B190/B200proved to be only partially able to correctly identify the chemotype of
individuals belonging to other pedigrees.
The allelic nature of the two genes, which code for the two main synthases of
C. sativa,appeared to be confirmed when their gene sequences were made available
in the early years of 2000 by Taura’s group. The THCAS coding sequence was
found to be 1635 bp long, with no introns and it shared 84% identity with the
CBDAS. The availability in the GenBank database of these two sequences
(accession nrs. E33090 and E55107; Sirikantaramas et al. 2004 ; Taura et al. 2007 )
has created the opportunity to design gene-specific markers, identifying the allelic
status of a plant at the locus determining chemotype well beforeflowering, and
much more economically than by gas-chromatographyc analysis. A new
multiple-PCR marker (B1080/B1192) based on the GenBank sequences of THCAS
(E33090) and CBDAS (E55107), and fully predictive of chemotypes I, II and III
simultaneously (Fig.15.3 c) was soon developed (Pacifico et al. 2006 ).
However, it soon appeared clear from the growing body of sequencing data on
differentCannabisaccessions, that the monogenic inheritance model did not rule
out the simultaneous presence in theCannabisgenome of more than the single
coding sequences identified by Taura’s group, necessary to determine the chemo-
type. Kojoma et al. ( 2006 ), identified a THCAS drug-type haplotype, which
Fig. 15.3 aBulk segregant analysis-deriving RAPD markers associated to the two main
chemotypes (Tchemotype I bulks;Cchemotype III bulks); the different pairs of samples
correspond to DNA bulks from different segregating progenies.bassay of the chemotypes with the
B190/B200marker, deriving from partial sequencing of the chemotype-associated RAPD
fragments;cassay with theB1180/B1192marker, based on the THCAS and CBDAS gene
sequences. The last two markers are designed as multiplex assays in such a way to be codominant,
due to the co-presence of both fragments upon amplification of chemotype II (heterozygous)
plants. Note that in the case shown, chemotype IV (high CBGA) and chemotype V (zero
cannabinoids) plants are read by the marker as CBDA, chemotype III plants, though this is not
always verified (see text for a discussion)
15 Genomics and Molecular Markers inCannabis sativaL. 333