Bush monkey flower (Mimulus aurantiacus) is an important horticultural plant native to North America. To meet market demands for new superior varieties, producers are extending their breeding programs of M. aurantiacus, introducing modern biotechnological approaches, such as doubled haploid (DH) production, which already have wide application in various breeding programs and genetic studies. The establishment of completely homozygous lines of a selected species in one generation without the need of self-pollination for several years enables numerous applications of haploids and DHs, such as the production of homozygous lines, induced mutations, chromosome reduction of polyploid species, gene or quantitative trait loci (QTL) mapping, genomics, and transformation experiments (Kasha and Maluszynski, 2003).
Double haploid approaches combine various in vivo, in vitro, or both stimuli, which induce haploid gametes to undergo sporophytic development, resulting in haploid or diploid regenerants in different ratios. The latter can originate from spontaneous “diploidization” of haploid structures through endoreduplication and nuclear fusion (Kasha et al., 2001) or from diploid heterozygous somatic cells and zygotic embryos. To assess the origin of diploids and to exclude undesired heterozygous regenerants from further genetic experiments and breeding programs, homozygosity testing must be performed. Biochemical and molecular marker systems are becoming increasingly important today for homozygosity testing and they are replacing other techniques, such as self-pollination with subsequent progeny testing and morphological markers. The most frequently used molecular markers for homozygosity testing used to be randomly amplified polymorphic DNAs (Eimert et al., 2003; Yahata et al., 2005a, b), but microsatellite markers are used much more nowadays because of their codominant nature and unambiguous results. Analysis of the genetic origin of haploid/diploid regenerants using microsatellite markers has been applied in various fruit crops, including clementine (Citrus clementina Hort. ex Tan.) (Germanà and Chiancone, 2003; Germanà et al., 2005), apple (Malus ×domestica Borkh.) (Höfer et al., 2002; Vanwynsberghe et al., 2005), pear (Pyrus communis L.) (Bouvier et al., 2002), and other crops such as wheat (Triticum aestivum L.) (Muranty et al., 2002) and maize (Zea mays L.) (Aulinger et al., 2003; Tang et al., 2006).
To evaluate the results of our haploid induction experiments conducted during the past 3 years, a codominant marker was needed. For homozygosity testing of putative DH plants produced, we first tested some published microsatellites for M. guttatus DC (Awadalla and Ritland, 1997; Kelly and Willis, 1998). Because we could not get any amplification products with M. aurantiacus cultivars or other species tested (M. guttatus and M. luteus L.), we tried to amplify intron 10 of topoisomerase 6 subunit B (top6B), as an alternative marker. Our results confirmed the previously reported cross-species applicability of the top6B locus, because successful amplification of this locus with degenerative primers in all tested Mimulus species and cultivars was achieved.
The objectives of our study were 1) to develop a simple codominant marker for homozygosity testing of putative DH plants of M. aurantiacus, 2) to examine the applicability of the marker for determination of different Mimulus species, and 3) to determine the origin of diploid regenerants from haploid induction experiments.
Aulinger, I.E., Peter, S.O., Schmid, J.E. & Stamp, P. 2003 Rapid attainment of a doubled haploid line from transgenic maize (Zea mays L.) plants by means of anther culture In Vitro Cell. Dev. Biol. Plant 39 165 170
Awadalla, P. & Ritland, K. 1997 Microsatellite variation and evolution in the Mimulus guttatus species complex with contrasting mating systems Mol. Biol. Evol. 14 1023 1034
Bouvier, L., Guérif, P., Djulbic, M., Durel, C.E., Chevreau, E. & Lespinasse, Y. 2002 Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers Euphytica 123 255 262
Eimert, K., Reutter, G. & Strolka, B. 2003 Fast and reliable detection of doubled-haploids in Asparagus officinalis by stringent RAPD-PCR J. Agr. Sci. 141 73 78
Eujayl, I., Sledge, M.K., Wang, L., May, G.D., Chekhovskiy, K., Zwonitzer, J.C. & Milan, M.A.R. 2004 Medicago truncatula EST-SSRs reveal cross-species genetic markers for Medicago spp Theor. Appl. Genet. 108 414 422
Germanà, M.A. & Chiancone, B. 2003 Improvement of Citrus clementina Hort. Ex Tan. microspore-derived embryoid induction and regeneration Plant Cell Rpt. 22 181 187
Germanà, M.A., Chiancone, B., Lain, O. & Testolin, R. 2005 Anther culture in Citrus clementina: A way to regenerate tri-haploids Austral. J. Agr. Res. 56 839 845
Höfer, M., Gomez, A., Aguiriano, E., Manzanera, J.A. & Bueno, M.A. 2002 Analysis of simple sequence repeat markers in homozygous lines of apple Plant Breed. 121 159 162
Jakse, J., Kindlhofer, K. & Javornik, B. 2001 Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers Genome 44 773 782
Kasha, K.J., Hu, T.C., Oro, R., Simion, E. & Shim, Y.S. 2001 Nuclear fusion leads to chromosome doubling during mannitol pretreatment of barley (Hordeum vulgare L.) microspores J. Expt. Bot. 52 1227 1238
Kasha, K.J. & Maluszynski, M. 2003 Production of doubled haploids in crop plants: An introduction 1 4 Maluszynski M., Kasha K.J., Forster B.P. & Szarejko I. Doubled haploid production in crop plants: A manual Kluwer Academic Publishers Dordrecht, The Netherlands
Kump, B. & Javornik, B. 1996 Evaluation of genetic variability among common buckwheat (Fagopyrum esculentum Moench) populations by RAPD markers Plant Sci. 114 149 158
Muranty, H., Sourdille, P., Bernard, S. & Bernard, M. 2002 Genetic characterization of spontaneous diploid androgenetic wheat and triticale plants Plant Breed. 121 470 474
Rozen, S. & Skaletsky, H.J. 2000 Primer3 on WWW for general users and for biologist programmers 365 386 Krawetz S. & Misener S. Bioinformatics methods and protocols: Methods in molecular biology Humana Press Totowa, NJ
Scott, K.D., Eggler, P., Seaton, G., Rossetto, M., Ablett, E.M., Lee, L.S. & Henry, R.J. 2000 Analysis of SSRs derived from grape ESTs Theor. Appl. Genet. 100 697 712
Sisko, M., Ivancic, A. & Bohanec, B. 2003 Genome size analysis in the genus Cucurbita and its use for determination of interspecific hybrids obtained using the embryo-rescue technique Plant Sci. 165 663 669
Stajner, N., Jakse, J., Kozjak, P. & Javornik, B. 2005 The isolation and characterization of microsatellites in hop (Humulus lupulus L.) Plant Sci. 168 213 221
Tang, F., Tao, Y., Zhao, T. & Wang, G. 2006 In vitro production of haploid and doubled haploid plants from pollinated ovaries of maize (Zea mays L.) Plant Cell Tissue Organ Cult. 84 233 237
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. 1997 The Clustal_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools Nucl. Acids Res. 25 4876 4882
Vanwynsberghe, L., De Witte, K., Coart, E. & Keulemans, J. 2005 Limited application of homozygous genotypes in apple breeding Plant Breed. 124 399 403
Yahata, M., Harusaki, S., Komatsu, H., Takami, K., Kunitake, H., Yabuya, T., Yamashita, K. & Toolapong, P. 2005a Morphological characterization and molecular verification of a fertile haploid pummelo (Citrus grandis Osbeck) J. Amer. Soc. Hort. Sci. 130 34 40
Yahata, M., Kunitake, H., Yabuya, T., Yamashita, K., Kashihara, Y. & Komatsu, H. 2005b Production of a doubled haploid from a haploid pummelo using colchicine treatment of axillary shoot buds J. Amer. Soc. Hort. Sci. 130 899 903