Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant

Nature Genetics volume 44, pages 467–470 (2012)Cite this article

Subjects

Abstract

Traditionally, hybrid seeds are produced by crossing selected inbred lines. Here we provide a proof of concept for reverse breeding, a new approach that simplifies meiosis such that homozygous parental lines can be generated from a vigorous hybrid individual. We silenced DMC1, which encodes the meiotic recombination protein DISRUPTED MEIOTIC cDNA1, in hybrids of A. thaliana, so that non-recombined parental chromosomes segregate during meiosis. We then converted the resulting gametes into adult haploid plants, and subsequently into homozygous diploids, so that each contained half the genome of the original hybrid. From 36 homozygous lines, we selected 3 (out of 6) complementing parental pairs that allowed us to recreate the original hybrid by intercrossing. In addition, this approach resulted in a complete set of chromosome-substitution lines. Our method allows the selection of a single choice offspring from a segregating population and preservation of its heterozygous genotype by generating homozygous founder lines.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Meiosis in wild-type (WT; above) and RNAi:DMC1 transformants (below).
Figure 2: Reverse-breeding strategy and genotypes of wild-type (WT) and reverse-breeding (RB) doubled-haploid offspring.

Similar content being viewed by others

References

  1. Chen, Z.J. Molecular mechanisms of polyploidy and hybrid vigor. Trends Plant Sci. 15, 57–71 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. van Dijk, P. & van Damme, J. Apomixis technology and the paradox of sex. Trends Plant Sci. 5, 81–84 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Dirks, R. et al. Reverse breeding: A novel breeding approach based on engineered meiosis. Plant Biotechnol. J. 7, 837–845 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ma, H. A molecular portrait of Arabidopsis meiosis. Arabidopsis Book 4, e0095 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Couteau, F. et al. Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis. Plant Cell 11, 1623–1634 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hartung, F. et al. The catalytically active tyrosine residues of both SPO11–1 and SPO11–2 are required for meiotic double-strand break induction in Arabidopsis. Plant Cell 19, 3090–3099 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Forster, B.P., Heberle-Bors, E., Kasha, K.J. & Touraev, A. The resurgence of haploids in higher plants. Trends Plant Sci. 12, 368–375 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Ravi, M. & Chan, S.W.L. Haploid plants produced by centromere-mediated genome elimination. Nature 464, 615–618 (2010).

    Article  CAS  PubMed  Google Scholar 

  9. Drouaud, J. et al. Sex-specific crossover distributions and variations in interference level along Arabidopsis thaliana chromosome 4. PLoS Genet. 3, e106 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Toyota, M., Matsuda, K., Kakutani, T., Terao Morita, M. & Tasaka, M. Developmental changes in crossover frequency in Arabidopsis. Plant J. 65, 589–599 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Alonso-Blanco, C. et al. Development of an AFLP based linkage map of Ler, Col and Cvi Arabidopsis thaliana ecotypes and construction of a Ler/Cvi recombinant inbred line population. Plant J. 14, 259–271 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Hauge, B.M. et al. An integrated genetic/RFLP map of the Arabidopsis thaliana genome. Plant J. 3, 745–754 (1993).

    Article  CAS  Google Scholar 

  13. Nadeau, J.H., Singer, J.B., Matin, A. & Lander, E.S. Analysing complex traits with chromosome substitution strains. Nat. Genet. 24, 221–225 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Koumproglou, R. et al. STAIRS: A new genetic resource for functional genomic studies of Arabidopsis. Plant J. 31, 355–364 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Marimuthu, M.P.A. et al. Synthetic clonal reproduction through seeds. Science 331, 876 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Mercier, R. & Grelon, M. Meiosis in plants: Ten years of gene discovery. Cytogenet. Genome Res. 120, 281–290 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Tester, M. & Langridge, P. Breeding technologies to increase crop production in a world. Science 327, 818–822 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Gleave, A.P. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207 (1992).

    Article  CAS  PubMed  Google Scholar 

  19. Clough, S.J. & Bent, A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  Google Scholar 

  20. Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K. & Scheible, W.-R. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ross, K.J., Fransz, P. & Jones, G.H. A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosome Res. 4, 507–516 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Ross, K.J. et al. Cytological characterization of four meiotic mutants of Arabidopsis isolated from T-DNA–transformed lines. Chromosome Res. 5, 551–559 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Tang, X. et al. Cross-species bacterial artificial chromosome–fluorescence in situ hybridization painting of the tomato and potato chromosome 6 reveals undescribed chromosomal rearrangements. Genetics 180, 1319–1328 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Warthmann, N., Fitz, J. & Weigel, D. MSQT for choosing SNP assays from multiple DNA alignments. Bioinformatics 23, 2784–2787 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Stam, P. Construction of integrated genetic linkage maps by means of a new computer package: JoinMap. Plant J. 3, 739–744 (1993).

    Article  CAS  Google Scholar 

  26. Lister, C. & Dean, C. Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana. Plant J. 4, 745–750 (1993).

    Article  CAS  Google Scholar 

  27. Singer, T. et al. A high-resolution map of Arabidopsis recombinant inbred lines by whole-genome exon array hybridization. PLoS Genet. 2, e144 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Bikard, D. et al. Divergent evolution of duplicate genes leads to genetic incompatibilities within A. thaliana. Science 323, 623–626 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Stoker, G. Stunnenberg, C. Pillen and H. Blankestijn for help in the greenhouse, J. van Ooijen for advice in using Joinmap, and L. Mlynarova, Y.-F. Lin and D.Z. Agudelo for their help with real-time PCR. We thank M. Koornneef, B. Zwaan and P.J. Flood for critical reading of the manuscript.

Author information

Author notes

  1. Erik Wijnker and Kees van Dun: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands

    Erik Wijnker, Joost J B Keurentjes & Hans de Jong

  2. Rijk Zwaan R&D Fijnaart, Fijnaart, The Netherlands

    Kees van Dun, C Bastiaan de Snoo, Cilia L C Lelivelt & Rob Dirks

  3. Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands

    Joost J B Keurentjes

  4. Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, Selangor, Malaysia

    Nazatul Shima Naharudin

  5. Department of Plant Biology, University of California, Davis, Davis, California, USA.,

    Maruthachalam Ravi & Simon W L Chan

  6. Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA.,

    Simon W L Chan

Authors
  1. Erik Wijnker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kees van Dun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C Bastiaan de Snoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cilia L C Lelivelt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joost J B Keurentjes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nazatul Shima Naharudin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maruthachalam Ravi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Simon W L Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hans de Jong
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Rob Dirks
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.v.D., C.L.C.L., H.d.J. and R.D. conceived the research. E.W. performed cytology and crosses, C.B.d.S. constructed vectors and performed genotyping and N.S.N. performed cytology and FISH. E.W. analyzed data with the help of C.B.d.S., J.J.B.K., M.R. and S.W.L.C. E.W. wrote the manuscript and made the figures with substantial contributions by J.J.B.K., M.R., S.W.L.C. and H.d.J. All authors except N.S.N. were involved in planning and design of experiments, and read and improved the manuscript.

Corresponding author

Correspondence to Erik Wijnker.

Ethics declarations

Competing interests

E. W., K.v.D., C.B.d.S., C.L.C.L. and R.D. are employees of Rijk Zwaan. H.d.J. has received research funding from Rijk Zwaan in recent years.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1, 3 and 4 (PDF 735 kb)

Supplementary Table 2

Genotypes of WT and RB haploids and reconstructed heterozygotes. (XLSX 134 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wijnker, E., van Dun, K., de Snoo, C. et al. Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant. Nat Genet 44, 467–470 (2012). https://doi.org/10.1038/ng.2203

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.2203

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing