A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis

Abstract

Doubled haploid technology using inducer lines carrying mutations in ZmPLA1/MTL/NLD and ZmDMP1,2,3,4 has revolutionized traditional maize breeding. ZmPLA1/MTL/NLD is conserved in monocots and has been used to extend the system from maize to other monocots5,6,7, but no functional orthologue has been identified in dicots, while ZmDMP-like genes exist in both monocots and dicots4,8,9. Here, we report that loss-of-function mutations in the Arabidopsis thaliana ZmDMP-like genes AtDMP8 and AtDMP9 induce maternal haploids, with an average haploid induction rate of 2.1 ± 1.1%. In addition, to facilitate haploid seed identification in dicots, we established an efficient FAST-Red fluorescent marker-based haploid identification system that enables the identification of haploid seeds with >90% accuracy. These results show that mutations in DMP genes also trigger haploid induction in dicots. The conserved expression patterns and amino acid sequences of ZmDMP-like genes in dicots suggest that DMP mutations could be used to develop in vivo haploid induction systems in dicots.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Mutation of Arabidopsis DMP genes results in multiple seed phenotypes.
Fig. 2: Haploid seed production and identification with the FAST-Red marker.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on request. The raw whole-genome sequencing data have been deposited in the NCBI Sequence Read Archive under the accession code PRJNA608056. Source Data for Figs. 1 and 2 are provided with the paper.

References

  1. 1.

    Gilles, L. M. et al. Loss of pollen‐specific phospholipase NOT LIKE DAD triggers gynogenesis in maize. EMBO J. 36, 707–717 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    Kelliher, T. et al. MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature 542, 105–109 (2017).

    CAS  Article  Google Scholar 

  3. 3.

    Liu, C. et al. A 4-bp insertion at ZmPLA1 encoding a putative phospholipase A generates haploid induction in maize. Mol. Plant 10, 520–522 (2017).

    CAS  Article  Google Scholar 

  4. 4.

    Zhong, Y. et al. Mutation of ZmDMP enhances haploid induction in maize. Nat. Plants 5, 575–580 (2019).

    Article  Google Scholar 

  5. 5.

    Yao, L. et al. OsMATL mutation induces haploid seed formation in indica rice. Nat. Plants 4, 530–533 (2018).

    CAS  Article  Google Scholar 

  6. 6.

    Liu, C. et al. Extension of the in vivo haploid induction system from diploid maize to hexaploid wheat. Plant Biotechnol. J. 18, 316–318 (2019).

    Article  Google Scholar 

  7. 7.

    Liu, H. et al. Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system. J. Exp. Bot. 71, 1337–1349 (2020).

    Article  Google Scholar 

  8. 8.

    Takahashi, T. et al. The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants. Development 145, dev170076 (2018).

    Article  Google Scholar 

  9. 9.

    Cyprys, P., Lindemeier, M. & Sprunck, S. Gamete fusion is facilitated by two sperm cell-expressed DUF679 membrane proteins. Nat. Plants 5, 253–257 (2019).

    CAS  Article  Google Scholar 

  10. 10.

    Chang, M.-T. & Coe, E. H. in Molecular Genetic Approaches to Maize Improvement (eds Kriz, A. L. & Larkins, B. A.) 127–142 (Springer Berlin Heidelberg, 2009).

  11. 11.

    Dwivedi, S. L. et al. Haploids: constraints and opportunities in plant breeding. Biotechnol. Adv. 33, 812–829 (2015).

    Article  Google Scholar 

  12. 12.

    Kalinowska, K. et al. State-of-the-art and novel developments of in vivo haploid technologies. Theor. Appl. Genet. 132, 593–605 (2019).

    CAS  Article  Google Scholar 

  13. 13.

    Coe, E. H. A line of maize with high haploid frequency. Am. Nat. 93, 381–382 (1959).

    Article  Google Scholar 

  14. 14.

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

    CAS  Article  Google Scholar 

  15. 15.

    Kelliher, T. et al. Maternal haploids are preferentially induced by CENH3-tailswap transgenic complementation in maize. Front. Plant Sci. 7, 414 (2016).

    Article  Google Scholar 

  16. 16.

    Watts, A., Kumar, V., Raipuria, R. K. & Bhattacharya, R. C. In vivo haploid production in crop plants: methods and challenges. Plant Mol. Biol. Rep. 36, 685–694 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Barret, P., Brinkmann, M. & Beckert, M. A major locus expressed in the male gametophyte with incomplete penetrance is responsible for in situ gynogenesis in maize. Theor. Appl. Genet. 117, 581–594 (2008).

    CAS  Article  Google Scholar 

  18. 18.

    Prigge, V. et al. New insights into the genetics of in vivo induction of maternal haploids, the backbone of doubled haploid technology in maize. Genetics 190, 781–793 (2012).

    CAS  Article  Google Scholar 

  19. 19.

    Dong, X. et al. Fine mapping of qhir1 influencing in vivo haploid induction in maize. Theor. Appl. Genet. 126, 1713–1720 (2013).

    CAS  Article  Google Scholar 

  20. 20.

    Liu, C. et al. Fine mapping of qhir8 affecting in vivo haploid induction in maize. Theor. Appl. Genet. 128, 2507–2515 (2015).

    CAS  Article  Google Scholar 

  21. 21.

    Kasaras, A. & Kunze, R. Expression, localisation and phylogeny of a novel family of plant-specific membrane proteins. Plant Biol. 12, 140–152 (2010).

    CAS  Article  Google Scholar 

  22. 22.

    Prigge, V. et al. Doubled haploids in tropical maize: I. Effects of inducers and source germplasm on in vivo haploid induction rates. Crop Sci. 51, 1498–1506 (2011).

    Article  Google Scholar 

  23. 23.

    Wu, P., Li, H., Ren, J. & Chen, S. Mapping of maternal QTLs for in vivo haploid induction rate in maize (Zea mays L.). Euphytica 196, 413–421 (2014).

    Article  Google Scholar 

  24. 24.

    Tian, X. et al. Hetero-fertilization together with failed egg–sperm cell fusion supports single fertilization involved in in vivo haploid induction in maize. J. Exp. Bot. 69, 4689–4701 (2018).

    CAS  Article  Google Scholar 

  25. 25.

    Li, L., Xu, X., Jin, W. & Chen, S. Morphological and molecular evidences for DNA introgression in haploid induction via a high oil inducer CAUHOI in maize. Planta 230, 367–376 (2009).

    CAS  Article  Google Scholar 

  26. 26.

    Zhao, X., Xu, X., Xie, H., Chen, S. & Jin, W. Fertilization and uniparental chromosome elimination during crosses with maize haploid inducers. Plant Physiol. 163, 721–731 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Kelliher, T. et al. One-step genome editing of elite crop germplasm during haploid induction. Nat. Biotechnol. 37, 287–292 (2019).

    CAS  Article  Google Scholar 

  28. 28.

    Nanda, D. K. & Chase, S. S. An embryo marker for detecting monoploids of maize (Zea Mays L.). Crop Sci. 6, 213–215 (1966).

    Article  Google Scholar 

  29. 29.

    Portemer, V., Renne, C., Guillebaux, A. & Mercier, R. Large genetic screens for gynogenesis and androgenesis haploid inducers in Arabidopsis thaliana failed to identify mutants. Front. Plant Sci. 6, 147 (2015).

    Article  Google Scholar 

  30. 30.

    Wang, B. et al. Development of a haploid-inducer mediated genome editing system for accelerating maize breeding. Mol. Plant 12, 597–602 (2019).

    Article  Google Scholar 

  31. 31.

    Rosso, M. G. et al. An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol. Biol. 53, 247–259 (2003).

    CAS  Article  Google Scholar 

  32. 32.

    Castel, B., Tomlinson, L., Locci, F., Yang, Y. & Jones, J. D. G. Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis. PLoS ONE 14, e0204778 (2019).

    CAS  Article  Google Scholar 

  33. 33.

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

    CAS  Article  Google Scholar 

  34. 34.

    Kumar, S., Stecher, G. & Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).

    CAS  Article  Google Scholar 

  35. 35.

    Chen, C., Chen, H., He, Y. & Xia, R. TBtools, a toolkit for biologists integrating various biological data handling tools with a user-friendly interface. Preprint at https://www.biorxiv.org/content/10.1101/289660v3 (2018).

  36. 36.

    Păcurar, D. I. et al. A collection of INDEL markers for map-based cloning in seven Arabidopsis accessions. J. Exp. Bot. 63, 2491–2501 (2012).

    Article  Google Scholar 

  37. 37.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  Article  Google Scholar 

  38. 38.

    Poplin, R. et al. Scaling accurate genetic variant discovery to tens of thousands of samples. Preprint at https://www.biorxiv.org/content/10.1101/201178v3 (2017).

  39. 39.

    Kurtz, S. et al. Versatile and open software for comparing large genomes. Genome Biol. 5, R12 (2004).

    Article  Google Scholar 

  40. 40.

    Tan, E. H. et al. Catastrophic chromosomal restructuring during genome elimination in plants. eLife 4, e06516 (2015).

    Article  Google Scholar 

  41. 41.

    Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    Article  Google Scholar 

Download references

Acknowledgements

We thank D. Ye and L. Chen for providing the ms1 and ms1-1 mutants, X. Yang for help with the analysis of haploid sequence data, Y. Leng for help with the analysis of seed development, and Z. Li for help with the phylogenetic analysis. This work was supported by the National Key Research and Development Program of China (2016YFD0101200 and 2018YFD0100201), Modern Maize Industry Technology System (CARS-02-04) and National Natural Science Foundation of China (91935303) (to S.C), as well as a China Scholarship Council grant (to B.C).

Author information

Affiliations

Authors

Contributions

Y.Z., B.C., C.L. and S.C. conceived of and designed the experiments. Y.Z., M.L. and B.C. performed most of the experiments. D.W., Y.J., X.Q., Z.L., C.C., Y.W., M.C., J.L., Z.X. and D.C. performed some of the experiments. Y.Z., B.C., S.C., C.L., M.W., W.L. and M.L. analysed the data. Y.Z., B.C., C.L., K.B. and S.C. discussed and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Chenxu Liu or Shaojiang Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Thomas Widiez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Tables 2, 4, 6 and 7.

Reporting Summary

Supplementary Table 1

ZmDMP-like genes in plants.

Supplementary Table 3

A summary of CRISPR–Cas9 mutagenesis.

Supplementary Table 5

Genotyping data of haploids.

Supplementary Table 8

SNP data of haploids.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhong, Y., Chen, B., Li, M. et al. A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis. Nat. Plants 6, 466–472 (2020). https://doi.org/10.1038/s41477-020-0658-7

Download citation

Search

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