Abstract
Maize mutants of the centromeric histone H3 (CENP-A/CENH3) gene can form haploids that inherit only chromosomes of the pollinating parent but the cytoplasm from the female parent. We developed CENH3 haploid inducers carrying a dominant anthocyanin colour marker for efficient haploid identification and harbouring cytoplasmic male sterile cytoplasm, a type of cytoplasm that results in male sterility useful for efficient hybrid seed production. The resulting cytoplasmic male sterility cyto-swapping method provides a faster and cheaper way to convert commercial lines to cytoplasmic male sterile compared to conventional trait introgression.
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Data availability
The sequences of the cenh3 edited alleles are shown on Extended Data Fig. 2. The number of embryos extracted, seedlings genotyped and confirmed haploids for both cyto-swapping experiments are provided in Extended Data Tables 1 and 2. The assays used for genotyping cenh3 alleles are in Supplementary Table 1. The genotyping results of cyto-swapped experiments are provided as Source Data. The data used for analyses of colchicine effect on HRR and seed set are in Supplementary Data.
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Acknowledgements
We thank S. Jensen for providing feedback on the manuscript, S. Rigoulot for performing protoplasts transfections and S. Bonefas for helping with maize crosses.
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Contributions
E.B. wrote the manuscript, supervised the experiments, carried out crosses, performed the T7EI assays and evaluated CMS-converted lines. R.S. performed the CMS cyto-swapping experiments. L.T. performed the Normal B to Normal A cyto-swapping experiments. R.E. supervised the Normal B to Normal A cyto-swapping experiment and contributed to the writing. S.D. performed the protoplast transfections and supervised the maize embryo bombardment. K.B. and K.M. performed the maize embryo bombardment. J.F. contributed with statistical analyses. M.D. selected genotyping assays and created genetic distance figures. S.G. provided pictures for Fig. 1. D.M. planned and supervised greenhouse plantings. R.C.M.L. supervised and executed samplings for genotyping. K.S. contributed with plant maintenance and crossings. D.D. contributed to the design of the CMS cyto-swapping experiment and selected lines for conversions. R.K.D. provided lines carrying cenh3-KD. T.K. conceived the experiments, carried out crosses and contributed to the writing.
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E.B. and T.K. are co-inventors on patent WO23225469, Conferring cytoplasmic male sterility, filed by Syngenta relating to work in this manuscript. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Heat map of genetic distances from 2346 markers among NSS inbreds (NB-SYN-INBXXX) and their cyto-swapped Double Haploid progeny (NA-SYN-INBXXX-X).
The scale represents the percent of alleles that are different. NA-cenh3-KD-11 and −12 are the conversion lines carrying the cenh3-KD allele and Normal A cytoplasm used in this experiment.
Extended Data Fig. 2 Alleles of CENH3 used in this study.
(a) The region of CENH3 targeted by crRNA140, the location of the 1 bp deletion in cenh3-KD8 and the sequences of the alleles selected for cyto-swapping to CMS-C. (b) The deduced amino acids sequences that could potentially be encoded by the deletion alleles (Figure made in Geneious 2023.0.4).
Extended Data Fig. 3 Steps taken to produce the CMS-C conversion and Maintainer lines.
The steps shown here are simplified, some crosses have been omitted. From top to bottom: An RNP complex was bombarded into maize calli carrying the R1-scm2 allele. Plants that contained edited alleles expected to result in CENH3 loss of function were crossed as males to a CMS-C line and selfed. The resulting progeny, free of T-DNA and homozygous for R1-scm2 and the recessive restorer allele rf4, consists of a male-sterile Conversion line with a CMS-C cytoplasm and a male fertile Maintainer line with a Normal cytoplasm. The latter is used to pollinate both the CMS-C and the Maintainer lines. Both lines are propagated as segregating families (wild type 1:1 cenh3 heterozygous).
Extended Data Fig. 4 Heat map of genetic distances from 198 markers among Doubled Haploids converted to CMS-C cytoplasm and RP.
The scale represents the percent of alleles that are different.
Supplementary information
Supplementary Information
Supplementary Tables 1–3.
Supplementary Data
Data for analyses of effect of colchicine on HRR and DHBC1 seed produced.
Source data
Extended Data Fig. 1
Genotypic data from 2,346 SNP markers.
Extended Data Fig. 4
Genotypic data from 198 SNP markers.
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Bortiri, E., Selby, R., Egger, R. et al. Cyto-swapping in maize by haploid induction with a cenh3 mutant. Nat. Plants 10, 567–571 (2024). https://doi.org/10.1038/s41477-024-01630-1
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DOI: https://doi.org/10.1038/s41477-024-01630-1
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