characterizes drug accessions upon sequencing the THCAS gene in thirteen dif-
ferent strains ofC. sativawith different THCA contents and CBDA/THCA ratios.
Some of thefiber-type accessions sequenced, however, also had a THCAS-like,
1635 bp-long sequence, fully translatable, characterized by a huge number of SNPs
(over 60) and aminoacid substitutions (about 38) compared to drug-type THCAS.
The presence of these sequences infiber-type accessions only, suggested that,
despite the presence of a full open reading frame, they were not expressed (not
transcribed or not translated, or translated in a non-functional enzyme) and therefore
did not contribute to thefinal chemotype (Kojoma et al. 2006 ). Nevertheless,
several breeders and geneticists observed that whilst it is possible to obtain a drug
strain which does not produce any detectable amount of CBDA, the complete
elimination of an exceedingly small amount of THCA fromfiber accessions or
varieties is apparently not attainable (E. de Meijer, personal communication),
suggesting that these sequences might not be entirely non-functional. From a
practical point of view, the sequence-based distinction between“drug-type”and
“fiber-type”THCAS, allowed Kojoma’s group to develop a PCR marker solely
associated to fully functional, drug-type THCAS (see also Rotherham and Harbison
2011 ). TheB1080/B1192marker, developed by Pacifico et al. (2006; see above),
was observed to be designed on significantly diverging regions from those
described by Kojoma et al. ( 2006 ), and for this reason they maintain full association
with chemotype in both homozygous and heterozygousCannabisplants.
The application of this information to forensic sciences has also led to new
marker-based tools; Staginnus et al. ( 2014 ) recently characterized two segregating
populations by a PCR-based specific marker, and confirmed that sequence poly-
morphisms detected in THCAS were completely linked to either THCA-
predominant or THCA-intermediate chemotypes of the plants. Kojoma’s
“drug-type”sequences were used to design chemotype-specific primers encom-
passing four SNPs which produced a band only in individuals endowed with at least
one copy of the THCAS sequence, and could therefore correctly recognize the 111
forensic casework samples chosen to test the reliability of the assay.
15.4.3 Single Nucleotide Polymorphisms
in Chemotype-Determining Genes
It is becoming increasingly clear that the“catalogue”of different THCAS and
CBDAS sequences in various accessions ofC. sativais far from being complete, as
distinct versions of putatively functional synthases have been identified over the last
10 years following the discovery of Kojoma’s sequences (van Bakel et al. 2011 ;
Onofri et al. 2015 ; Weiblen et al. 2015 ; McKernan et al. 2015 ). Today, the new
challenge seems to be that of associating chemotypes to sequences and developing
a vision that is able to combine the simplicity of the results of genetic analysis,
suggesting monogenic inheritance, with the underlying complexity of the molecular
structure in the genome region including the chemotype-determining genes.
334 C. Onofri and G. Mandolino