Download Identification of molecular markers for selection of supermale (YY

J Appl Genet 48(2), 2007, pp. 129–131
Short communication
Identification of molecular markers for selection
of supermale (YY) asparagus plants
Piotr Gebler1, £ukasz Wolko2, Miko³aj Knaflewski1
1
2
Department of Vegetable Crops, August Cieszkowski Agricultural University, Poznañ, Poland
Department of Biochemistry and Biotechnology, August Cieszkowski Agricultural University, Poznañ, Poland
Abstract. The research was aimed to elaborate a method for selection of male plants (XY, YY) and female ones
(XX) as well as for identification of supermale genotypes (YY) among male phenotypes. The population obtained
by self-pollination of andromonoecious plants was analysed. In order to identify the bands differentiating the
male from the female genotypes, Bulk Segregant Analysis (BSA) was carried out. Primers identified by BSA
analysis were used for RAPD amplification on the template of the male and female individuals. Among the products obtained by the use of primer OPB-20, some bands were linked with sex. A band of about 700 bp was found
in all female plants, and in 4 phenotypically male specimens. In the male plants, the band showed a much lower
intensity, compared with the female specimens. It seems that this fragment can be linked to the X chromosome in
the investigated specimens. In the female specimens with XX karyotype, template duplication occurs and hence
the band intensity is twice as high as in the XY karyotype. Three male plants did not include the OPB-20-700
fragment so they could potentially have the supermale (YY) karyotype. If the obtained marker proved its usefulness for identification of supermale plants, it could become a valuable tool facilitating breeding work.
Keywords: asparagus, molecular markers, supermale plants, RAPD.
In asparagus there are sex chromosomes X and Y.
The female and male plants have chromosome
patterns XX and XY, respectively. Nowadays ‘all
male’ asparagus cultivars are bred world-wide.
They are obtained as a result of crossing a YY
supermale plant with an XX female plant according to the formula XX × YY = XY. To obtain
supermale plants, two methods can be applied.
One of them is self-pollination of flowers
of andromonoecious plants (Sneep 1953; Corriols
et al. 1983; Corriols 1984). The other method consists in application of pollen cultures, in which
haploid plants are produced by means of
androgenesis (Doré 1973, 1974; Falavigna 1979;
Torrey and Peirce 1983, 1984; Falavigna et al.
1990, 1996). Regardless of the applied method the
population including supermales can be obtained
in a relatively short time. Identifying the
supermales, however, is more time-consuming
and laborious, because each male plant should be
crossed with a female plant and only in hybrids
with all male progeny the paternal plants are
supermales. This method takes at least 3 years.
Therefore developing a method permitting fast selection of supermale plants would enable a considerable shortening of the breeding period of ‘all
male’ cultivars. A series of efforts have been made
to identify genetic markers linked to the sex chromosome in asparagus (Maestri et al. 1991; Jiang
and Sink 1997; Biffi et al. 1995; Jiang and Sink
1997; Reamon-Buttner et al. 1998; ReamonButtner and Jung 2000; Jamsari et al. 2004). However, the identified markers have worked only in
the selection of the plant line for which they have
been elaborated. The aim of the study was to identify a sex-linked molecular marker that would enable fast identification of supermale specimens in
asparagus.
Received: December 7, 2006. Accepted: January 16, 2007.
Correspondence: £. Wolko, Department of Biochemistry and Biotechnology, August Cieszkowski Agricultural University,
Wo³yñska 35, 60–637 Poznañ, Poland; e-mail: [email protected]
130
P. Gebler et al.
Figure 1. RAPD products generated with primer OPB-20. Female plants: 1–9; Male plants: 10–17; M = weight marker
pGem5/MspI (765 bp, 489 bp, 404 bp)
The investigations were carried out on
1–2-year-old plants obtained from seeds produced
by self-pollination of flowers of andromonoecious
plants. The andromonoecious plants were selected
from ‘Backlim’, ‘Gynlim’, ‘Horlim’, and ‘Thielim’,
included in the collection of asparagus cultivars at
the Marcelin Experimental Station, Department of
Vegetable Crops, August Cieszkowski Agricultural University, Poznañ.
The DNA purification from plant samples was
made according to the method described by Williams et al. (1990), using CTAB detergent with
modifications. The DNA samples prepared in this
way, were used for RAPD amplification. RAPD
reaction was carried out in the following conditions: denaturation for 5 min at 95°C, followed by
20 cycles of 92°C for 1.5 min, 35°C for 1.5 min
and 72°C for 2 min, and then 20 subsequent cycles
of 92oC for 1.5 min, 38oC for 1.5 min, 72oC for
2 min, and the final elongation at 75°C for 5 min.
The Operon QIAGEN kits of 10-nucleotide primers were applied for the RAPD reaction. In total,
60 primers derived from OPA, OPB and OPC kits
were tested. For amplification, 2 × PCR Master
MIX (Fermentas) was used, containing: Taq DNA
polymerase in reaction buffer (0.05 unit in 1 µL of
mixture) MgCl2 (4 mM), and dNTP (0.4 mM).
In order to identify RAPD primers differentiating male and female specimens, a BSA (Bulk
Segregant Analysis) analysis was carried out. Two
bulk probes, including a DNA mixture from
10 male or from 10 female plants, were compared
with each other. In that way, 60 RAPD primers
were tested. On the basis of BSA analysis,
12 primers producing distinctive bands were selected for a further study on individuals, namely
OPA-07, 09, 12, 18, 19, OPB-09, 20, and OPC-07,
13, 16, 18, 20. These primers were used for RAPD
amplification with DNA from individuals obtained from seeds of andromonoecious plants. For
investigations, we selected 18 plants with a defined sex phenotype but without a diagnosed genotype, including 10 female and 8 male
specimens.
In the case of 11 primers neither polymorphism
was observed nor the differences in the pattern of
bands showed any correlation with the sex gene.
Among products obtained by the use of primer
OPB-20 (Figure 1), some bands linked to sex were
found. The band of about 700 bp, obtained by using primer OPB-20, occurred in all females and in
4 male phenotype probes. In the male probes
in which OPB-20-700 is present, it shows a much
lower intensity in comparison with female phenotypes. Therefore, the OPB-20-700 fragment seems
to be linked to the X chromosome. So in female
probes with XX karyotype the template is duplicated and hence the intensity of the band is doubled in relation to XY karyotype. Individuals no.
17, 11, and 5 do not have the OPB-20-700 fragment, so they seem to have the supermale
karyotype (YY). The band of 400 bp in length also
differentiates male from female individuals but
it does not show any clear linkage to the sex determination.
The experiment described above was carried
out on plants produced by self-pollination of flowers of andromonoecious plants, which include
male chromosomes but develop dioecious flowers. Therefore, the presence of individuals with
supermale genotype among male plants was expected. We do not have any confirmed supermale
plant to be used as a control of this marker. Therefore, to prove the potential usefulness of the
OPB-20-700 fragment as a marker that enables the
identification of supermales, it would be indispensable to confirm the genotype of the individuals being investigated. If the individuals marked as
the ones having the supermale karyotype produce
only male progeny after crossing with female
plants, this will prove that the OPB-20-700 fragment is useful as a marker.
At present the breeding research that aims
at confirmation of the results obtained by the
RAPD method is being carried out. If the
OPB-20-700 fragment proves to be useful
as a marker, its sequencing should be made. On the
Markers for supermale asparagus plants
basis of the identified sequence it would be then
possible to design specific PCR primers for identification of the X chromosome in a repeatable way.
This would enable a great simplification and activation of breeding work as well as producing asparagus supermale individuals.
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