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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. REFERENCES Biffi R, Restivo FM, Caporali A, Marziani GP, Spada A, Falavigna A, 1995. A restriction fragment length polymorphism probe for early diagnosis of gender in Asparagus officinalis L. HortScience 30: 1463–1464. Corriols L, 1984. Are asparagus all-male hybrids attractive? Asparagus Res Newsletter 2: 16–19. Corriols L, Doré C, Cassini R, 1983. Asparagus breeding: national French work. Asparagus Res Newsletter 1: 12. Doré C, 1973. Androgense in vitro par culture d’anthres d’Asparagus officinalis. tat actuel des recherches. Eucarpia 4e runion sur la selection de l’Asperge, Versailles: 155-162. Doré C, 1974. Production de plantes homozygotes males et femalles α partir d’anthers d’Asperge. C. R. Acad Sci (Paris) D 278: 2135–2138. Falavigna A, 1979. Pure lines of A. officinalis obtained by in vitro anther culture in Italy. Proc 5th Int. Asparagus Symp., Geisenheim: 91–99. Falavigna A, Casali PE, Taccomi MG, 1990. Potential of in vitro anther culture technique for asparagus. Acta Hortic 271: 39–46. 131 Falavigna A, Casali PE, Taccomi MG, 1996. Advances in asparagus breeding following in vitro anther culture. Acta Hortic 415: 137–142. Jamsari A, Nitz I, Reamon-Buttner SM, Jung C, 2004. BAC-derived diagnostic markers for sex determination in asparagus. Theor Appl Genet 108: 1140–1146. Jiang Ch, Sink KC, 1997. RAPD and SCAR markers linked to the sex expression locus M in asparagus. Euphytica 94: 329–333. Maestri E, Restivo FM, Marziani-Longo GP, Falavigna A, Tassi F, 1991. Isozyme gene markers in the dioecious species Asparagus officinalis L. Theor Appl Genet 81: 613–618. Reamon-Buttner SM, Jung C, 2000. AFLP-derived STS markers for the identification of sex in Asparagus officinalis L. Theor Appl Genet 100: 432–438. Reamon-Buttner SM, Schondelmaier J, Jung C, 1998. AFLP markers tightly linked to the sex locus in Asparagus officinalis L. Mol Breed 4: 91–98. Sneep S, 1953. The significance of andromonoecy for the breeding of Asparagus officinalis L. Euphytica 2: 89–95. Torrey D, Peirce L, 1983. Production of haploid plantlets from anthers of asparagus. HortScience 18: 569. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV, 1990. DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18: 6531–6535.