(500 and 730 bp in size) which were detected in all male plants but were absent in
female plants. These two DNA fragments were cloned and used as probes in gel
blot analysis of genomic DNA. When the male and female DNAs were allowed to
hybridize with the 500 bp probe, no differences in patterns were observed between
these plants. In contrast, when the DNAs were allowed to hybridize with the 730 bp
probe, much more intense bands specific to male plants were detected, in addition to
less intense bands that were common to both sexes. The 730 bp DNA fragment was
named MADC1 (male-associated DNA sequence in Cannabis sativa). The
sequence of MADC1 did not include a long open reading frame and it exhibited no
significant similarity to previously reported sequences. In a study by Mandolino
et al. ( 1999 ), RAPD analysis revealed the occurrence of a 400 bp band which was
consistently found only in male plants. Following sequence characterization of this
MADC2, a low homology (54.8–59.8%) was found to retrotransposon-like ele-
ments in plants but not to MADC1. Interestingly, the sequence was shown to be
present in both male as well as female plants, suggesting it was not specific to the
male chromosome. Primers developed from this region were used to specifically
distinguish male from female plants based on different-sized bands produced fol-
lowing PCR. Sakamoto et al. ( 2005 ) conducted further RAPD analysis to identify
additional male-specific bands in hemp (MADC3–771 bp in size and MADC4–
576 bp in size) which were characterized as retrotransposable elements and
reported to be present on the Y chromosome as well as on other chromosomes in
male plants. Torjek et al. ( 2001 ) reported on additional male-specific sequences
MACS 5 and MADC6 which were not homologous to any previous sequence.
During our research on the development of a new PCR-based test to distinguish
male from female plants of marihuana and hemp at the seedling stage of
growth (Chen et al. 2015 ), we analyzed DNA samples of leaves from growers and
breeders across North America and a few from Europe. The PCR analysis con-
sistently showed the presence of 2 bands in all male plants (560 bp and 390 bp in
size) and only one band in female plants (560 bp) (Fig.19.13). In a few plants that
were hermaphrodites (showing maleflowers in addition to primarily femaleflow-
ers), all were observed to be genetically female. In a population of seedlings derived
from germinated seed, we observed a 4:6 segregation of male: female plants
(Fig.19.13). This PCR-based method was developed into the GreenScreen Plant
Sex ID kit (www.screenyourgreen.com/).
19.14 Future Directions
One of the obstacles to obtaining a complete understanding of the genetic com-
plexity of cannabis is the current lack of genetic information on commercially used
strains of marihuana that are deposited in a verified database (Welling et al. 2016 ).
Given the vast number of strains with unique names developed by seed companies,
breeders and home enthusiasts, this would be a vast undertaking. However, the
inclusion of information of the genetic background and parentage of the most
19 Assessing Genetic Diversity inCannabis sativa... 413