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Sakamoto K, Shimomura K, Komeda Y, Kamada H, and Satoh S. 1995. A male-associated dna sequence in a dioecious plant, Cannabis sativa L. Plant Cell Physiol. 36(8): 1549-1554
Male-associated DNA sequences were analyzed in a dioecious plant, Cannabis sativa L. (family: Moraceae), which is known to have sex chromosomes. DNA was isolated from male and female plants and subjected to random amplification of polymorphic DNA. Two out of 15 primers yielded fragments of 500 and 730 bp which were detected in all male plants but not in any of the female plants tested. These two DNA fragments were cloned and used as probes in gel blot analysis of genomic DNA. When the male and female DNAs were allowed to hybridize with the 500-bp probe, no differences in patterns were observed between male and female plants. By contrast, when these DNAs were allowed to hybridize with the 730-bp probe, much more intense bands specific to male plants were detected, in addition to less intense bands that were common to both sexes. The 730-bp DNA fragment was named MADCI (male-associated DNA sequence in Cannabis sativa). The sequence of MADCI did not include a long open reading frame and it exhibited no significant similarity to previously reported sequences.
The flowers of most plant species have stamens and a pistil. Such plants are classified as hermaphrodite plants because they have bisexual flowers. Some plant species, such as cucumber and maize, are classified as monoecious plants, with each plant having both male and female flowers. In such cases, the male flowers have well developed stamens and an undeveloped pistil, while the reverse is true for the female flowers. Moreover, some species, such as Asparagus,. Rumex, Humulus, Sitene and Cannabis, are classified as dioecious because individual plants have either male or female flowers exclusively. Cannabis, Rumex and Silene are reported to have sex chromosomes (X and Y chromosomes) (Parker 1990, Chattopadhyay and Sharma 1991) and the sex of plants is known to be genetically determined. The analysis of the sex chromosomes in dioecious plants should provide significant information about sex differentiation in plants.
Cannabis sativa L. is a dioecious plant with two heteromorphic sex chromosomes (Hirata 1924). Yamada (1943) revealed that the female plants have two X chromosomes whereas the male plants have one X chromosome and one Y chromosome, with the latter being much larger than the X chromosome and the autosomes. The roles of the sex chromosomes in sex determination have been studied in haploid, diploid and triploid plants (Warmke and David- son 1944, Nishiyama et al. 1947). Warmke and Davidson reported that the X and Y chromosomes of Cannabis sativa carry female- and male- determining genes, respectively, and that the autosomes are not involved in sex determination.
In the present study, we assumed that a male-determining gene or a female-suppressing exists on the Y chromosome only (not on the X chromosome), and we compared male and female DNAS, searching for specific DNA sequences that exist only in males by the technique known as random amplification of polymorphic DNA (RAPD; Williams et al. 1990).
Materials and Methods
Plant materials - Seeds of Cannabis sativa L. var. sativa (CBDA strain) were obtained from a population that had been grown in an experimental field at the Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences (Tsukuba, Japan). The plants were grown in earthenware pots at 25'C under a photoperiodic regime of 16 h of light and 8 h of darkness daily. Light from fluorescent lamps (FL40SS D; Toshiba, Tokyo, Japan) was adjusted to 40,m mol photons m-2 s-1 at plant level. The humidity was maintained at 70%. When plants reached 100-150 cm in height, they were transferred to a photoperiodic regime of 8 h of light and 16 h of darkness daily for induction of flowering. After two weeks, the sexes of the flowers were determined and then the fully expanded young leaves were collected, frozen in liquid nitrogen and stored at - 80'C for analysis.
Preparation of DNA - DNA was extracted separately from the leaves of five male and five female plants by the method of Shure et al. (1983) with modifications. Frozen leaves were ground to a fine powder in liquid nitrogen. The resultant powder was mixed with 8 volumes of extraction buffer [50 mM Tris-HCl (pH 7.5), 7 M urea, 350 mM NACI, 20 mM EDTA, 2% N-lauroylsarkosine (sodium salt), 5% phenol], mixed with SDS (final concentration 0.2%), shaken gently for 20 min and centrifuged (3,000 x g, 15 min). The supernatant was mixed with an equal volume of a mixture of phenol, chloroform and isoamyl alcohol (75 : 24 : 1, v/v), shaken for 20 min and centrifuged (3,000 x g, 15 min). The upper phase was collected, and extraction with the mixture of phenol, chloroform and isoamyl alcohol was repeated two more times. The final upper phase was collected, mixed with 1/4 volume of 10 M lithium chloride and incubated at 4'C for 12 h. Then the solution was brought to room temperature, cooled again to 4'C and centrifuged (8,000 x g, 30 min, 4'C). The resultant supernatant was collected and mixed with 0.1 volume of 3 M sodium acetate (pH 4.8) and 2.5 volumes of ethanol. The DNA was pelleted by centrifugation (8,000 x g, 30 min, 4'C) and dissolved in TE buffer [ 1 0 mM Tris-HCI (pH 8.0), 1 mM EDTA]. The DNA was quantified spectrophotometrically with a DNA/RNA Calculator (UV-260; Pharmacia, Uppsala, Sweden) and used for the polymerase chain reaction (PCR). For the gel blot analysis, DNA was further purified by CsCl density gradient centrifugation (Ausubel et al. 1990).
Polymerase chain reaction (PCR) - Each 5 ml of the reaction mixture for PCR contained 5 ng of DNA in 1ml of TE buffer, 0.4ml of 1 M Tris-HCl (pH 9.0), 0.2 ml of 0.5 M (NH4)2S04, 0.2 ml of 2.5 mM dATP, 0.2 ml of 2.5 mM dCTP, 0.2 ml of 2.5 mM dGTP, 0.2 ml of 2.5 mM dTTP, 1ml of 17.5 mM MgCl2, 0.2 ml of a 10 mM solution of primer, 1 ml of distilled water, and 0.2 ml of AmpliTaq DNA Polymerase (5 units ml-1; Perkin-Elmer, New Jersey, U.S.A.). After incubation at 92'C for 3 min, 45 cycles of PCR were carried out (95'C for 30 s, 34'C for 15 s, 74'C for 1 min), with a final incubation at 72'C for 8 min. The primers used were the 15 arbitrary 10-meric oligonueleotides shown in Table 1.
Gel electrophoresis and recovery of DNA fragments - The products of PCR were separated by electrophoresis on 2.0% (w/v) agarose gels and visualized by staining with ethidium bromide (Sambrook et al. 1989). The DNA fragments were recovered with a Mermaid kit (BIO 101, Inc., La Jolia, CA, U.S.A.).
Preparation of DNA probes - A fragment isolated after PCR was cloned into the pCR vector (Invitrogen, San Diego, CA, U.S.A.). The cloned fragment was recovered by digestion with the restriction endonuelease EcoRI and labeled with [a-32P]dCTP by the random-priming method (Feinberg and Yogelstein 1983, 1984).
Gel blot analysis of genomic DNA - Ten micrograms of DNA isolated from leaves of individual male and female plants were separately digested with restriction endonucleases (EcoRI, BamHI and HindIII). The digests were fractionated on a 1.0% (w/v) agarose gel and transferred to a GeneScreen Plus membrane (Du Pont, Boston, MA, U.S.A.) as recommended by the manufacturer. For the repeated analysis of blots, each membrane was washed first with 0.4 M NaOH at 42'C for 30 min and then with 0.1% SSC that contained 0.1% SDS and 0.2 M Tris-HCl (pH 7.5) at 42'C for 30 min.
Sequence analysis of the DNA fragment - The DNA fragment was cloned into the M13 vector and a singlestranded DNA template was obtained (Sambrook et al. 1989). DNA sequencing was performed by the dideoxy chain-termination method (Sanger et al. 1977) using a Taq Dye Primer Cycle Sequencing kit (Perkin-Elmer, New Jersey, U.S.A.) and a DNA sequencer (model 373A; Perkin-Elmer, New Jersey, U.S.A.). The GENETYX- MAC program package was used for all computer analysis. Examination of similarities was performed with the EMBL Nucleotide Sequence Database.
PCR with arbitrary primers - DNA that had been separately isolated from leaves of five male and five female plants was used as the template for PCR. PCR was performed with arbitrary 10-meric oligonucleotides as primers (Table 1). The products of PCR were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. When primers 1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 13, 14, and 15 were used, some differences in banding patterns were detected among the five individuals of both sexes. However no sex-specific band that was common to five individuals of one sex was detected (data not shown). Several differences in the patterns of amplified fragments were observed when the two remaining primers (nos. 8 and 11) were used (Fig. 1, 2). Each of these two primers gave a band that was detected in the analysis of all male plants but not in that of any of the female plants. The sizes of the fragments responsible for these bands were about 500 and 730 bp in the case of primer no. 8 and primer no. 11, respectively. The DNA fragments were recovered from gels and cloned into a plasmid vector. The cloned DNAs were used in the following experiments.
Gel blot analysis of genomic DNA - DNAs isolated from leaves of each individual male and female plant were separately digested with EcoRI, BamHI and HindIII. The digests were fractionated on agarose gels, and then fragments were denatured and transferred to a membrane. When the male and female DNAs were allowed to hybridize with the 500-bp probe, no differences in patterns were observed between male and female plants with all the restriction endonucleases tested (Fig. 3). However, when these DNAs were allowed to hybridize with the 730-bp probe, some bands specific to male plants were detected in addition to bands that were common to both sexes with all these restriction endonucleases (Fig. 4). In particular, HindIII yielded two intense bands that were detectable only in the case of male plants. The same results were obtained when the DNA prepared from another set of male and female plants was used for blotting.
The 730-bp DNA fragment was named MADC1 (male-associated DNA sequence in Cannabis sativa). MADC1 was digested simultaneously with XbaI and XhoI, and three fragments (of 220 bp, 346 bp and 164 bp, respectively) were obtained and used as probes. The results shown in Figure 5 demonstrate that many more bands common to both sexes were detected with the 220-bp probe than with the entire sequence of MADC1 as probe [Fig. 5-(1), Fig. 4). By contrast, when DNAs were allowed to hybridize with the 164-bp probe, the blot gave a pattern that was specific to male plants [Fig. 5-(3)]. Furthermore, when the 346-bp probe (middle region of MADC1) was used, the blot gave a pattern intermediate between that generated by the 220-bp probe and that generated by the 164-bp probe [Fig. 5-(2)].
Analysis of sequence - The nucleotide sequence and open reading frames are shown in Figures 6A and 6B. MADC1 consisted of 729 base pairs and it did not include a long open reading frame. Moreover, no conspicuous amino-acid sequences were encoded in the sequence. When a homology search was performed using the 729-bp nucleotide sequence and corresponding deduced amino acid sequences, no significant similarity was found to sequences reported from any organism to date.
Two male-specific fragments (500 and 730 bp) were detected in Cannabis sativa by the RAPD technique. Though some differences in the patterns of bands were detected using the other 13 primers, no sex-specific band common to five individuals of one sex was detected. Therefore, the two male-specific bands appeared not to be a consequence of individual variation (Fig. 1, 2). When the male and female DNA was allowed to hybridize with the cloned 500-bp fragment as probe, no differences in patterns were observed between male and female plants (Fig. 3). The reason for this result may be that the differences between male DNA and female DNA are slight and only exist in the region that corresponds to the sequences used as primers. However, when the male DNA and female DNA were allowed to hybridize with MADC1 or with sub-fragments of MADC1, some more intense bands specific to male plants were detected, in addition to less intense bands that were common to both sexes [Fig. 4, Fig. 5-(3)]. MADC1 DNA might be amplified in male plants. However, because less intense bands common to both sexes were also detected, the same or similar sequences might exist in the DNA of both sexes. The intense male-specific bands were also observed in the analysis of another population, the Tochigishiro cultivar (data not shown), so the sequence might be a characteristic of the species Cannabis sativa.
In Silene, another dioecious plant, identification of a male-associated region of DNA by the RAPD technique was reported recently (Muleahy et al. 1992, Hormaza et al. 1994). However, no report has yet been published, to our knowledge, of the cloning and sequencing of a male-specific region of a plant DNA.
The nucleotide sequence of MADC1 does not include a long open reading frame. Moreover, it seems possible that it is highly amplified. Thus, the sequence is not likely to correspond to a transcribable gene. It could be a linker or a sequence involved in the construction of the sex chromosomes. We suggest that amplification of the male-associated sequence might occur on the Y chromosome but this hypothesis must be confirmed by in situ hybridization on chromosomes. This study and further investigations into the molecular structure of sex chromosomes should contribute to the elucidation of the mechanisms of sex differentiation in plants.
This study was supported in part by a Grant-in-Aid for Special Research on Priority Areas (07281101, Genetic Dissection of Sexual Differentiation and Pollination Process in Higher Plants) from the Ministry of Education, Science and Culture, Japan.
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(Received May 8, 1995; Accepted September 18, 1995)
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