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MENDELNET 2016
PROGRESS IN EARLY SEX DETERMINATION OF
CANNABIS PLANT BY DNA MARKERS
PETER MENDEL1, AJINKYA BHARAT LALGE1, TOMAS VYHNANEK1, VACLAV
TROJAN1, PETR KALOUSEK1, HUGO MAASSEN2, LADISLAV HAVEL1
1
Department of Plant Biology
Mendel University in Brno
Zemedelska 1, 613 00 Brno
CZECH REPUBLIC
2
Department of Phyto Engineering
Bedrocan International
2009, 9640 CA Veendam
NETHERLANDS
[email protected]
Abstract: The cannabis plant is a tall annual crop of economic importance. It is mostly dioecious, but
fiber hemp varieties have been bred to be monoecious. Separating male and female plants at early
developmental stage is useful due to the influence of gender at agriculturally significant traits. Several
experiments have been focused on developing a reliable molecular marker for sex determination in
cannabis plants. Our study compares three DNA markers for the detection of male genotype in totally
twelve samples of industrial hemp and medicinal cannabis plants. Genotype scoring of SCAR119
marker appeared to be the most reliable, followed by MADC2 and SCAR323, when compared to the
observed phenotype of plants. The results confirmed the insights given in previous studies. Research
and Polymerase Chain Reaction (PCR) analysis should continue in order to find more advanced DNA
markers for sex determination of cannabis plants.
Key Words: cannabis, sex determination, DNA markers
INTRODUCTION
Cannabis (Cannabis sativa L.) is a tall upright annual herb. It is generally dioecious i.e. producing
separate male and female plants but fiber hemp varieties have been specifically bred to be monoecious
(hermaphrodite) (Debruyne et al. 1994, Srivastava and Yadav 2013). The sex of most dioecious plants
can only (reliably) be determined at the time of flowering. The significance of separating male and
female plants at seedling stage lies in the fact that in many dioecious plants gender influences the
economic value, breeding schemes and opportunities for commercial use of genetically modified
materials (Parker and Clark 1991).
The discussed demand for tools supporting sex determination in plants gave rise to a series of
molecular studies investigating DNA markers that could be used for that purpose. A molecular marker
(DNA marker) is a DNA sequence observed in at least two versions that are easy to distinguish (Brown
2002), which reveals individual polymorphisms. The preferred marker should demonstrate the widest
possible range of variation in the analyzed trait, and it should not be affected by environmental factors.
An effective marker should guarantee reproducibility, and it should be easy to detect. Molecular markers
facilitate analyzes of variations between individuals, regardless of their development stage (SztubaSolińska 2005), which is particularly useful in sex determination studies of plants. Some researchers
have suggested that effective markers for plants should be relatively short to support sex determinations
in herbarium specimens with damaged DNA. Shorter sequences increase the probability of successful
amplification (Korpelainen et al. 2008).
This study is focused on testing the reliability and reproducibility of three developed molecular
markers linked to male sex in the cannabis plant (Cannabis sativa L.). Standard technical hemp varieties,
as well as medicinal ones were included.
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MATERIAL AND METHODS
DNA isolation and samples used
Four well known varieties of industrial hemp and six experimental varieties of medicinal cannabis
were included in this experiment (Table 1). The medicinal cannabis samples were all provided by
Bedrocan International in form of DNA. For the variety Bialobrezskie, three plants from an experiment,
where plants were treated with phytohormones were selected: a NAA (naphtylacetic acid) – auxin
analogue variant, a BAP (benzylaminopurine) – cytokinin analogue variant and an untreated control
group. Chemicals were applied every two weeks by spraying it on the leaves in six different doses, each
for different group: 5, 10 and 20 mg/l for NAA and 10, 25 and 50 mg/l for BAP. After 11 weeks of
application, one plant from the most concentrated auxin group as well as one from highest cytokinin
dosing, showed male flowering structures. DNA was isolated from these two plants as well as from one
plant of the untreated control group. These three plants were included in the sex determination
experiment.
Total genomic DNA was isolated from 0.1 g of fresh leaves homogenized by mortar and pestle
and liquid nitrogen. A DNeasy Plant Mini Kit (Quiagen) was used for the isolation process.
Concentration and purity of isolated DNA was measured using Picopet 1.0 spectrophotometer
(Picodrop).
Table 1 Overview of used samples
Sample
Variety/ genotype
marking
CAR
Carmagnola
Material
Source/ provider
seeds
Hempoint Ltd.
KHT
Kompolti Hybrid TC
leaves
Hempoint Ltd.
UNI
Unikó
leaves
Hempoint Ltd.
B-C
Bialobrezskie
leaves
Hempoint Ltd./ untreated control
B-M1
Bialobrezskie
leaves
male plant treated with 20 mg/l NAA
B-M2
Bialobrezskie
leaves
male plant treated with 50 mg/l BAP
BK1
medicinal cannabis
(unspecified)
medicinal cannabis
(unspecified)
medicinal cannabis
(unspecified)
medicinal cannabis
(unspecified)
medicinal cannabis
(unspecified)
medicinal cannabis
(unspecified)
DNA
Bedrocan International
DNA
Bedrocan International
DNA
Bedrocan International
DNA
Bedrocan International
DNA
Bedrocan International
DNA
Bedrocan International
BK4
BK5
BK7
BK8
BK9
PCR conditions
Three different primer pairs for sex determination were tested (Table 2). The first two markers
(SCAR – sequence-characterized amplified region) were developed from RAPD primers by Tӧrjék et
al. (2002), the other marker (MADC – male-associated DNA from Cannabis sativa) is based on the
research of Mandolino et al. (1999).
PCR was performed in a total volume of 25 μl consisting of 0.5 U Taq polymerase (Promega),
1˟ aliquot buffer, 0.1 mM of each dNTP(Promega), 0.3 M of each primer and 20 ng of template DNA
in a T3 thermocycler (Biometra) for the SCAR markers and gradient thermal cycler QB-96 (Quanta
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Biotech) for the MADC marker. We used the same PCR protocols as the aforementioned authors,
without any further optimization for the SCAR (Tӧrjék et al. 2002) and the MADC (Mandolino et al.
1999) markers.
Electrophoresis was performed in a 1.5 % agarose gel on a Blue Marine 200 apparatus (Serva),
with Tris-acetate-EDTA buffer (TAE) and ethidium-bromide used for for staining. The presence and
size of PCR products was visualized by using a UV transluminator and VisionCapt software (Vilber
Lourmat).
Table 2 Primers used for sex determination
Marker name
Amplification product
Primer sequence 5’-3’
SCAR119_F
SCAR119_R
SCAR323_F
SCAR323_R
MADC2_F
MADC2_R
male sex linked DNA
(119 bp)
male sex linked DNA
(323 bp)
male sex linked DNA
(390 bp)
TCAAACAACAACAAACCG
GAGGCCGATAATTGACTG
GAGCGGACATCATTGCCT
ATCACCCCACCGTTTAGG
GTGACGTAGGTAGAGTTGAA
GTGACGTAGGCTATGAGAG
RESULTS AND DISCUSSION
MADC marker amplification products
Total genomic DNA of all twelve cannabis plant samples was amplified by specific primers. A
single DNA band of size about 390 bp was expected for all putative male plants, while all female and
monoecious plants were expected to have two products of about 560 and 870 bp. This appeared to be
the case with ten of the samples, two deviations – in the case of Carmagnola and Kompolti Hybrid TC
have possible explanations (Figure 1).
Figure 1 Visualisation of PCR products for sex determination of cannabis varieties (MADC2 marker)
SM
CAR KHT UNI B-C B-M1 B-M2 BK1 BK4 BK5 BK7 BK8 BK9
SM
Legend: SM – 100 bp size marker, CAR – Carmagnola, KHT – Kompolti Hybrid TC, UNI – Unikó, B-C – Bialobrezskie
monoecious control, B-M1 – Bialobrezskie male plant treated with NAA, B-M2 – Bialobrezskie male plant treated with BAP,
BK1, BK4, BK5, BK7, BK8, BK9 – unknown genotypes of medicinal cannabis (Phyto Engineering Department, Bedrocan
International)
As mentioned by Mandolino et al. (1999), MADC2 is probably a non-coding genome region and
it is not confirmed whether the sequence is a part of the genes for sex determination. Most likely, it is
not located solely on the male chromosome, as in previous studies MADC2 failed to discriminate sex
phenotype in some cases (Sakamoto et al. 1995). Our results support this hypothesis, as the first two
samples should be phenotypically female/ monoecious, but show products supposedly corresponding to
the male genotype. In addition, DNA of the first variety (Carmagnola) was isolated from seeds. Sex
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determination in cannabis is complex and can even be reversed or modified by chemical treatment and
environmental factors (Chailakhyan 1979, Mohan Ram and Sett 1979). At the same time, male flowers
are able to develop on female plants under extreme conditions (Clarke 1997). BK8, although being a
medicinal cannabis plant, was observed to form male flowering structures and was, based on the
examination of the plant morphology, classified as a male and possibly dioecious plant. This theory can
help us explain the fact, that during our experiment with phytohormones two unexpected staminate
plants with male phenotypes appeared (they produced a lot of pollen), while the used variety
Bialobrezskie is normally monoecious, as stated in a list (2014) by the Czech Central Institute for
Supervising and Testing in Agriculture. Two slightly visible bands of higher molecular weight can be
seen on the gel (Figure 1) in case of the second male plant. This suggests some gradual development
from a previously monoecious plant. Another hypothesis is genetic recombination between the sex locus
and the marker, on which both authors (Mandolino et al. 1999, Tӧrjék et al. 2002) - speculated. The
research group of Techen et al. (2010) was dealing with sex determination of cannabis and certain types
of MADC and SCAR markers as well, being able to reliably identify female individuals in all cases.
However, the experiment was carried out in very early seedling stage and it is not exactly known whether
they included monoecious plants, in which stage of onthogenesis the development of male phenotype
really begins and what mechanisms are underlying it.
Comparison with SCAR markers
The same set and order of DNA samples (Table 1) was used to test the SCAR markers. In case of
SCAR119, our sex determination results seem to be generally in accordance with MADC2 and with the
information from Tӧrjék et al. (2002) – that male plants are presented with single band of 119 or 323 bp
(for SCAR323). And while some female plants may show the same product as well, it is much less
intensive. This, however, appeared to be in contrast with our results for SCAR323 – the high intensity
for male might be corresponding in case of Carmagnola seeds, previously mentioned BK8 and two
phenotypic males (B-M1, B-M2), but there was still quite a robust product of 323 bp in the case of the
monoecious Bialobrezskie plant (B-C) and some of the medicinal genotypes (BK4 and BK7). An overall
evaluation of phenotype versus genotype scoring of all varieties and DNA markers used in this study is
shown in Figure 2.
Figure 2 Final comparison of DNA markers results for all varieties
Legend:
- plant with female phenotype,
- plant with male phenotype,
- monoecious plant, GREEN colour – sample
with genotype scoring in accordance to phenotype, RED colour – samples with incorrect genotype indication
CONCLUSION
Three molecular markers were tested for their ability to detect male plants in cannabis. DNA
material of various origin was used, twelve different varieties of cannabis plants in total.
The SCAR119 marker appeared to be the most reliable, with genotype scoring results
corresponding to phenotype in case of all twelve samples. The MADC2 marker showed only one
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contradictive sample result. The SCAR323 marker appeared to be the most controversial, with three
deviant results.
This study confirmed that sex determination of the cannabis plant is a complex process, for which
further research is needed to develop a reliable molecular tool to distinguish male and female plants at
an early developmental stage.
ACKNOWLEDGEMENTS
The research was financially supported by the IGA FA MENDELU No. IP 2/2016. All authors thank to
Hana Gabrielová and Hugo Maassen for providing research material and cooperation, and to Peter van
Peer for grammar correction.
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