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Potential of marker-assisted selection in hemp genetic improvement


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The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based markers have also been exploited in the field of forensic science, in an attempt to discriminate licit from illicit crop. The main applications of the molecular markers to the breeding, however, have been achieved with the development of markers closely linked to the male sex and to some of the most relevant chemotypes. Active research is carried out by several groups in the field of identification and characterization of the genes involved in fiber quality and quantity, and in the determination of monoecy, another very important target of hemp breeding. Besides, markers associated to new, potentially useful chemotypes are being developed, for the marker-assisted breeding of pharmaceutical Cannabis.
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Euphytica 140: 107–120, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands. 107
Potential of marker-assisted selection in hemp genetic improvement
G. Mandolino&A.Carboni
Istituto Sperimentale per le Colture Industriali, Via di Corticella 133, 40128 Bologna, Italy;
author for correspondence: e-mail:
Key words: Cannabis, breeding, forensic, marker-assisted selection, molecular markers
The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-
1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP
and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based
markers have also been exploited in the field of forensic science, in an attempt to discriminate licit from illicit crop.
The main applications of the molecular markers to the breeding, however, have been achieved with the development
of markers closely linked to the male sex and to some of the most relevant chemotypes. Active research is carried out
by several groups in the field of identification and characterization of the genes involved in fiber quality and quantity,
and in the determination of monoecy, another very important target of hemp breeding. Besides, markers associated
to new, potentially useful chemotypes are being developed, for the marker-assisted breeding of pharmaceutical
Recently, a great expansion of applications and de-
velopments in the field of agricultural biotechnology
has occurred. Many of these developments rely on ge-
nomics’ advancements, and a remarkable part of the
achievements of this newly born science have been
transferred and exploited in the field of plant breeding.
However, the extent to which the biotechnologies were
exploited in the different crops, depended not only on
economic reasons (e.g. the area dedicated to the crop,
the added value of the crop itself, the economy relying
on the products obtained from that crop), but also on the
importance of that crop in the most advanced countries.
In this sense, the case of hemp is somehow peculiar. In
the United States, one of the most advanced countries
from the point of view of the development and applica-
tions of agricultural biotechnologies, hemp cultivation
is simply not allowed (Morris, 2002), and in several
countries the situation was no different, at least until
Despite the difficulties that hemp cultivation en-
countered in some countries, several advanced tech-
niques were exploited in the study of this non-food
species. Some of the most recent advancements will be
reviewed in other contributions of this special issue; in
this paper, we will focus on the state of the art of the
applications of molecular markers and genomics to the
study and genetic improvement of Cannabis sativa L.
A brief outline of the marker systems
used in Cannabis studies
Several papers reviewed the application and the re-
quirements of marker-assisted selection (MAS) in crop
plants (Mohan et al., 1997). Molecular markers em-
ployed in MAS should: (i) co-segregate or be tightly
linked (ideally, less than 1 cM) to the trait object of
selection; (ii) it should lend itself to a mass-screening
for the identification of the marker genotype in breed-
ing lines and populations; (iii) its validity should be
recognized in a laboratory-independent manner, i.e.
it should be reliable and reproducible in different
Historically, the earliest molecular markers to be
extensively used in population studies and in plant
breeding have been isozymes (Lewontin and Hubby,
1966). Isozyme variation has been exploited to a good
extent in plant breeding. The identification of isoen-
zymatic forms have been associated to specific traits
such as disease resistance (e.g. to root knot disease
in tomato; Rick and Fobes, 1974); the distribution of
the different variant isoforms in natural and domesti-
cated populations has been used to infer the genetic
structure and heterozygosity level of plant populations
(Brown and Weir, 1983). Besides, the codominant na-
ture of isoenzyme markers (i.e. the heterozygous plants
can be distinguished by both homozygous types for
the contemporary presence in a starch gel of both the
isoforms) made them particularly useful in the estima-
tion of heterozygosity and in studies of gene flow from
crop species to their wild relatives (e.g. Bartsch et al.,
1999). Isozyme markers are quite reproducible, but the
number of known loci corresponding to isoenzymatic
systems, is not very high; besides, obtaining a read-
able pattern of enzymatic activity may be technically
difficult in some plant material.
In Cannabis research, isozymes markers have been
exploited only to a limited extent. In recent studies
on the chemotaxonomy of the genus, in the frame of a
germplasm survey, more than 150 Cannabis accessions
were analyzed for isozyme variation (Hillig, 2004).
The second marker type used in plant breeding was
restriction fragment length polymorphisms (RFLPs).
RFLP markers rely on differences, at the genomic
DNA level, in the target sequences of the restriction
endonucleases; such differences lead to variant DNA
fragment length upon restriction, usually visualized
by agarose gel separation, followed by hybridization
of the immobilized fragments to a labeled (usually
radioactively) probe and autoradiography. An RFLP
marker is codominant and identifies one single locus at
a time; RFLP molecular maps were developed since the
1980s for several important crop species (Beckmann
and Soller, 1983; Tanksley et al., 1989). However, the
earliest applications of molecular biology techniques
to the study of Cannabis sativa date back only to
the mid-1990s; at that time, the main molecular tools
used in plant breeding and genetics were PCR based.
As a consequence, RFLP technology has not been
widely exploited in hemp. As far as we know, no RFLP
markers were developed for hemp, and the molecular
maps available today do not contain any of these
Since the introduction of PCR, the strategy of pro-
duction of molecular markers and of genetic analysis
changed. The earliest PCR-based markers to be ex-
tensively applied to plant breeding and MAS were
the RAPD markers (Williams et al., 1990). In this
case, PCR amplification is mediated by short decamer
primers of random sequence. Such primers find with
a certain frequency annealing sites on the opposite
strands of the target DNA molecule, so that an ampli-
fication product can be produced from the intervening
sequence. The annealing sites of the decamer primers
can be variable, and consequently some of the amplified
fragments will be polymorphic in the different DNAs.
The nature of RAPDs (and of many PCR-based mark-
ers) is dominant and “multilocus”. As it will be detailed
in the next sections, RAPD markers have been success-
fully applied in Cannabis germplasm analysis and MAS
research. The inherent limited between-laboratories re-
producibility of many RAPD markers, however, makes
in most cases necessary the isolation of the RAPD
bands found associated to a trait of interest. The rel-
evant RAPD fragments are gel-isolated, cloned and se-
quenced; specific 20-mer primers are then designed,
amplifying only the sequence found by genetic analysis
to be linked to the trait (SCAR markers: Sequence Char-
acterized Amplified Region). A further improvement
in marker producing techniques came from the devel-
opment of amplified fragment length polymorphisms
(AFLPs; Vos et al., 1995). The restriction digestion
of genomic DNA is followed by a PCR amplification
of the fragments obtained, mediated by labeled primers
able to anneal to the ends of the fragments, and having a
variable number of extra nucleotides randomly chosen,
so to amplify only a subset of the total fragments ob-
tained. The labeled amplification products are then run
in polyacrilamide gels. These markers are much more
reproducible than RAPD markers, are multilocus and
dominant; in recent years, the increasingly widespread
availability of automatic sequencers/genetic analyzers
machines, allowed the scoring of fluorescently labeled
AFLP fragments by capillary electrophoresis. The frag-
ments are identified as peak signals detected by cou-
pling charge devices (CCD), upon laser eccitation of
the fluorochromes. AFLP markers have been widely
used in hemp research, and one of the molecular maps
produced has been composed with these markers (see
Microsatellites (or simple sequence repeats, SSR)
are short sequences of two, three or more nucleotides
that are repeated for a variable number of times in the
genome. These markers are codominant; primers are
designed on the basis of the DNA sequences flank-
ing the repeat stretch, able to amplify the intervening
sequence by PCR. In general, one single locus is iden-
tified by each PCR reaction, but the number of alleles
that can be identified is very high, as the variability in
the repeated motif number is high in the plant genomes
(Morgante and Olivieri, 1993). As it will be discussed
in the next section, microsatellites have been only very
recently identified and used in C. sativa.
The forensic applications of DNA markers
The aims of forensic scientists working on Cannabis
were mainly two. First, they intended to develop meth-
ods capable of recognizing the presence of cannabis
(an illicit material in most countries), distinguishing it
from other plant sources. Secondly, there was an in-
terest to exploit molecular markers in analyzing sus-
pect plant material, so to reconstruct phylogenies of
the different drug strains, and possibly to infer, from
their relative distribution, the routes of diffusion of
such illicit crops. It must be therefore acknowledged,
that molecular tools have been exploited in Cannabis
by forensic scientists well before than by plant breed-
ers. The first aim has been accomplished by exploiting
sequences from chloroplast DNA (ctDNA), and par-
ticularly a short intergenic sequence located between
the chloroplast genes for the transport RNAs for the
aminoacids leucine and phenylalanine. It was found
that when specific primers flanking this sequence were
used, amplified products occurred only if the template
was made of C. sativa DNA; this simple test was devel-
oped in a patented product to be used by the law rein-
forcements organs (Linacre and Thorpe, 1998; Wilkin-
son and Linacre, 2000). Within this region, only limited
nucleotide polymorphisms was detected; this is not sur-
prising, as it is known that chloroplast DNA has both
avery low structural evolution and nucleotide syn-
onymous substitution rate. In fact, among the entries
present in NCBI for this sequences, 34 are actually
the same sequence obtained from different Cannabis
sources, by different authors, and are practically coin-
cident, with only a nucleotide missing in one sequence
compared to the others (Kohjyouma et al., 2000).
Another approach to identify C. sativa at the DNA
level exploited the properties of the Internal Tran-
scribed Spacers I and II (ITS1 and ITS2) of the nu-
clear ribosomal genes. Once amplified and sequenced,
this DNA region was found to distinguish univocally
C. sativa from any other plant species, including the
closely related hop; a limited number of single nu-
cleotide polymorphisms (SNPs) were found examining
five different Cannabis accessions (Siniscalco Gigliano
et al., 1997). As an alternative to the discrimination
based on sequencing, a cleaved amplified polymorphic
sequence (CAPS) marker test of the amplified ITS1
was also devised, allowing identification of hemp sam-
ples, but showing no within-species polymorphisms
(Siniscalco Gigliano and Di Finizio, 1997).
The approaches described earlier have been used to
distinguish Cannabis from other plants; it is our opin-
ion, however, that the real task, also from the forensic
point of view, remains the discrimination of drug from
non-drug strains. In a scenario in which C. sativa cul-
tivation for fiber, oil or pharmaceutical purposes be-
came widespread, such methods of discrimination of
licit from illicit crops are likely to be of limited use,
and to generate confusion, rather than to solve it. How-
ever, forensic researchers also contributed insights in
the plant genetic structure that are useful for hemp
breeders too. Protocols were established for the ex-
traction of DNA for AFLP analysis from marihuana
samples (Coyle et al., 2003), and RAPD (Gillan et al.,
1995; Jagadish et al., 1996) and ISSR (Kojoma et al.,
2002) markers were also used in an attempt to establish
a more causative relation between the cannabinoid type
of the plant and the markers identified. These attempts
however were not fully successful, and no correlations
of molecular markers with the gas chromatographic
or HPLC cannabinoid’s profiles of different Cannabis
germplasm were detected.
Microsatellites and the genetic variability
in Cannabis
Until recently, no information was available about
microsatellite loci in C. sativa.In2003, three different
groups of forensic researchers reported the isolation,
sequencing and use of different microsatellites in
the analysis of Cannabis germplasm (Alghanim and
Almirall, 2003; Gilmore and Peakall, 2003; Hsieh
et al., 2003).
Microsatellite markers were most useful in de-
scribing the hemp germplasm. It was found that the
most common repeated motif, the dinucleotide GA/CT,
is also the most frequently detected not only in the
close relative hop, but in general in the plant kingdom
(Alghanim and Almirall, 2003; Jake et al., 2001; Toth
et al., 2000). Di- and tri-nucleotide repeats were the
most frequent, with an allele number ranging from 2 up
to 28. The highest number of alleles detected was found
for an exanucleotide repeat (CACCAT), for which 30
alleles were detected in a survey of 108 plant samples
(Hsieh et al., 2003); in this work, genotypes are reported
showing up to four alleles of different size, suggesting
that this particular SSR was multilocus. Table 1 lists
the microsatellite loci described so far in Cannabis.
In most cases, amplification by primers flanking the
repeats yielded one or two bands; all the papers de-
scribing these loci, however, only examined Cannabis
germplasm (Gilmore et al., 2003); therefore, no data
Table 1. The microsatellites known in Cannabis sativa L.
Size range Number of
Locus Repeat (bp) alleles detected Reference
Dinucleotide repeats
ANUCS201 (GA)26 161–223 18 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS202 (GA)20 147–185 14 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS203 (CT)50 169–267 28 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS204 (CT)26 128–184 14 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS205 (CT)21 172–242 18 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS206 (AT)11 159–167 4 Gilmore & Peakall, For. Sci. Intl., 2003
C08-CANN2 (GA)21 171–203 9 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
H11CANN1 (CT)18 285–297 7 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
H09-CANN2 (GA)15 204–224 6 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
Trinucleotide repeats
ANUCS301 (TTA)15 209–261 13 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS303 (GTG)7141–156 5 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS305 (TGG)10 141–162 7 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS307 (ACC)6105–108 2 Gilmore & Peakall, For. Sci. Intl., 2003
B02-CANN2 (AAG)10 163–172 3 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
E07-CANN1 (CTA)9105–111 3 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
B05-CANN1 (TTG)9235–244 4 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
D02-CANN1 (GTT)7105–111 3 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
H06-CANN2 (ACG)7266–273 3 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
Pentanucleotide repeats
ANUCS501 (TTGTG)480–95 3 Gilmore & Peakall, For. Sci. Intl., 2003
Exanucleotide repeats
CS1F/CS1R (CACCAT) 18–240 30 Hsieh et al., For. Sci. Intl., 2003
Heterogeneous repeats
ANUCS302 (CAA)7-(CAA)4140–173 10 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS304 (TCT)8TCA(TCT)7167–230 15 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS306 (GAT)3-(GAT)692–95 2 Gilmore & Peakall, For. Sci. Intl., 2003
ANUCS308 (TAA)3-(AT)5177–203 8 Gilmore & Peakall, For. Sci. Intl., 2003
C11-CANN1 (GAT)8(GGT)7150–175 5 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
B01-CANN1 (GAA)13A(GAA)3323–339 5 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
D02-CANN2 (CTT)6ATT(CTT)10 221–236 4 Alghanim & Almirall, Anal. Bioanal. Chem., 2003
about the heritability of the alleles identified were pro-
vided in these studies.
Irrespective of the potential application in the foren-
sic field, some of these studies also provided insights on
the genetic structure of hemp. The variability found was
very high; in one study, out of 93 plants examined, only
four (belonging to the same drug accession) were not
distinguishable from each other (Gilmore et al., 2003),
while all the other presented different microsatellite
combinations at the different loci identified. The av-
erage allele number found in all the three studies was
9, but because the relatively limited number of plants
examined, it is likely that the identified loci have more
alleles than detected. The heterozygosity, or gene di-
versity, was found to be 0.83 in one study (93 plants,
15 loci) and 0.568 in another (average of 11 loci for
41 plant samples). Alghanim and Almirall (2003) cal-
ibrated their neighbor joining tree analysis performed
with SSR allele frequencies data, on the basis of previ-
ously known associations between the Cannabis sam-
ples they examined; they found a good agreement with
AFLP data. The extent of variability found was always
very high, except for samples probably deriving from
clonal propagation of single plants. Given the high level
of diversity found, the allele frequencies were on av-
erage quite low, usually below 0.30, except for a few
alleles occurring at high frequencies in cultivars with
a high extent of inbreeding, such as Fibrimon and a
drug strain (Gilmore and Peakall, 2003). In this work,
15 accessions were examined, and the molecular data
were used to estimate variance partition between and
within accessions. The majority of the observed marker
variation (73%) was attributable to individual differ-
ences within accessions, and only 21% of the varia-
tion was due to between-accession diversity. Besides,
when the accessions were divided into the two groups
“drug” and “non-drug”, it was found that only 6% of
the variation was attributable to the chemotype, and
therefore it was concluded by the authors that there
wasnoclear split or defined boundary between drug
and non-drug materials. Conceptually similar results
had been obtained also by our group in a survey of six
different varieties or inbred lines (10 plants per vari-
ety) examined by RAPD markers (102 loci; Forapani
et al., 2001); despite the fact that this analysis was con-
ducted using dominant, multilocus markers, there was
a basic agreement with Gilmore and Peakall’s findings,
essentially on two facts. First, the very high degree of
variation within the cultivars or accessions, account-
ing for over 50% of the total observed variation, that
was found still high even in inbred lines obtained af-
ter two cycles of inbreeding (31% of polymorphic loci
in an inbred female line, while for Fibranova, a cross-
bred variety, the value found was above 78%; see also
Faeti et al., 1996). Secondly, despite several specific
markers were found showing significantly higher fre-
quencies in certain cultivars, and therefore potentially
useful for variety discrimination, out of 102 markers
scored, 68 had a calculated FST value below the aver-
age (0.48); this finding indicated that the majority of
the loci had low discriminating power, and confirmed
by independent means the existence of a widely shared
gene pool in Cannabis, with limited cultivar boundaries
and relatively poor loci segregation between different
populations (Forapani et al., 2001). This holds true also
for drug and non-drug material, as later confirmed by
Gilmore and Peakall by SSR markers (2003).
The results reviewed show that different studies
point out that no wide split between drug and non-
drug accession is possible on the basis of the analysis
of the genetic structure of the different varieties and
accessions. While some cvs, like monoecious fiber cvs
or drug inbred lines, do have a genetic base narrower
than most fiber hemp accessions, no safe identification
(for the purpose of illicit cultivations repression) of a
single or limited number of drug or fiber plants is possi-
ble on the basis of the general distribution of molecular
markers. This can only be accomplished based on the
causal genetic determinants of the drug or non-drug
phenotype (i.e. of the chemotype); studies like those
described earlier, however, have a great interest from
the population genetics and genomic point of view.
Molecular maps
The extremely high degree of variation and het-
erozygosis found by different authors, especially in
dioecious hemp, suggested that many markers could be
present at the heterozygous state, and therefore could
potentially segregate in F1s, allowing the construction
of linkage maps.
Two non-saturated molecular maps of Cannabis
have been published so far. The first one derives from
a cross between monoecious plants found during a
screening for phenotypic traits in the accession CAN
19/86 (from Southern Italy, kindly provided by Dr.
Graner, IPK germplasm bank, Gatersleben). Some
of the plants having a female habit turned out to be
monoecious, and were used as pollen parents in a
cross with Carmagnola female plants (males were
eradicated before they were recognizable by using a
male-specific marker, see later). Other monoecious
plants of the 19/86 accession were also isolated to
produce S1-selfed seed. The F1 plants were raised,
their sexual phenotype scored at maturity, and screened
by RAPD markers (Carboni et al., 2000; Mandolino
and Ranalli, 2002). In the 19/86 ×Carmagnola cross,
182 markers out of 441 were polymorphic between
the two parentals (genotypes Aa and aa, respectively),
and segregated in the progeny. Among the 259 non-
polymorphic loci, 31 segregated 3:1 in the progeny
and therefore had genotype Aa in both parents. For a
limited number of non-polymorphic, non-segregating
loci, a complete genotyping was obtained from the
data relative to the segregation of the same locus in the
Figure 1. RAPD pattern of a monoecios plant belonging to the CAN 19 accession (lane S), and of part the selfed progeny obtained from it (lanes
1–17). Note the segregation of the markers present in heterozygosity in the parental plant. The figure is the negative of an ethidium-bromide
stained gel. In the first and last lanes, the molecular weight standards.
selfing S1 progeny (Figure 1). The markers formed 11
linkage groups in Carmagnola; the monoecious trait,
was found segregating 1:1 in the F1 offspring, but it
was not placed on the map at standard LOD scores,
along with several other markers.
The second map available for C. sativa, has been
constructed from a cross between male and female
plants of another germplasm accession, CAN 18 (Peil
et al., 2000), and should therefore enclose the male Y
chromosome. This AFLP map shows another charac-
teristics quite commonly found in AFLP maps of plant
genomes, i.e. the tendency of the markers to cluster in
“hot spot” along the linkage groups. The early map was
composed of five linkage groups, while a later version
enclosed 10 linkage groups (122 markers; Peil et al.,
2001). This latter work also shed some light on the
structure and properties of the Y chromosome, as it
will be discussed in the next section.
The high between-individual variability for molec-
ular markers found in hemp by different authors, limits
the practical utility of a genetic map for C. sativa;it
is possible that markers appearing closely associated
with important traits in one map will not be of general
use in MAS when used in different populations. This
is a general problem of many molecular markers, but
it might reveal particularly important in hemp. Despite
of this limitation, different mapping populations could
be used for the construction of dense, saturated maps
of specific regions of the genome, surrounding differ-
ent genes of interest (e.g. monoecy, chemotype). From
such regions of high marker density, it will probably be
possible to identify and isolate the genes by positional
cloning, and to obtain markers to be used in MAS.
DNA markers and sex phenotype
In nature, C. sativa is a dioecious plant; however, mo-
noecious varieties were also developed, and proved to
have distinctive advantages, such as their high seed
yield, a higher uniformity compared to dioecious va-
rieties, and ease of mechanical harvesting. Drawbacks
of monoecy are a narrower genetic base, necessary to
maintain the monoecious trait in a significant propor-
tion of the plants, and the necessity of strict isolation
and seed batch control, due to the lower competitive-
ness of monoecious pollen compared with pollen from
male plants, with consequent high probability of un-
intended pollination from dioecious males. In dioe-
cious varieties, it is in some cases necessary to identify
and score the male plants for fiber quality, and to al-
low the pollination only to the best-scoring males (the
Bredemann principle). Therefore, the practice of hemp
breeding requires in several occasions the identifica-
tion of the sexes (male, female, monoecious), with the
male sex being especially important for the different
strategies of improvement in dioecious and for quality
controls in monoecious hemp.
It is now a well-established fact that C. sativa is
endowed with heteromorphic chromosomes. The male
sex is characterized by a Y chromosome, reported to
be larger than the X chromosome, and presumably re-
sponsible for the extra 47 Mbp characterizing the male
genome, as measured by flow citometry (1636 Mbp
for females and 1683 Mbp for males; Sakamoto et al.,
1998). These authors also found that the satellite and
the long arm of Y chromosome were the earliest struc-
ture to condensate at the metaphase stage, suggesting
that its sequence composition might be peculiar and
different from the short Y arm, the X chromosome and
the autosomes. This characteristics is paralleled by dif-
ferent reports of DNA markers found to be specific of
male plants, or showing male–female polymorphism.
Sakamoto et al. (1995) found two male-associated
RAPD markers after a screening of five male and five
female plants with only 15 random primers. One of
these markers (730 bp long) included a 164 bp sequence
that, when used as an hybridization probe, yielded
male-specific patterns in EcoRI, BamHI and HindIII
digests of male and female DNAs. The whole male-
specific RAPD fragment was sequenced and named
MADC1; it did not include a long ORF, and no sig-
nificant homologies with other sequences present in
gene banks were identified. Sakamoto et al. did not de-
velop from this marker a SCAR marker or a rapid test,
suitable for identification of male and female plants
in MAS. Mandolino et al. (1997, 1998), upon RAPD
screening of Cannabis germplasm, identified a 400 bp
marker almost exclusively associated to the male phe-
notype (Figure 2a). This marker was cloned and se-
quenced, and specific 20-mer primers were designed.
These primers were able to amplify a 391 bp fragment
in all male plants, and two larger DNA fragments in fe-
male and monoecious plants (Mandolino et al., 1999).
It was therefore a marker exploiting the organization of
the genome regions surrounding the primer’s annealing
sites, rather than identifying a sequence solely present
in male plants. This sex-associated SCAR marker was
unable to distinguish male and female DNAs when it
was used as an hybridization probe in Southern blots of
DNA digested with several restriction enzymes. Dou-
ble digestion of genomic DNA and hybridization to
the isolated and cloned 400 bp fragment (Figure 2b)
suggested the possibility that the sequence (named
MADC2) corresponding to the marker was present in
more than one copy in the hemp genome (Figure 2c).
The two sequences, MADC1 (Sakamoto et al.,
1995) and MADC2 (Mandolino et al., 1999), shared
some features, like the G +C content (39.9% for
MADC1 and 40.4% for MADC2) and the absence of
significant ORFs. Like MADC1, MADC2 has limited
homology to other known sequences. However, all the
homologies reported by BLAST analysis, were with re-
peated regions or retrotransposon-like sequences; this
is also in agreement with the fact that MADC1 is a
LINE-like retrotrasposon, physically located on the Y
chromosome, as evidenced later by FISH (Sakamoto
et al., 2000). It was known that DNA from sex chro-
mosomes in different dioecious species are rich in
retrotransposon-like sequences (Clark et al., 1993), and
this also seems to be the case in C. sativa. The location
of MADC2 on the Y chromosome was suggested not
by direct evidence, but from genetic analysis; in fact, it
was shown that in different F1 progenies of a cross
between male and female plants, no recombination
between the SCAR marker developed from MADC2
and the sexual phenotype occurred (Mandolino et al.,
2002). Therefore, this marker satisfied the requirement
of general association to the male phenotype, irrespec-
tive of the population screened; as a consequence, it was
ideally suited for the development of a quick method
of sex identification, based on direct PCR of leaf frag-
ments. This “boil and amplify” method (Mandolino
and Ranalli, 2002) allows the 100% safe identification
of male plants in both dioecious and monoecious vari-
eties, at stages as early as necessary, with no need of
previous preparation of intact genomic DNA; only a
few milligrams of plant tissue are necessary to identify
the male plants.
Male-associated markers have been described also
using AFLPs (Flachowsky et al., 2001; Peil et al.,
2003). In this case, however, there have apparently been
problems in the correct scoring of the sexual pheno-
type, especially in the case of field-grown plants. The
reasons for these difficulties are not clear; the two ac-
cessions used for the study (CAN 17 and CAN 18) are
from IPK germplasm bank, and are originated from
Hungary and Germany, respectively. It is possible that,
at least in some cases, sex in these accessions was sub-
ject to incomplete expression, or partial reversion of the
phenotype; besides, it is known that the ploidy level can
influence the expression of the sexual phenotype, de-
spite the presence of a Y chromosome. Though these
phenomena have indeed been described in hemp, it is
difficult to explain why they should only occur in field-
grown plants. Therefore, the usefulness of these mark-
ers in MAS is still uncertain. In a later work, Peil et al.
(2003) assigned 43 more AFLP markers to the Y chro-
mosome, on the basis of the complete linkage of these
markers with the male sex in a progeny of 80 plants de-
riving from a male ×female cross; besides, the obser-
vation of the skewed segregation ratios of several other
markers led the authors to hypothesize the existence, on
the Y chromosome of male plants, of a region (pseudo-
autosomal region, PAR), able to recombine to some
extent with the corresponding portion of the X chro-
mosome, similarly to what observed in other dioecious
species with sex chromosomes (Di Stilio et al., 1998).
Other research groups described markers linked to the
male sex (NCBI nucleotide sequences AF364954 and
Figure 2. (a) RAPD pattern generated by the primer OPA08 (Operon Technologies, USA). The arrow indicate the 400 bp male-associated
marker, present in the male plants (lanes 1–5) and absent in the female plants (lanes 6–10). M, molecular weight standard. (b) Hybridization
pattern obtained using the cloned 400 bp RAPD fragment visible in a) on digested DNA from male and female plants restricted with HindIII
(H), XhoI (X) and with both enzymes (H +X). Note the absence of male–female polymorphism. (c) Restriction map of the genome region
containing the sequence corresponding to MADC2.
AF364955, MADC5 and MADC6, by Torjek et al.),
and also female-associated RAPD markers were re-
ported (Shao et al., 2003), though in the latter case
no chromosome-specific location can be hypothesized,
and it was not specified whether this marker was able
to distinguish female from monoecious plants.
The monoecious trait is another very important
character for which MAS would be extremely useful.
As stated earlier, it is already available a male-
identifying marker that can be fruitfully used in keep-
ing under strict control the number of “contaminat-
ing” male plants present in a monoecious stand or
seed lot, allowing for instance the introduction of the
“elite” or “superelite” terms for seed batches in a very
precise and controllable manner. The marker identi-
fying male plants can be therefore already exploited
in MAS and in seed quality certification of monoe-
cious varieties. However, the availability of a marker,
presumably autosomic, tightly linked to the monoe-
cious trait itself would be extremely important. Unfor-
tunately, no reports of such a marker have yet been
made until today; as described in the fifth section, the
monoecious trait did not integrate, under the mapping
conditions used, in the map of CAN 19 accession ob-
tained so far (Carboni et al., 2000). The identification of
monoecious-associated markers is also complicated by
the strong environmental influences altering the expres-
sion of the male flowers in monoecious plants, so that
under different conditions the ratio female:monoecious
plants in a monoecious seed lot can vary quite strongly.
In the CAN 19 (monoecious) ×Carmagnola (female)
cross described in fifth section, the monoecious trait
segregated 1:1, and the segregating groups can be used
for the identification of monoecious-associated mark-
ers through the bulk segregant analysis strategy (Paran
and Michelmore, 1993). Alternatively, these segregat-
ing materials, in which the genome background is
randomized into the two groups, could be used for
isolating the pertinent genes, via reverse genetics ap-
proaches (see also Moliterni et al., in this special is-
sue). The monoecious trait is presently one of the main
Figure 3. Chemotype distribution of the old Italian fiber cultivar “Eletta Campana”. This tripartite distribution is typical of old ecotypes before
the counter-selection for THC begun, and directly visualizes the distribution of BDand BTalleles.
targets for biotechnology applied to hemp research, and
it is likely that we will assist to major breakthroughs
in the next future; the availability of a male- and of a
monoecious-specific marker, possibly combined in a
single PCR assay, would allow the complete identifi-
cation of the sexual phenotype of plants belonging to
any variety.
Molecular markers for the chemotype
Today, it is generally accepted that three major “chemo-
types” (i.e. “chemical phenotypes”, plants character-
ized by a defined cannabinoid profile) exist in C. sativa;
these chemotypes were formally defined about 30 years
ago (Small and Beckstead, 1973) on the basis of the
content ratio of the two major cannabinoids found in
hemp, 9-tetrahydrocannabinol (THC) and cannabid-
iol (CBD), expressed as percent of the inflorescence
dry matter. The three chemotypes (I, prevalent THC;
II, THC and CBD; and III, prevalent CBD) were found
in several ecotypes and varieties, though some of the
most inbred materials, such as the French monoecious
cultivars, only showed two chemotypes (Fournier and
Paris, 1980). The tripartite distribution of chemotypes
(Figure 3) had suggested a simple genetic determinism,
based on one or few genes (Becu et al., 1998). However,
only recently accurate progeny analysis experiments
demonstrated the existence of one locus, named B, with
at least two alleles, BTand BD, each responsible for the
synthesis of the two most common cannabinoids, THC
and CBD, and probably coding for the respective syn-
thases (De Mejier et al., 2003; Mandolino et al., 2003).
Figure 4. The biosynthetic pathway of the most common cannabinoids in Cannabis plants. For each step, the relative enzyme has been indicated
(if known), and the state of the alleles at the B locus is proposed, accounting for the chemical phenotype. The inset shows the B190/B200
markers, obtained amplifying Cannabis DNA with the RAPD-deriving SCAR primers. Note the codominancy of this marker.
The model illustrating the enzymes involved
and the alleles reputed responsible for the differ-
ent steps of cannabinoid biosynthesis, is shown in
Figure 4. The condensation of geranygeraniol diphos-
phate with olivetolic acid (catalyzed by geranylgeran-
iol:olivetolate transferase, GOT; Fellermeier and Zenk,
1998) is the step leading to the first Cannabis’ exclu-
sive product, cannabigerol (CBG); this particular com-
pound was also described as the prevalent cannabinoid
in some plants (Fournier et al., 1987). These “mutants”
could not therefore be considered belonging to any of
the three formerly known chemotypes, and were as-
signed to a new chemotype (prevalent CBG, or chemo-
type IV; Figure 5). CBG is today widely accepted as the
common precursor for the synthesis of both THC and
CBD (Fellermeier et al., 2001). A further chemotype
was found, with an undetectable amount of cannabi-
noids. This “zero cannabinoid” type, (we propose for
it the creation of a chemotype V; see Figure 5 for its
gas-chromatogram) has been described by some au-
thors in different germplasm (G. Grassi and I. Virovets,
personal communication), though it is not yet clear
whether this absence was due to a metabolic block
at the level of GOT, or rather to a very limited for-
mation of glandular trichomes, the site of synthesis
and accumulation of cannabinoids (Kim and Mahlberg,
2003). Usually, CBG is detected in very small amounts
in Cannabis’ extracts, probably because it is almost
completely utilized as substrate by the downstream
synthases (THC- and CBD-synthases), transforming
it into THC, CBD or other less common end prod-
ucts (such as cannabichromene, CBC; Figure 4). The
two synthases are respectively coded by BDand BT,
the two alleles at the Blocus, and have very sim-
ilar Kmand Vmax (Taura et al., 1995, 1996). This
peculiarity explains the fact that, when both enzymes
are present (i.e. when the genotype at the Blocus is
BDBT), the almost equal efficiency of oxidocycliza-
tion of CBG into the respective end products, leads to
a ratio close to the unity in the THC:CBD ratio. This
produces the distribution along the median line of the
THC vs. CDB scatter plots observed for chemotype II
plants, and for all the F1 progenies of pure chemotype
One of the main targets for fiber hemp has been
for a long time the eradication of hemp plants bearing
the BTallele, and consequently synthesizing more or
less “illegal” amounts of THC. There have been, in the
Figure 5.The gas chromatographic profiles of the different chemotypes of Cannabis sativa (see text for details).
hemp breeding community, proposals of total elimina-
tion of THC, irrespective of its amount (even if well
below the 0.2% needed to get the EU subsidies), and
rumors of creation of transgenic hemp plants in which
THC synthesis was completely suppressed by antisense
technology. It is our opinion that these approaches are
unnecessary, and that standard selection practices, with
the possible help of MAS, can be sufficient to limit the
THC content well below the 0.2% of inflorescence dry
weight. However, in some fiber ecotypes, like the one
for which chemotypes distribution is shown in Figure 3
(an old Italian fiber ecotype, Eletta Campana), the num-
ber of plants that should be considered homozygous
for THC was indeed not negligible; besides, the ab-
solute amount of THC in the inflorescences, though
not at the levels of the drug strains, is high enough
to make this cultivar ineligible for EU subsidies. At
the beginning of the 1990s, this situation was common
for many dioecious fiber cultivars, and therefore the
necessity arose, for an effective presence on the mar-
ket, to “clean” the seed batches from THC-producing
plants, without altering the overall genetic background.
This has been the aim of the work leading to the iden-
tification of chemotype-associated molecular markers,
made in our Institute in collaboration with E. de Meijer,
breeder at Horta Pharm BV (The Netherlands) before,
and presently at GW Pharmaceuticals (UK). Different
segregating F2s were obtained from initial crosses be-
tween inbred lines with contrasting chemotypes (I and
III, i.e. almost pure THC and almost pure CBD); the ge-
netic analysis of the gas-chromatographic data demon-
strated that the F1 offspring was completely hybrid
(chemotype II), while all three chemotypes were again
present in the F2 generations, in a 1:2:1 proportion
(pure THC:mixed THC +CBD:pure CBD) within each
progeny; this finding was in agreement with the hypoth-
esis of one gene and two codominant alleles (BDand
BT)for chemotype determination. This hypothesis is
not the only possible, but it is the simplest explaining of
the presently available data. The F2 segregating groups
were screened by RAPD markers using the bulk seg-
regant approach, and several CBD- or THC-associated
markers were identified. All these markers behaved as
dominant, except one (named B190/B200; Figure 4 in-
sert), deriving from a CBD-associated RAPD fragment,
that once transformed into a SCAR marker, turned out
to be codominant, and therefore able to genotype com-
pletely at the Blocus the plants. The efficiency of cor-
rect identification of the chemotypes was 88% for pure
THC plants, 95% for mixed chemotype plants, and 98%
for pure CBD plants (de Meijer et al., 2003). However,
these markers, very useful within the pedigrees created
from the starting inbred lines, were not equally effective
in unrelated materials, like the dioecious fiber varieties
Carmagnola, Fibranova or Eletta Campana. Besides,
despite the very good degree of association with the
chemotype shown by marker B190/B200,itcannot be
taken into consideration for the marker-assisted identi-
fication of illicit crops and for legal purposes (P. Cantin,
personal communication). In this case, in fact, a marker
must be 100% linked to the chemotype, for its exploita-
tion as an effective and reliable drug repression tool.
The only marker with these characteristics is of course
the gene itself. In the NCBI database, there are the se-
quences corresponding to the genes for the THC- and
CBD-synthases (entry numbers AB057805, E55107,
E55108, E55090 and E55091); these sequences have
been patented by a research group of the Taisho Phar-
maceuticals Company, Japan.
The sequences of the genes coding for THC- and
CBD-synthase show very high similarities; the identity
along the 1635 bp coding sequence is 89.3%. The ma-
jor difference is apparently a missing nucleotide triplet
in the positions 757–759 of the THC-synthase se-
quence. The translated protein sequence is 545 and 544
aminoacids, for CBD- and THC-synthase, respectively.
The THC-synthase has a missing aminoacid (SER) in
position 253 of the sequence. Out of the 545 aminoacids
stretch, only 87 (16%) are different between the two
enzymes (including the missing one); about half of
these variations, however, are between aminoacids of
the same type. The aminoacid changes are quite evenly
distributed throughout the sequence, the longest variant
stretch consisting of six aminoacids in positions 491–
496. These differences are large enough to allow the
construction of specific primers, able to identify in the
different chemotypes the allelic complement of each
plant. In our laboratory, we devised a three-primers
system able to amplify, in a single PCR reaction of leaf
tissue fragments, the DNA sequences identifying the
allelic status at the Blocus (A. Carboni, unpublished).
Genetic analysis of heterozygous (BDBT) plants from
different crosses, revealed that the THC:CBD ratio
may vary slightly but consistently and heritably around
the value of 1 (de Meijer et al., 2003). This sug-
gests the possibility that several isoenzymatic forms of
THC- and CBD-synthases exist in different germplasm.
Confirmation of this hypothesis, presently in progress
through the sequencing in our laboratory of these pos-
sible variants, could lead to the identification of fur-
ther alleles of potential interest at the Blocus. Besides,
the identification, either by progeny analysis or by
direct sequencing, of the alleles responsible for the
synthesis of the several cannabinoids described in C.
sativa,would open the possibility of assisted selection
in Cannabis, bred not only as a fiber crop, but also
for its pharmaceutical applications (see G. Guy and R.
Pertwee contributions in this special issue). Chemotype
IV and V, having CBG or no cannabinoids, are of re-
markable interest for both fiber and pharmaceutical
purposes; the identification of the alleles at the Blo-
cus responsible for the accumulation of CBG or for the
absence of cannabinoids (Figure 4), would open the
waytothe development of molecular markers for these
chemotypes. The knowledge of the genes and alleles re-
sponsible for the different chemotypes could also lead
to the manipulation of the pathway in both plants and
cell cultures; the availability of chemotype-specific cell
cultures in which the cannabinoid biosynthesis is made
active by manipulations of the key enzymes of their
pathway, could lead to the development of bioreactors
useful for the in vitro large-scale production of specific
cannabinoids for the pharmaceutical industry.
However, C. sativa remains primarily a fiber crop,
and a great deal of work is being done for improving
our knowledge of gene expression during fiber devel-
opment. The physical properties of the fibers depend
on their chemical characteristics and on the way they
are assembled into bundles. Fiber quality is strongly
influenced by the chemical composition of elementary
fibers and cell wall. In the latest years, the comple-
tion of the genome sequence of Arabidopsis thaliana
made available a great deal of information on gene
sequences. About 15% of the about 25.000 Arabidop-
sis genes probably codes for functions correlated to
the biosynthesis, assembling and modification of the
cell wall (Carpita et al., 2001). Only a very limited
number of the genes involved have been so far iden-
tified, though an increasing number of xylem-specific
EST sequences is available from studies on pine and
poplar, Arabidopsis and Zinnia (Boudet et al., 2003).
A high cellulose content, a low degree of lignification
and a reduced number of cross linking between the
pectines and the structural components of the wall, are
the main characteristics reputed to be important to ob-
tain a easily extractable fiber and a good quality, for
both textile and paper industry. The identification of the
genes responsible for these characteristics will lead to
the development of new varieties through genetic engi-
neering strategies, and/or through the use of molecular
markers associated to the allelic variants conferring the
desirable traits during the selection or the germplasm
screening work. Because the high number of genes
probably involved, the technology of choice to iden-
tify them might be genome-wide microarray analysis
(M. Toonen, unpublished data).
The renewed interest in C. sativa as a multi-purpose
crop has been the main driving force for the increas-
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the breeding of this species by several research group;
the biotechnological products obtained from these re-
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... Previously described microsatellites validated in (Köhnemann et al. 2012, Houston et al. 2015, Presinszka et al. 2015, Dufresnes et al. 2017, Mandolino and Carboni 2004 and sourced from (Alghanim and Almirall 2003, Gilmore and Peakall 2003, Gao et al. 2014 were chosen due to their repeatedly demonstrated ability to differentiate between Cannabis sativa L. subspecies. ...
... This study found the mean number of alleles per locus was 10.3. Across three microsatellite studies, the average number of alleles found was 9 (Mandolino and Carboni 2004). The alleles per locus are higher in this study's results; this could be due to the method of microsatellite allele quantification, the microsatellites used, or it could be due to sampling and replication 43 differences. ...
... In addition, there are differences among studies which may or may not play a role in the number of alleles found. Overall, Gilmore and Peakall found more alleles on average than Alghanim and Almirall's work, but more alleles were detected with greater sample size (Mandolino and Carboni 2004). ...
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Cannabis sativa L. is a crop plant that is native to Asia. It is a primarily dioecious annual agriculturally used for its seed, fiber, and flowers. After recent legislative action legalized hemp nationally for research and cultivation in the US, distinct classifications had to be made. Cannabis sativa L. cultivars are presently separated into two major categories by the FDA: medical marihuana and hemp. Within the hemp classification, there is CBD-type hemp and industrial hemp. These classifications are based on Cannabis sativa L.’s chemical composition, which varies throughout the plant’s tissue. The desired cannabinoid chemicals for CBD-type hemp and medical Cannabis (high THC) are found in high concentrations in the female flowers. To differentiate CBD-type hemp from medical marihuana, the USDA’s current regulations limit tetrahydrocannabinol (THC) concentration to no more than 0.3% total. Genetic research on the plant has been, until recently, limited to legal applications of differentiating industrial hemp from medical marihuana. This study has been completed to ascertain Cannabis sativa L.’s current chemical composition and genetic diversity among Maryland growers. Hemp growers located across Maryland provided the field sites and samples. Chemical composition was determined from an analysis of flowers through a partnership with a Morgan State University chemistry lab. The determination of genetic diversity was completed on hemp leaves through the analysis of 12 standard and novel microsatellites. Cannabis sativa L. samples were taken from eight field sites across twenty eight distinct cultivars. Cultivars #5 and Cherry had the highest CBDA content among the strains studied. Microsatellite analysis determined that the most genetically variable cultivars were A-B1 and #5. With this research, CBDA type hemp growers in Maryland will be able to determine some strains which have high concentrations of their desired cannabinoids and which cultivars are more genetically variable.
... This barrier is difficult to overcome, and would require fundamental restructuring of regulations on both national and international levels [14]. Thanks to recent advances in molecular markers and genetic engineering methods (e.g., CRISPR), it is possible to detect and/or produce cannabis with very low levels of THC [43,44]. Though there could still be complications differentiating ornamental from medicinal varieties, cannabis cultivars that produce low cannabinoid levels might help the ornamental industry to overcome legal barriers and prevent unwanted diversion. ...
... Various molecular genetic markers have also been employed in the cannabis field to analyze genetic variations, sex determination, chemotype inheritance, and genetic mapping (reviewed by Hesami et al. [30]). For instance, Mandolino and Carboni [44] employed molecular markers to study chemotype inheritance in cannabis. They discovered a chemotype with an undetectable amount of phytocannabinoids (almost zero phytocannabinoids) and classified it as chemotype V [44]. ...
... For instance, Mandolino and Carboni [44] employed molecular markers to study chemotype inheritance in cannabis. They discovered a chemotype with an undetectable amount of phytocannabinoids (almost zero phytocannabinoids) and classified it as chemotype V [44]. Johnson and Wallace [64] employed genotyping by sequencing (GBS) to evaluate chemotype inheritance in cannabis. ...
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The characteristic growth habit, abundant green foliage, and aromatic inflorescences of cannabis provide the plant with an ideal profile as an ornamental plant. However, due to legal barriers, the horticulture industry has yet to consider the ornamental relevance of cannabis. To evaluate its suitability for introduction as a new ornamental species, multifaceted commercial criteria were analyzed. Results indicate that ornamental cannabis would be of high value as a potted-plant or in landscaping. However, the readiness timescale for ornamental cannabis completely depends on its legal status. Then, the potential of cannabis chemotype Ⅴ, which is nearly devoid of phytocannabinoids and psychoactive properties, as the foundation for breeding ornamental traits through mutagenesis, somaclonal variation, and genome editing approaches has been highlighted. Ultimately, legalization and breeding for ornamental utility offers boundless opportunities related to economics and executive business branding.
... Since then, large-scale cultivation as ∆ = 0.15 pg/2C [50]. Early sex determination is usually carried out using male-associated DNA markers [61][62][63][64][65][66][67], but the accuracy and reproducibility of some of them have been questioned [67,68]. Based on the above, there is no doubt that developing a method of sex detection through flow cytometry, as previously suggested [50], would be of great interest. ...
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Cannabis sativa has been used for millennia in traditional medicine for ritual purposes and for the production of food and fibres, thus, providing important and versatile services to humans. The species, which currently has a worldwide distribution, strikes out for displaying a huge morphological and chemical diversity. Differences in Cannabis genome size have also been found, suggesting it could be a useful character to differentiate between accessions. We used flow cytometry to investigate the extent of genome size diversity across 483 individuals belonging to 84 accessions, with a wide range of wild/feral, landrace, and cultivated accessions. We also carried out sex determination using the MADC2 marker and investigated the potential of flow cytometry as a method for early sex determination. All individuals were diploid, with genome sizes ranging from 1.810 up to 2.152 pg/2C (1.189-fold variation), apart from a triploid, with 2.884 pg/2C. Our results suggest that the geographical expansion of Cannabis and its domestication had little impact on its overall genome size. We found significant differences between the genome size of male and female individuals. Unfortunately, differences were, however, too small to be discriminated using flow cytometry through the direct processing of combined male and female individuals.
... According to scientific studies, there are three cannabis species with distinct phenotypic differences, namely C. sativa L., C. indica Lam (Lamarck), and C. ruderalis [7,8]. However, the majority of classifications performed to date evidence the existence of C. sativa Within a given cannabis species, cultivars are categorised into groups based on their chemotype, from I to V, according to the number and ratio of main cannabinoids [16]. These compound profiles can be employed both as quality markers and fingerprints for cannabis standardization. ...
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Cannabis (Cannabis sativa L.), also known as hemp, is one of the oldest cultivated crops, grown for both its use in textile and cordage production, and its unique chemical properties. However, due to the legislation regulating cannabis cultivation, it is not a well characterized crop, especially regarding molecular and genetic pathways. Only recently have regulations begun to ease enough to allow more widespread cannabis research, which, coupled with the availability of cannabis genome sequences, is fuelling the interest of the scientific community. In this review, we provide a summary of cannabis molecular resources focusing on the most recent and relevant genomics, transcriptomics and metabolomics approaches and investigations. Multi-omics methods are discussed, with this combined approach being a powerful tool to identify correlations between biological processes and metabolic pathways across diverse omics layers, and to better elucidate the relationships between cannabis sub-species. The correlations between genotypes and phenotypes, as well as novel metabolites with therapeutic potential are also explored in the context of cannabis breeding programs. However, further studies are needed to fully elucidate the complex metabolomic matrix of this crop. For this reason, some key points for future research activities are discussed, relying on multi-omics approaches. ******************************************************************************* Keywords: cannabis; genomics; metabolomics; multi-omics; transcriptomics
... Recent increase in the agricultural areas that are used for hemp production requires automated approaches that can be used for confirmatory differentiation between male and female plants prior to their flowering. PCR-based markers have been identified specific to male plants (Mandolino and Carboni 2004) and are routinely used by service laboratories; however, these tests are costly and time consuming. ...
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Main conclusion Hand-held Raman spectroscopy can be used for highly accurate differentiation between young male and female hemp plants. This differentiation is based on significantly different concentration of lutein in these plants. Abstract Last year, a global market of only industrial hemp attained the value of USD 4.7 billion. It is by far the fastest growing market with projected growth of 22.5% between 2021 and 2026. Hemp (Cannabissativa L.) is a dioecious species that has separate male and female plants. In hemp farming, female plants are strongly preferred because male plants do not produce sufficient amount of cannabinoids. Male plants are also eliminated to minimize a possibility of uncontrolled cross-fertilization of plants. Silver treatments can induce development of male flowers on genetically female plants in order to produce feminized seed. Resulting cannabinoid hemp production fields should contain 100% female plants. However, any unintended pollination from male plants can produce unwanted males in production fields. Therefore, there is a growing demand for a label-free, non-invasive, and confirmatory approach that can be used to differentiate between male and female plants before flowering. In this study, we examined the extent to which Raman spectroscopy, an emerging optical technique, can be used for the accurate differentiation between young male and female hemp plants. Our findings show that Raman spectroscopy enables differentiation between male and female plants with 90% and 94% accuracy on the level of young and mature plants, respectively. Such analysis is entirely non-invasive and non-destructive to plants and can be performed in seconds using a hand-held spectrometer. High-performance liquid chromatography (HPLC) analysis and collected Raman spectra demonstrate that this spectroscopic differentiation is based on significantly different concentrations of carotenoids in male vs female plants. These findings open up a new avenue for quality control of plants grown in both field and a greenhouse.
... Chemotype IV also has low THC contents but with the potent percentage of CBG. Furthermore, the chemotypes producing very little to almost zero cannabinoid compounds (neutral) are grouped as chemotype V -was first described by Mandolino et al. (Cascini et al., 2012;Hartsel et al., 2016;Mandolino and Carboni, 2004). Apart from cannabinoid (THC, CBD) content, drug and fiber-type plants have significant genetic variation. ...
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Cannabis sativa L. has been one of the oldest medicinal plants cultivated since 10,000 years for several agricultural and industrial applications. However, the plant became controversial due to some psychoactive components that have adverse effects on human health. In this review, we analyzed the trends in cannabis research for the past two centuries. We discussed the historical transitions of cannabis from the category of an herbal medicine to an illicit drug and back to a medicinal product post-legalization. In addition, we address the new-age application of immuno-suppressive and anti-inflammatory extracts for the treatment of COVID-19 inflammation. We further address the influence of the legal aspects of cannabis cultivation for medicinal, pharmaceutical, and biotechnological research. We reviewed the up-to-date cannabis genomic resources and advanced technologies for their potential application in genomic-based cannabis improvement. Overall, this review discusses the diverse aspects of cannabis research developments ranging from traditional use as an herbal medicine to latest potential in COVID, legal practices with updated patent status, and current state of art genetic and genomic tools reshaping cannabis biotechnology in modern age agriculture and pharmaceutical industry.
Cannabis sativa L. (hemp) develops plants with either male or female flowers, and growers of hemp greatly prefer female flowers which bear the glandular trichomes that contain cannabinoids. Feminized (all female) seeds are highly desired, which are produced by crossing a female plant with a masculinized female plant. Masculinization is achieved through the inhibition of ethylene and/or addition of gibberellins before flower initiation in female plants. The hemp industry uses silver thiosulfate (STS) to masculinize hemp, but spraying silver poses environmental concerns. This study compared STS to three other ethylene-inhibiting agents: aminoethoxyvinylglycine (AVG), cobalt nitrate (CBN), and 1-methylcyclopropene (1-MCP). Treatments of STS and CBN also included gibberellic acid as a synergist. Plants treated with STS exhibited superior masculinization and pollen dispersal compared to plants treated with AVG, CBN or 1-MCP. Only plants treated with STS or AVG produced pollen in sufficient quantities for collection. This pollen was assayed for germination potential initially and after storage for up to five weeks at 22.2, 7.2, or 1.1°C. Pollen from plants treated with AVG remained viable for four weeks at 1.1°C, whereas STS-treated plants produced pollen that was viable for three weeks at 1.1°C. Due to phytotoxicity problems with AVG, STS remains the best treatment to masculinize female hemp plants when breeding for feminized seeds. In a separate study, flower tissues of hemp had considerably higher total cannabinoid concentrations compared to leaf tissues but significantly lower ratios of cannabidivarin (CBDV) to cannabidiol (CBD). To reduce variability, at least 1 g samples of fresh leaf or flower tissue should be extracted with 10 mL of methanol. Rapid throughput testing of cannabinoids as part of a breeding program should use flower tissue, preferably at the time typical of harvest.
In Cannabis sativa L. the presence of delta 9-tetrahydrocannabinolic acid (THCA) above legal limit is a challenging issue that still restricts the industrial exploitation of this promising crop. In recent years, the interest of entrepreneurs and growers who see hemp as a dynamic and profitable crop was joined by the growing knowledge on C. sativa genetics and genomics, accelerated by the application of high throughput tools. Despite the renewed interest in the species, much remains to be clarified, especially about the long-standing problem of THCA in hemp inflorescences, which could even result in the seizure of the whole harvest. Although several hypotheses have been formulated on the accumulation of this metabolite in industrial varieties, none is conclusive yet. In this work, individuals of a population of the hemp cultivar 'FINOLA' obtained from commercial seeds were investigated for total THC level and examined at molecular level. A marker linked to THCA synthase was found at a high incidence in both male and female plants, suggesting a considerable genetic variability within the seed batch. Full-length sequences encoding for putatively functional THCA synthases were isolated for the first time from the genome of both female and male plants of an industrial hemp variety and, using transcriptional analysis, the THCA synthase expression was quantified in mature inflorescences of individuals identified by the marker. Biochemical analyses finally demonstrated for these plants a 100% association between the predicted and actual chemotype.
After decades of prohibition of the cultivation and breeding of hemp ( Cannabis sativa < 0.3% ∆9‐tetrahydrocannabinol [THC]), there is untapped potential for genetic improvement of this crop to provide food, feed, fiber, and medicinal compounds. Successful breeding efforts will require the development and characterization of germplasm resources, optimization of crossing methods, better understanding of sex determination, high‐throughput phenotyping platforms, and deployment of genomic tools for rapid selection. This review provides a brief overview of these topics and some key opportunities for genetic improvement of hemp to support an emerging industry utilizing this newly legalized crop.
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Terpenes and terpenoids form a large class of organic compounds, produced by numerous plants, including Cannabis, as secondary metabolites, together with cannabinoids, flavonoids, sterols, etc. They give the scent of the plants, as well as the fragrance of their extracts. In Cannabis sativa 110 terpenes and 121 terpenoids were described up to now, with myrcene and β-caryophyllene as the most prevalent mono and sesquiterpene, respectively. More than 30 genes are known to control the terpenes synthesis, through terpene synthases, less known. From here, a great variability of the relative content of tepenes has been described among Cannabis chemotypes, contributing to the modulation of some pharmacological actions, among the plant extracts. Cannabis terpenes have therapeutical potency, through independent actions, as well as through synergism or potentiation of the phytocannabinoids actions: antimicrobial and antiinflammatory properties, recently described analgesic effect, anticarcinogenic, antimutagenic, antiallergic, antihyperglycemic, antidepressive, anxiolytic, sedative effects, immunostimulating action, bronchodilator, increase of memory and concentration, active action on the main dermatological inflammatory diseases. The mini-review brings uptodate basic information referring to the chemical structure and classification of Cannabis terpenes, biosynthesis pathways, relative content variability, therapeutical effects.
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We examined the abundance of microsatellites with repeated unit lengths of 1–6 base pairs in several eukaryotic taxonomic groups: primates, rodents, other mammals, nonmammalian vertebrates, arthropods, Caenorhabditis elegans, plants, yeast, and other fungi. Distribution of simple sequence repeats was compared between exons, introns, and intergenic regions. Tri-and hexanucleotide repeats prevail in protein-coding exons of all taxa, whereas the dependence of repeat abundance on the length of the repeated unit shows a very different pattern as well as taxon-specific variation in intergenic regions and introns. Although it is known that coding and noncoding regions differ significantly in their microsatellite distribution, in addition we could demonstrate characteristic differences between intergenic regions and introns. We observed striking relative abundance of (CCG) n • (CGG) n trinucleotide repeats in intergenic regions of all vertebrates, in contrast to the almost complete lack of this motif from introns. Taxon-specific variation could also be detected in the frequency distributions of simple sequence motifs. Our results suggest that strand-slippage theories alone are insufficient to explain microsatellite distribution in the genome as a whole. Other possible factors contributing to the observed divergence are discussed.
The authors report on a method to identify unknown samples of plant material as Cannabis sativa L. The method involves polymerase chain reaction (PCR) amplification of the internal transcribed spacer I (ITS1) of the nuclear ribosomal deoxyribonucleic acid (n-rDNA) in five different accessions of C. sativa from various geographical areas, as well as in one accession of Humulus lupulus L., which belongs to the only other genus of the family Cannabaceae. The use of ITS1, amplified and successively digested with appropriate restriction endonucleases, has allowed the construction of a Cannabis fingerprint that can be used in forensic investigations for the identification of samples suspected of being Cannabis. The method reported in the present paper has the merit of making it possible to process very small amounts of material; it is also not expensive, does not require access to a sequencing facility and does not utilize sophisticated apparatus.
The authors report on a method to identify unknown samples of plant material as Cannabis sativa L. The method involves polymerase chain reaction (PCR) amplification of the internal transcribed spacer I (ITS1) of the nuclear ribosomal deoxyribonucleic acid (n-rDNA) in five different accessions of C. sativa from various geographical areas, as well as in one accession of Humulus lupulus L., which belongs to the only other genus of the family Cannabaceae. The use of ITS1, amplified and successively digested with appropriate restriction endonucleases, has allowed the construction of a Cannabis fingerprint that can be used in forensic investigations for the identification of samples suspected of being Cannabis. The method reported in the present paper has the merit of making it possible to process very small amounts of material; it is also not expensive, does not require access to a sequencing facility and does not utilize sophisticated apparatus.
A dioecious plant, Cannabis sativa has two sex chromosomes (X and Y). The genome sizes of the diploid female and male plants were determined to be 1636 and 1683 Mbp, respectively, by flow cytometry. By the karyotype analysis, the X and Y chromosomes were found to be submetacentric and subtelocentric, respectively. The Y chromosome had the largest long arm with a satellite in the terminal of its short arm. Conspicuous condensation was specifically observed in the long arm and satellite of the Y chromosome during the prometaphase to metaphase stages. These results indicate that the Y chromosome, especially in its long arm, specifically differentiates in Cannabis sativa and might contribute to the sex determination.
Recently a new class of genetic polymorphism, restriction fragment length polymorphisms (RFLPs), has been uncovered by the use of restriction endonucleases which cleave DNA molecules at specific sites and cloned DNA probes which detect specific homologous DNA fragments. RFLPs promise to be exceedingly numerous and are expected to have genetic characteristics - lack of dominance, multiple allelic forms and absence of pleiotropic effects on economic traits - of particular usefulness in breeding programs. The nature of RFLPs and the methodologies involved in their detection are described and estimated costs per polymorphism determination are derived. The anticipated costs of applying RFLPs to genome mapping are considered in terms of the number of RFLPs required for a given degree of genome coverage, the number of probe × enzyme combinations tested per polymorphism uncovered, and the total number of individuals and polymorphisms scored for mapping purposes. The anticipated costs of applying RFLPs to genetic improvement are considered in terms of the number of individuals and the number of polymorphisms per individual that are scored for the various applications. Applications considered include: varietal identification, identification and mapping of quantitative trait loci, screening genetic resource strains for useful quantitative trait alleles and their marker-assisted introgression from resource strain to commercial variety, and marker-assited early selection of recombinant inbred lines in plant pedigree breeding programs and of young sires in dairy cattle improvement programs. In most cases anticipated costs appear to be commensurate with the scientific or economic value of the application.
Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.
Samples of the seeds and seedlings of Cannabis sativa, and its dried leaves and flowerheads (marijuana), could be reliably distinguished by RAPD-PCR (Random Amplified Polymorphic DNA using the Polymerase Chain Reaction). DNA was best extracted from fresh tissues using buffers and the detergent cetyltrimethylammonium bromide; poorly dried tissue or inviable seed yielded coloured samples of degraded DNA. DNA was isolated from 51 C. sativa and two Humulus lupulus (hops) samples. Of the C. sativa samples 43 were from Australia (ten from Canberra gardens, eight from a New South Wales crop and 25 from two Queensland crops) and eight were from Papua-New Guinea (P-NG). A total of 102 different bands were obtained using four 10-nucleotide primers with arbitrarily chosen sequences. Banding patterns were compared by calculating pairwise distances using various algorithms, and presented using the neighbour-joining tree and multidimensional scaling methods. These showed a clear difference between C. sativa and H. lupulus, and separated the samples of the latter into three distinct groups; one group comprised all the P-NG samples, another the Canberra samples, and the third, the three crop samples.
The objective was to study the genetic structure and degree of variability of hemp (Cannabis sativa L.) varieties. Six varieties of hemp were analyzed by random amplified polymorphic DNA (RAPD) analysis, using 10 plants per variety. The varieties were a dioecious landrace, a dioecious selection from it, a cross-bred cultivar, a monoecious variety, a drug strain, and an inbred female line. The genetic complexity of each cultivar was investigated by determining the number of bands produced by the primers used, the number of fixed 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 97.1%; the single cultivar polymorphism ranged from 31.1 to 85.5%. Heterozygosity ranged from 0.05 (inbred female line) to 0.26 (cross-bred Fibranova). The average heterozygosity calculated over all 102 loci and all plants studied was 0.29. The Fst (Wright's fixation index) value calculated for all loci was 0.48, and only 33.3% of the scored loci had higher values and can be considered informative for cultivar identification. A Fisher's test based on allele frequencies suggested complete differentiation among all varieties, with the exception of the Italian dioecious varieties Carmagnola and CS, for which no discriminating alleles were found. The correlations among the molecular data and the genetic structure of the different cultivars and the consequences in relation to variety discrimination in hemp are discussed.