecular markers, given that the degenerated primers used anchor to
simple repeats such as (CA)n. They were observed to generate a specific pattern of
bands useful in estimating the genetic variation among different samples of
C. sativa, although the study was based on a very small number of samples (9
individuals from three different strains; Kojoma et al. 2002 ).
A more numerous collection was recently considered by Zhang et al. ( 2014 ). In
their study the authors performed a parallel analysis of ISSR markers which cap-
tures and quantifies the genetic variations in a population, and chromosome markers
reflecting the results of accumulated changes at different evolutionary stages. The
set of 27 samples of Chinese nativeCannabiscultivars was subdivided infive
groups according to the genetic distances calculated (average genetic distance
0.3297) by ISSR genotyping and this subdivision was reflected in the karyotype
characterizations of the different samples.
Incidentally, ISSR markers also found successful application in a very different
task, i.e. the assessment of genetic stability of plantlets originating from different
conditions: from synthetic seeds from a mother plant after in vitro storage, and from
plants regenerated through organogenesis after several passages ofin vitroculture
(Lata et al. 2011 , 2010 ; see also Chap. 16 of this volume).
15 Genomics and Molecular Markers inCannabis sativaL. 323