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The sexual differentiation of Cannabis sativa L.: A morphological and molecular study

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  • CREA - Research Centre for Cereal and Industrial Crops (Bologna)
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The sexual differentiation of Cannabis sativa L.: A morphological and molecular study

Abstract and Figures

Cannabis sativa L. is a dioecious species with sexual dimorphism occurring in a late stage of plant development. Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (XY) and female is the homogametic one (XX). The sexual phenotype of Cannabis often shows some flexibility leading to the differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). Sex is considered an important trait for hemp genetic improvement; therefore, the study of the mechanism of sexual differentiation is of paramount interest in hemp research. A morphological and molecular study of Cannabis sativa sexual differentiation has been carried out in the Italian dioecious cultivar Fibranova. Microscopic analysis of male and female apices revealed that their reproductive commitment may occur as soon as the leaves of the fourth node emerge; the genetic expression of male and female apices at this stage has been compared by cDNA-AFLP. A rapid method for the early sex discrimination has been developed, based on the PCR amplification of a male-specific SCAR marker directly from a tissue fragment. Five of the several cDNA-AFLP polymorphic fragments identified have been confirmed to be differentially expressed in male and female apices at the fourth node. Cloning and sequencing revealed that they belong to nine different mRNAs that were all induced in the female apices at this stage. Four out of them showed a high degree of similarity with known sequences: a putative permease, a SMT3-like protein, a putative kinesin and a RAC-GTP binding protein.
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Euphytica 140: 95–106, 2004.
C
2004 Kluwer Academic Publishers. Printed in the Netherlands.
95
The sexual differentiation of Cannabis sativa L.: A morphological
and molecular study
V.M. Cristiana Moliterni
1,
, Luigi Cattivelli
2
,P.Ranalli
1
& Giuseppe Mandolino
1
1
Istituto Sperimentale per le Colture Industriali, via di Corticella 133, 40128 Bologna, Italy;
2
Istituto Sperimentale
per la Cerealicoltura, via S. Protaso 308, 29017 Fiorenzuola d’Arda, Italy
(
author for correspondence: e-mail: vmc.moliterni@isci.it)
Key words: Cannabis sativa, cDNA-AFLP, dioecy, sex linked markers, sexual differentiation
Summary
Cannabis sativa L. is a dioecious species with sexual dimorphism occurring in a late stage of plant development.
Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (XY) and female
is the homogametic one (XX). The sexual phenotype of Cannabis often shows some flexibility leading to the
differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). Sex is considered an
important trait for hemp genetic improvement; therefore, the study of the mechanism of sexual differentiation is of
paramount interest in hemp research. A morphological and molecular study of Cannabis sativa sexual differentiation
has been carried out in the Italian dioecious cultivar Fibranova.
Microscopic analysis of male and female apices revealed that their reproductive commitment may occur as soon
as the leaves of the fourth node emerge; the genetic expression of male and female apices at this stage has been
compared by cDNA-AFLP. A rapid method for the early sex discrimination has been developed, based on the PCR
amplification of a male-specific SCAR marker directly from a tissue fragment.
Five of the several cDNA-AFLP polymorphic fragments identified have been confirmed to be differentially
expressed in male and female apices at the fourth node. Cloning and sequencing revealed that they belong to nine
different mRNAs that were all induced in the female apices at this stage. Four out of them showed a high degree
of similarity with known sequences: a putative permease, a SMT3-like protein, a putative kinesin and a RAC-GTP
binding protein.
Abbreviations: AFLP: Amplified Fragment Length Polymorphism; LINE: Long Interspersed Elements; LTR: Long
Terminal Repeat; SCAR: Sequence Characterized Amplified Region
Introduction
Cannabis sativa belongs to the family of Cannabaceae,
order Rosales (APGII, 2003). It is a naturally dioecious
species with male and female individuals showing
unisexual flowers and characterized by sexual dimor-
phism: male plants are generally taller and slender than
female plants and have a shorter life cycle. Unisexual
flowers are bored in inflorescences that are terminal
at an earlier stage, and terminal or lateral in a later
stage.
Male inflorescence consists of hanging panicles
sometimes branched, generally with few or no leaves
and composed by a variable number of flowers. Male
flower has a perianth of five sepals that encloses the an-
droecium, composed by five stamens bored by subtle
stalks. The anthers at maturity undergo dehiscence lon-
gitudinally, releasing the pollen grains that are mostly
wind dispersed (Mohan Ram and Nath, 1964). Female
inflorescence is a raceme developing at the apex of
the plant or at the axils of leaves or lateral branches.
The female flower has a very simple structure as it
96
is composed by a green bract that completely wraps
the rudimental perianth and the ovary. This latter is
uniloculate and has a short style that distally differen-
tiates a bifid stigma.
The chromosome set of Cannabis sativa is
composed by nine pairs of autosomes and one pair
of sexual chromosomes: X and Y. The male sex is
endowed with an XY pair, and the female one with
an XX pair, similarly to what found in other dioecious
species such as Humulus lupulus, Silene latifolia,
Coccinia indica, Rumex hastatulus;however, sex de-
termination in Cannabis has been supposed to be based
on a X:autosome dosage rather than on an active-Y
mechanism (Westgaard, 1958; Grant et al., 1994).
The Y chromosome in Cannabis is subtelocentric and
characterized by a satellite at the extremity of the short
arm; besides, the long arm is particularly developed
and probably responsible for the difference found be-
tween the male and the female genome sizes (1683 and
1636 Mbp, respectively; Sakamoto et al., 1998). The
X chromosome is submetacentric, and bears a satellite
at the end of short arm. There are no specific reports
about the chromosome set of monoecious plants.
As already reported for many other plant sexual
chromosomes, Cannabis sativa Y chromosome is
strongly heterochromatic and rich of repetitive se-
quences that are likely cause of its marked metaphasic
condensation. A high percent of the repeated DNA
is made of LINE-like sequences (Boecke, 1989),
probably representing traces of transposable elements
showing a low level of transcription for the presence of
still active ORFs, coding for enzymes involved in the
transposition mechanism. Cannabis sativa LINE ele-
ments (LINE-CS) are represented in the X chromosome
and in the autosomes too, but their concentration at the
end of Y chromosome is particularly high. This obser-
vation led to the hypothesis that these sequences might
have a role in maintaining the structure of Y chromo-
some and that they can contribute to the morphological
and structural differentiation of the sex chromosomes,
by creating heteromorphic regions in which the recom-
bination is prevented (Sakamoto et al., 2000; Peil et al.,
2003).
The phenotypic expression of sex in hemp shows
some flexibility. Anomalies in flower development
are sometimes observed, such as the appearance of
hermaphrodite flowers or the development of mixed
inflorescences (bearing both male and female flowers),
like those occurring in the monoecious phenotypes.
Monoecious varieties have been developed from some
of these mutations, and need a strict selection to be
maintained in the variety during the seed multiplica-
tion, due to the recessive nature of the trait.
In some hemp genotypes it is possible to obtain to-
tal or partial reversion of the sex. It is known that the
treatment with masculinizing or feminizing chemical
agents is effective in determining the formation of the
opposite sex reproductive organs even in plants that are
already sexually well differentiated. Chemicals that in-
hibit the biosynthesis or the activity of ethylene, such
as aminoetoxyvinylglycine, silver thiosulphate and sil-
ver nitrate, have a masculinizing effect, while the pre-
cursors or activators of the biosynthesis of ethylene,
like etephon, have a feminizing effect (Mohan Ram
& Sett, 1982a, 1982b). The ability to undergo sexual
reversion is thought to have a genetic base: some eco-
types such as the Italian Carmagnola are very resistant
to any sex reversion treatment, while plants belong-
ing to Fibranova cv. are quite prone to sex reversion
(G. Grassi, E. de Meijer, personal communications).
In Italian open field conditions, the life cycle of a
typical dioecious variety has a 5–6 months duration,
and sexual maturity is attained after 3–4 months, when
the earliest unisexual flowers appear. Sexual dimor-
phism of dioecious hemp is generally apparent only
in a much later stage of development, just before the
onset of flowering, when a marked elongation of the
last internodes occurs in male plants causing them to
become taller and slender than female plants.
Sex is considered an important trait for hemp ge-
netic improvement. The Bredemann’s strategy of se-
lection for fibre quality implies a relatively early quali-
quantitative analysis of fibre in male plants before
pollen dispersion (Bredemann, 1938). This analysis
is followed by the elimination of lower-quality male
plants, not intended for pollination. Therefore, the pos-
sibility of early sex identification and the study of the
mechanism of sexual differentiation in dioecious va-
rieties are of paramount interest in hemp research.
Attempts have been made in the pre-genomic era by
multivariate analysis of morphological traits followed
by correlation to the sex expression (Lacombe, 1980).
Since the Nineties, DNA markers were developed,
capable of discriminating the male plants from the
female and the monoecious ones (Mandolino et al.,
1998, 1999). Such markers (see also the paper by
G. Mandolino and A. Carboni in this special issue) can
be fruitfully used in the selection schemes for hemp
breeding, and in the assessment of the number of male
plants in monoecious seed lots.
Sex linked markers, provided that they are tightly
and reliably associated to the sexual phenotype, can
97
be of great importance in the study of the earliest
stages of Cannabis sexual differentiation. Beyond the
relationship between the presence of the Y chromo-
some and the male sex, very little is known about the
molecular mechanisms underlying sexual differentia-
tion of hemp. We have investigated the onset of sexual
differentiation both at the histological and molecular
level. The characterization of different developmental
stages in male and female plants was carried out by op-
tical microscopy; differentially expressed sequences at
the same developmental stage in the two sexes were
also identified by cDNA-AFLP analysis. The possible
role of these differently expressed sequences in male
and female plants at the onset of sexual differentiation is
discussed.
Materials and methods
Plant material
All the experiments were carried out using cv.
Fibranova, an Italian cross-bred variety, characterized
by high fiber content; Fibranova is dioecious and shows
asex ratio close to 1. Seeds were sown in peat paper
pots, and plants grown in the greenhouse at a 16 h
photoperiod, with a daily thermo period of 11–27
C,
throughout the plant’s life cycle.
Sex identification
Early sex identification was carried out according
to Klimyuk et al. (1993) with minor modifications
(Mandolino and Ranalli, 2002). Tissue fragments
3–4 mm long were picked up from young leaves at
the 2–3
node and collected in sterile 1.5 ml vials;
40 µlofa0.25 M NaOH solution were added to each
vial that was then placed in boiling water (100
C) for
50 s. An amount of 40 µlofa0.25 M NaCl solution
and 20 µlofadetergent-buffer solution (0.5 M Tris
pH 8, 0.25% Triton X-100) were added; the vials
were then centrifuged for few seconds at maximum
speed and placed in boiling water (100
C) for 2 min.
Each tissue fragment was transferred in a PCR vial
and 25 µlofthe PCR reaction mixture for the male
specific marker amplification were added (Mandolino
et al., 1999). Amplicons were run in a 1% agarose gel
in TAE 1X buffer and visualized by U.V. exposition
(305 nm), after ethidium bromide staining. All the
buffers and disposables used in this procedure were
sterile.
Histological methods
Apices from male and female plants grown in the
above described conditions were collected at the
emergence of the leaves of the second, fourth, sixth
and eight nodes. The collected samples were sub-
merged in the fixing solution (glutaraldehyde 3%
in KH
2
PO4/Na
2
PO4 0.1 M buffer, pH 7); this step
sometimes needed a brief treatment in a vacuum jar, to
allow the air to flow out of the stem vessels. Fixing was
obtained after a 1–3 weeks incubation at 4
C. Samples
were then rinsed several times in phosphate buffer
0.1 M, pH 7.0 at 4
C, dehydrated in a graded ethanol
series (ethanol/phosphate buffer, ethanol/water), and
pre-infiltrated in a mixed solution of ethanol and
Technovit 7100 resin (Heraeus Kulzer) for4hat4
C.
The infiltration and embedding steps were carried out
for 36 h at room temperature. Longitudinal sections
2–4 µm thick, obtained with an ultra microtome
(Reichert Jung) fitted with a glass blade, were then
stained with toluidine blue. A toluidine blue 0.1%,
borax 1% aqueous water solution was dropped on the
sections adhering on a glass microscope slide, and was
then dried on a hot plate. Differential staining was ob-
tained by adding few drops of an acid–alcohol solution
(ethanol 70%, HCl 1.5%) and rinsing with water; a drop
of water allowed the cover glass to adhere to the stained
sections that were then observed and photographed
with an optical microscope (Leitz Orthoplan).
RNA extraction and cDNA synthesis
Pooled apices, respectively from undifferentiated
plants at the second node, male plants at fourth node
and female plants at fourth node, were frozen and
grinded under liquid nitrogen. Extraction Buffer
(50 mM Tris-HCl, pH 9.0, 100 mM NaCl, 10 mM
EDTA, 2% SDS) was warmed at 37
C and added
at a 5:1 ratio (v/w) to the powdered sample. Ho-
mogenate was transferred in a sterile polypropylene
tube, extracted twice with an equal volume of a
phenol/chloroform/isoamyl alcohol mixture (25:24:1),
and once with two volumes of a chloroform/isoamyl
alcohol mixture (100:1). Oligo-dT cellulose (Roche)
50 mg/ml and NaCl 0.4 M (final concentrations)
were added to the supernatant. Samples were shaken
(30–50 rpm) for 30 min and then centrifuged for few
minutes at low speed. The cellulose was rinsed twice
with 10 ml of washing Buffer 1 (10 mM Tris-HCl, pH
7.5, 400 mM NaCl, 0.2% SDS) and twice with 10 ml of
washing Buffer 2 (20 mM Tris-HCl, pH 7.5, 100 mM
98
NaCl); the tubes were centrifuged at 800 × g for
5 min. after each washing. Cellulose was transferred
to a 10 ml sterile chromatography column, and rinsed
three times with 10 ml of washing Buffer 2. The
mRNA was eluted from cellulose by applying to the
column 10 mM Tris-HCl, pH 7.5, pre-heated at 65
C.
The eluted mRNA was precipitated with two volumes
of ethanol, and resuspended in DEPC treated water
(diethylpyrocarbonate 0.1%). mRNA (1 µg) was used
as template for the synthesis of double stranded cDNA.
First strand was synthesized by using the Superscript II
Reverse Transcriptase (400 U, Life Technologies) in a
final volume of 30 µl. RNAase H (1U, Life Technolo-
gies) and DNA Polymerase I (40U, Life Technologies)
were added to the first strand reaction mixture, for
the synthesis of the double stranded cDNA in a final
volume of 150 µl. The mRNA template was finally
digested by RNAase A (90 µg, Life Technologies) at
37
C for 15 min. The reaction mixture was extracted
with chlorophorm/isoamyl alcohol (100:1) and the
double stranded cDNA purified by Microcon YM-100
columns (Amicon). One tenth of the recovered volume
was quantified by gel electrophoresis, using lambda
DNA as standard.
CDNA-AFLP analysis and band elution
The cDNA-AFLP procedure (Bachem et al., 1996)
was adapted for Cannabis sativa following Hartings
(1999), with minor modifications. Double stranded
cDNA (20 ng) was digested by Mse I (5U) and BstY
I (0.5U) (Life technologies) in a final volume of 40
µl, for 1 h at 37
C. Mse I adapter (50 pmoles) and
BstY I adapter (20 pmoles) were added to the digested
cDNA and the ligation was carried out using theT4
DNA Ligase (5U, Amersham Pharmacia) for 3 h at 37
Cinafinal volume of 50 µl. The ligation mixture
was diluted to 100 µl, and 5 µl were used as template
in the pre-amplification step. The pre-amplification (30
pmoles of the BstY I and Mse I anchor primers, 1.5 mM
MgCl
2
, 0.2 mM each dNTPs, 1 U Taq Polymerase and
1X Buffer, Life Technologies) was carried out for 20
cycles using the following profile: 94
C, 30 s; 56
C,
60 s; 72
C, 60 s. One tenth of the secondary template
produced was checked on a 0.8% agarose gel in TBE
0.5 X. Selective amplifications were carried out with 60
combinations of two anchor primers for BstY I, bearing
an extension of one selective base (C or T), with 30 an-
chor primers for Mse I bearing a three selective bases
extension (sequences of adapters and primers used
are reported in Table 1). The BstY I selective primers
Table 1. Sequences of the adapters and primers used for cDNA-AFLP
fingerprinting (Hartings, 1999)
Mse I adapter; top strand 5
-GACGATGAGTCCTGAG-3
Mse I adapter; bottom strand 5
-TACTCAGGACTCAT-3
BstY I adapter; top strand 5
-CTCGTAGACTGCGTACC-3
BstY I adapter; bottom strand 5
-GATCGGTACGCAGTCTAC-3
Mse I anchor primer 5
-GATGAGTCCTGAGTA-3
BstY I anchor primer 5
-GTAGACTGCGTACCGATC-3
Mse I selective primer 5
-GATGAGTCCTGAGTANNN-3
BstY I selective primer 1 5
-GTAGACTGCGTACCGATCT-3
BstY I selective primer 2 5
-GTAGACTGCGTACCGATCC-3
The variable selective nucleotides are represented by N.
were labelled with Redivue-[γ -
33
P]dATP (Amersham
Pharmacia) using the T4 Polynucleotide Kinase (Life
Technologies), following the protocol suggested by the
manufacturer. PCR amplification was carried out as fol-
lows: 94
C, 30 s; 65
C, 30 s (0.7
C/cycle) for 13
cycles, and 94
C, 30 s; 56
C, 30 s; 72
C, 60 s for 13
cycles. Amplicons were size-fractionated on 6%, 0.35
mm thick polyacrylamide sequencing gels (urea 8 M)
at 40 W for 2.5 h in the Sequigen GT apparatus (Bio-
Rad). The gels were dried at 60
C for 1 h, coated with
transparent film and exposed to autoradiography film
(Biomax MR, Kodak) for 24–48 h at 20
C. The bands
of interest were excised from the gel and collected in
sterile vials; 50 µlofTEbuffer 0.1 X were added, and
vials incubated at 65
C for 15 min for band recovery.
Reverse Northern hybridization
The recovered bands (10 µlofthe eluted volume)
were re-amplified with the same primer combinations
and PCR profile used for their selective amplification.
PCR products were checked on 2% agarose gels in
TBE 1X and blotted on nylon membranes to obtain
two identical blots of all the polymorphic fragments
eluted. mRNA (1 µg) from males and females apices
was transcribed to labelled cDNA in a mixture con-
taining 10 mM DTT, 0.3 M each dNTP mix (with no
dCTP), 40 µCi Redivue- [α-
32
P] dCTP (Amersham
Pharmacia), 1 µg oligo-dT
18
, 400 U Superscript II RT
enzyme in the 1X first strand RT Buffer (Life Technolo-
gies), in a final volume of 30 µl. Labelled cDNA was
purified on Sephadex G-50 packed columns (Sigma-
Aldrich) and quantified by the Cerenkov method. The
two identical blots were hybridized with the same quan-
tity (total cpm) of labelled cDNA from males and from
females apices, for8hat65
C. The membranes were
rinsed with SSC 2X and 1X, 0.1% SDS solutions for
99
20 min at 65
C and then exposed to autoradiography
film (Biomax MR, Kodak) for 7–10 days.
Northern hybridization
Messenger RNA belonging to males and females apices
was separated on 1% agarose gel (40 mM MOPS,
10 mM sodium acetate, 1 mM EDTA, 0.1% DEPC,
2.2 M formaldehyde) and blotted on nylon membranes
according to Sambrook et al. (1989). AFLP fragments
were re-amplified from the eluted cDNA band as de-
scribed above, and purified on a 1.15% agarose gel
in TAE modified buffer 1X (40 mM TRIS-Acetate,
1mMdisodium-EDTA pH 8.0), with the Montage
DNA gel Extraction Kit (Millipore). Eighty nanograms
of the purified fragments were radioactively labelled
with the random oligonucleotide priming method, ac-
cording to Sambrook et al. (1989), using 40 µCi of
Redivue- [α
32
P]dCTP (Amersham Pharmacia), in a
final volume of 25 µl. Labelled probes were purified
on Sephadex G-50 (Sigma Aldrich) packed columns
and quantified. Northern hybridization was carried out
according to Sambrook et al. (1989); hybridized blots
were then exposed to autoradiography film (Biomax
MR, Kodak) for 1–3 days. Quantitation of mRNA im-
mobilized on nylon membranes was made by radio la-
belled oligo-dT
18
probes hybridization (Harley, 1997).
Eighty nanograms of Oligo-dT
18
were labelled in a
mixture containing 50 µCi of Redivue- [γ -
32
P] dATP
(Amersham Pharmacia), 6U of T4 Polynucleotide
Kinase and 1X enzyme buffer (Life Technologies),
in a final volume of 50 µl, at 37
C for 1 h. The
labelled probe was precipitated in absolute ethanol
(2.5 volumes) and ammonium acetate 10 M (2 volumes)
for 45 min at 20
C, centrifuged at 10.000 rpm and
the pellet resuspended in 500 µlofOligo Buffer (5X
Denhardt, 0.8 M NaCl). Filters were pre-hybridized
with Oligo Buffer at 30
C for 6 h and hybridization
was carried out over night. Filters were rinsed in SSC,
0.1% SDS solutions, at low but increasing stringency
(SSC 6X, 5X, 4X) at 30
C, and autoradiographed.
Cloning, reverse Northern hybridization
and sequencing of the subclones
Positive, differentially hybridizating probes were lig-
ated to the pCR 2.1-TOPO vector and cloned in the
TOP 10 F
cells One Shot chemically competent, by the
TOPO TA Cloning kit (Invitrogen). Twenty mini preps
per transformation were performed by the CONCERT
Rapid Plasmid Miniprep System (Life Technologies).
Cloned fragments were excised from vectors by Eco
RI digestion and separated onto agarose gel. Two iden-
tical gels of the excised fragments were blotted onto
nylon filters and hybridized with radio labelled cDNA
from males and females, following the reverse northern
procedure described above.
Recombinant subclones obtained from each trans-
formation reaction were sequenced by ABI PRISM
310 Genetic Analyzer (PE Applied Biosystem). The
sequences obtained were analyzed by the BLAST
algorithm (Altschul et al., 1990).
Results
Microscopic analysis of apex differentiation
Microscopic analysis of male and female apices was
performed at various developmental stages (emergence
of the leaves of the second, fourth, sixth and eight
nodes). At the last stage examined, the plants had
started flowering, and the inflorescence buds were al-
ready macroscopically visible. The microscopic anal-
ysis of the inflorescence buds and mature flowers was
also carried out. In most of the male and female apices
analyzed at the fourth node stage, meristematic buds
were observed at the axils of the leaves of the earlier
nodes (third and/or second node; Figure 1a). No floral
primordia were visible at this stage, but in the apices ob-
served at the subsequent stages of differentiation (sixth
and eighth node stages), meristematic buds were clearly
more developed and showed a stronger mitotic activity
(Figure 1b). From these observations, we argued that
the meristem primordia produced at the fourth node
stage could in most cases develop into an inflorescence
bud, in response to external or internal cues. No meris-
tem buds were observed at the axils of the leaves when
apices at the second node were analyzed (Figure 1c),
therefore it seems possible that the earliest step of apex
sexual commitment could occur at the emergence of
the fourth node’s leaves in Fibranova cv., under the en-
vironmental conditions used. This stage was therefore
chosen for the subsequent study of differential gene ex-
pression in male and female plants; as a control, apices
picked up at the second node were used, as they rep-
resent a fully vegetative and undifferentiated meristem
tissue.
Differential gene expression analysis
Sixty different primers combinations were used to
screen double stranded cDNA belonging to male and
100
Figure 1. (a) Longitudinal section of the apex at the fourth node (25×). (b) Detail of a meristematic bud at the axil of a sub apical leaf (40×).
(c) Longitudinal section of the apex at the second node (25×).
female apices at the fourth node. Four thousands eight
hundred bands were obtained, with an average of 80
bands per primer combination. Nine hundreds out
of the 4800 fragments were polymorphic, and there-
fore putatively belonging to differentially expressed
mRNAs, as they were present only in male or in female
cDNAs and were absent, or present below detectability
levels, in the control cDNA sample (second node stage;
Figure 2). Among the different combinations tested,
those obtained using the B0+T primer, produced a
higher number of polymorphic bands (509) compared
to those obtained by the B0+C primer (384). In
general, more polymorphic fragments were produced
in the male cDNAs (average value of 9.6 bands per
primer combination) than in the female ones (average
6.3 bands).
All the polymorphic fragments were eluted from
the gel and blotted onto nylon membranes, in order
to check by reverse northern hybridisation their true
differential expression. Most of the isolated AFLP
fragments resulted to be either equally expressed
or not detectable when probed with labelled cDNA
from males and female’s fourth node apices. Only 22
fragments were confirmed to be actually differentially
expressed. Five of these fragments, showing a clear
differential expression and a high hybridization signal,
were used as probes in northern analysis.
Northern data confirmed the differential expres-
sion of the five fragments at the fourth node stage;
the mRNAs corresponding to the isolated fragments
resulted all more expressed in female apices compared
to the male ones (Figure 3). The five AFLP differ-
entially displayed fragments, named C1, C2, C3, T1
and T2, were sub-cloned in pCR2.1 vector. About 20
preps from each subcloned fragment were further con-
trolled by reverse Northern analysis. Reverse Northern
revealed that only a subset of the subclones obtained
from each AFLP polymorphic fragment corresponded
to differentially expressed sequences in male and fe-
male apices at the fourth node, suggesting that in some
cases the original amplified AFLP fragment could con-
tained a mixture of different sequences.
Sequence analysis
All the subclones corresponding to male/female differ-
entially expressed mRNAs were sequenced. Sequence
analysis further confirmed the heterogeneous compo-
sition of some of the AFLP fragment isolated: the five
differentially expressed AFLP fragments were in fact
separated into nine different clones belonging to nine
different mRNAs, all induced in the female apices at
the fourth node. Clones derived from the same AFLP
fragment have been designated by different letters (e.g.
T1A, T1B, etc.); their main characteristics are sum-
marised in Table 2. The clones showing the high-
est level of similarity with known sequences were:
the C2.A clone (283 bp), within a 205 bp region of
the cDNA for an SMT3-like protein (86% similarity);
the C3.A clone (435 bp), within a 130 bp region of the
cDNA for a kinesin nine heavy chain (84% similarity);
the T2.A clone (282 bp), within a 231 bp long region of
101
Figure 2. AFLP pattern produced by eight BstY I+1/Mse I+3 primer combination on cDNAs from apices at the second node stage (lane 1),
male apices (lane 2) and female apices (lane 3) at the fourth node stage.
Figure 3. Northern analysis of five polymorphic cDNA AFLP frag-
ments: (a) C1; (b) C2; (c) C3; (d) T1; (e) T2. Male and female mRNA
from apices at the fourth node are blotted respectively on the left and
right lane, and quantified in (f).
the cDNA for a putative permease (80% similarity); the
T2.B clone (284 bp), that produced significant matches
(86% similarity) within a 203 bp region of a Rac-GTP
binding protein-like. Two examples of best matches are
graphically represented in Figure 4.
Discussion
Under the condition described, the beginning
of the sexual differentiation of Cannabis sativa
(cv Fibranova), occurs 50–60 days after seed ger-
mination; at this stage the plant reaches a height of
160–180 cm. However, the reduction of the photope-
riod length, or the exposition to low temperatures,
causes the lifecycle to shorten, and the development
of mature flowers to occur even 15–20 days after the
plant emergence. These observations confirmed that
the sexual commitment in hemp plants may take place
in a very early phase of the vegetative development,
102
Table 2. Results of BLAST analysis for the nine clones differentially expressed at the fourth node (see text for details)
Clone Primer combination Best match (blastn) Score E-value Best match (blastx) Score E-value Putative function
T2.A (282bp) B0+T/M0+CGT AF466198.1 putative permease 97.6 7e
18
AF466198 putative permease 159 1e
38
Permease
C2.A (283bp) B0+C/M0+AGT AF451278.1 SMT3 ubiquitin 184 4e
44
AF451278 SMT3 protein 149 1e
35
Ubiquitin like protein
like protein
C3.A (435bp) B0+C/M0+CAC AF272756 kinesin 9 heavy chain 101 8e
19
AF272756 kinesin heavy chain 156 4e
38
Kinesin heavy chain
T2.B (435bp) B0+T/M0+CGT ––AL163816 Rac-GTP binding protein like 100 3e
21
Rac-GTP binding
protein-like
C3.B (476bp) B0+C/M0+CAC DCU47095 putative ribosomal 135 5e
29
AP003241 putative ribosomal protein 60S 94 2e
23
Ribosomal protein
protein
C1.A (218bp) B0+C/M0+ACG NM119070.1 acid phosphatase 42 0.24 NM119070 acid phosphatase like protein 103 6e
22
Acid phosphatase
like protein protein
C1.B (212bp) B0+C/M0+ACG AF108891 ADP ribosilation factor 98 5e
18
AY062539 Calcium dependent protein kinase 50 2e
09
T1.A (106bp) B0+T/M0+AAC –––PPA1LYCES acid phosphatase precursor 40 0.006 Acid phosphatase
precursor
T2.C (280bp) B0+T/M0+CGT ZMRNARPP2 ribosomal protein 91 5e
16
––Ribosomal protein
103
Figure 4. (a) Alignment between the C2.A clone and the AF451278 (ubiquitin-like protein SMT3 of Phaseolus vulgaris, partial coding sequence)
nucleotide sequences and (b) aminoacid sequences (C2.A frame +2), performed by the MultAlin tool (Corpet, 1988).
and suggested to undertake a microscopic study of the
changes in apex morphology during development, in
support of gene expression analysis. Microscopic anal-
ysis was carried out in male and female apices picked up
at various stages of development, from the emergence
of the leaves of the second node, until the production of
unisexual flowers. The choice to use the node number
to define the plant developmental stage, instead of
other morphologic parameters as the plant height or
the time after seed germination, was suggested by the
observation that these parameters are more flexible and
more conditioned by environmental factors and genetic
variability (Mediavilla et al., 1998). The microscopic
analysis of male and female apices under non-inductive
conditions (long photoperiod) revealed the formation
of meristematic buds at the axils of the stem leaves
as soon as the fourth node stage is reached, at a plant
height of 15–20 cm. These undifferentiated meristem-
atic buds could develop in an inflorescence bud under
the opportune endogenous stimulus, as pointed out by
the results of the microscopic analysis of the apices at
the subsequent stage of development (data not shown).
Apices then could be committed to reproductive devel-
opment as early as the leaves of the fourth node emerge.
Our interest was therefore focused on the male and
female apices at the fourth node, and in particular on
the genes that are expressed at this early stage of apex
differentiation, and possibly responsible of its sexual
commitment.
Sexual dimorphism in Cannabis naturally occurs
late during its lifetime, immediately before the pro-
duction of the unisexual flowers, when the vegetative
development is almost completed. Therefore, the in-
vestigation of the early stages of sexual differentiation
requires a method for the precise identification of the
sex. A rapid method for sex determination in Cannabis
has been developed, based on the PCR amplification of
the 391 bp male-specific SCAR marker directly from
a leaf tissue fragment. This method is suitable for a
precise, early and rapid identification of male plants
and was of great importance in the setting of the ex-
perimental design for the analysis of differential gene
expression in the early phase of sexual differentiation;
it can be also useful during the fiber hemp MAS pro-
grams.
Gene expression in male and female apices at the
fourth node stage was compared by cDNA-AFLP, car-
ried out testing 60 primer combinations. Nine hundreds
polymorphic fragments were produced and tested by
reverse Northern and Northern hybridization; however,
only five fragments were confirmed as truly differen-
tially expressed. CDNA-AFLP is a high-throughput
technique: the restriction with two enzymes allows to
reduce the size of the fragments to be analyzed in a
104
range of length (50–1000 bp) that could be resolved in
a sequencing gel; the use of specific primers increases
the reproducibility, and the two PCR amplifications are
likely to increase the detectability of the genes with a
low expression rate. The best enzymes to be used as
rare cutter in the cDNA-AFLP should have one restric-
tion site per transcript, in order to obtain as a product
of the double digestion with rare and frequent cutter,
one fragment per transcript. The most frequently used
rare cutters are Eco RI, BamH I and Pst I enzymes, al-
though it has been demonstrated that all of them have a
restriction site only in half of the transcripts (Bachem
et al., 1996), causing the reduction of the transcripts
set to be analysed. BstY I, the rare cutter enzyme used
in this work, has two variable positions in its restric-
tion site, this allows to increase the probability for a
given cDNA to be digested, but also the number of
the fragments produced. In fact, we obtained an aver-
age of 80 bands per amplification, in comparison to a
mean of 50–70 bands obtained with standard combina-
tion of rare and frequent cutter enzymes used in plant
cDNA-AFLP analysis (Bachem et al., 1998). The high
number of false positives obtained might be explained
considering the high degree of intra-specific genetic
variability, characteristic of a dioecious species with an
obligate allogamous reproduction system. The degree
of genetic polymorphism in Cannabis sativa, assessed
for cv. Fibranova by the occurrence and frequency of
RAPD marker, resulted close to 85% (Faeti et al., 1996;
Forapani et al., 2001). As this study represents the first
report on gene expression analysis in Cannabis sativa,
there are no useful works to assess the impact of ge-
netic variability and plant heterogeneity on gene ex-
pression analysis. It is however conceivable that a high
degree of genetic polymorphism might affects the effi-
ciency of techniques designed to identify differentially
expressed genes, based on restriction analysis, such as
the cDNA-AFLP.
Four of the identified clones putatively belong to the
mRNA for a permease, for an SMT3-like protein, for
the heavy chain of a kinesin 9 protein and for a Rac-
GTP binding protein. Rac proteins are generally in-
volved in the signal transduction pathways that could be
activated by external or internal cues; in plants they are
more represented in the meristematic tissues (Valster
et al., 2000). Recently, it has been demonstrated for
Rac-GTP binding protein a signalling role in auxin-
regulated gene expression in Arabidopsis (Tao et al.,
2002). There is some evidence of the auxin role in the
differentiation of the female sex in Cannabis sativa,as
they accumulate in female plant during development
and reach high levels just before the transition to flow-
ering (Galoch, 1980). The SMT3 protein belongs to
the family of ubiquitins that are involved in the post-
translational modification of the most plant’s proteins.
The family of kinesins comprises proteins that mediate
the cytoskeleton movements. In mammals and plants,
kinesins are involved in the vescicles trafficking, and
in the formation of mitotic spindle (Baskin, 2000). The
permeases are a heterogeneous family of membrane’s
proteins that mediate the ions or metabolites exchange
between cells or within cell. The high expression of
these genes in the female apices at the fourth node sug-
gests that, at this stage, some metabolic processes are
more active in the females than in males, either due
to their specific activation in the female plants, or to
their repression in the male plants. The induction of
Rac-GTP binding proteins could be functional to the
activation of auxin-induced genes, probably involved
in the female sex differentiation. (Galoch, 1980).
It has been proposed that, from the evolutionary
point of view, dioecy derived from the hermafroditism
through an intermediate state of ginodioecy, and that
the male genotype derived from the hermaphrodite
one by the repression of the female characters; the
male and female sexes would then differ by the pres-
ence of female-suppressing factors in the male plants
(Charlesworth and Guttman, 1999). This theory would
be confirmed by the actual transition from an inter-
mediate state of hermafroditism to a definitive condi-
tion of dioecy, observed in many dioecious species dur-
ing the ontogenesis of unisexual flowers (Silene latifo-
lia, Fragaria spp., Asparagus officinalis and Vitis spp.;
Dellaporta, 1993); another evidence supportive of this
hypothesis is the possibility, in some dioecious species,
of partial or complete sex reversion (Grant et al., 1994).
It is therefore conceivable that in Cannabis sativa, the
repression of female characteristics in the male plants
apices implies the down-regulation of the genes cod-
ing for enzymes involved in metabolic pathways more
strictly related to the differentiation of the female sex,
as suggested by the results presented here.
The study of sexual differentiation in dioecious
species has been often approached by the identifica-
tion of sex-specific DNA markers that could be often
mapped on sexual chromosomes (Donnison and Grant,
1999; Peil et al., 2003). These markers often belong to
regions rich in repetitive DNA, and to LINE-like or
non-LTR retrotransposons repetitive sequences (Scutt
et al., 1999; Sakamoto et al., 2000; Mandolino et al.,
2002). In Silene latifolia, the analysis of subtracted li-
braries from different stages of male or female flower
105
differentiation, allowed the isolation of Men 1–10 and
MROS 1–4 genes, all male specific (except MROS 3),
and generally coding for functions related to the male
reproductive organs development or to pollen matura-
tion (Scutt et al., 1999). Our work represents an al-
ternative approach since gene expression in male and
female apices was analyzed when there were no floral
buds visible yet, and therefore differences in gene ex-
pression between the two sexes could be related to the
onset of apex sexual differentiation. Though it is diffi-
cult to make a direct correlation between the induction
of the differentially expressed clones and sexual dif-
ferentiation, the strategy used allowed the isolation of
gene sequences differentiating male and female plants
at an early stage of development, and represents the first
step on identification of sex-associated gene expression
in Cannabis sativa.
Finally, this research reported for the first time a
combined morphological and molecular description of
a critical stage of Cannabis sexual differentiation. The
sequences identified so far will be checked at various
stage of sexual development and their full length se-
quences will be tracked down, allowing the cloning
and the characterization of sex-related genes in hemp.
Acknowledgments
The authors would like to thank Prof. Piera Medeghini
Bonatti (University of Modena and Reggio Emilia)
for passing on the theoretical and practical knowledge
about plant sample microscopic observation, and for
the technical supplies.
This research was realized in the framework of the
project “Hemp for textiles: from the production to end
uses, supported by the Italian Ministry of Agriculture.
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