Doubled haploid technology can accelerate plant breeding and its two main steps are haploid induction and subsequent doubled haploid production from fertile haploid plants. Although haploid female fertility is present to some extent in plants, the lack of haploid male fertility is a bottleneck. Herein, we demonstrate that mutations in the parallel spindle genes are sufficient to restore haploid male fertility in Arabidopsis with no impact on haploid female fertility.
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The authors declare that all data supporting the findings of this study are available within the manuscript.
The R scripts used for statistical analysis are also available within the manuscript.
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We acknowledge the Iowa State University Office of Biotechnology Flow Cytometry Facility for assistance with ploidy analysis and the Arabidopsis Biological Resource Centre (Columbus, OH) for T-DNA insertion lines. This work was supported by the Foundation for Food & Agriculture Research under award number CA19-SS-0000000128 to T.L. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the Foundation for Food & Agriculture Research. T.L. also acknowledges the support from Iowa State University Plant Sciences Institute.
The authors declare no competing interests.
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Extended Data Fig. 1 Cartoon of proposed cytological mechanism restoring HMF in ps mutants.
Only three chromosomes (violet, blue and gray colors) are shown for simplicity. Homologous chromosomes (light colors) are present only in diploid (a). Spindle fibers are shown in orange color. Dotted lines denote the poles to where chromatids will migrate. At the end of meiosis I, chromosomes are equally distributed in diploids (a) but unequally distributed in haploids (b and c) resulting in unbalanced meiotic products in WT haploids (b). In ps mutant haploids, however, the unequal distribution in meiosis I is ‘corrected’ by parallel spindles during metaphase II leading to dyad formation with balanced meiotic products (c). Figure created with BioRender.com.
Extended Data Fig. 2 Ploidy analysis by flow cytometry in ps mutant and WT haploids.
Representative histograms from ploidy analysis of leaf sample of the putative haploids derived from crosses with cenh3 haploid inducer and respective mutants as indicated. 1C, 2C, 4C and 8C refer to the copy numbers of chromosomes in the nuclei analyzed and they were assigned by comparing to a known diploid WT control.
Extended Data Fig. 3 ps mutant and WT haploids are morphologically indistinguishable.
Representative pictures of 4-week old ps mutant and WT haploids displaying indistinguishable morphology from one another during their vegetative growth. Scale bar = 3 cm.
Extended Data Fig. 4 Representative histograms from ploidy analysis of leaf tissue of putative doubled haploids.
(a) A pool of 4 plants were analysed for each of the indicated genotypes and a total of 32 plants were used. (b) Each pool was co-analysed together with a 2n WT sample as an internal control. 2C, 4C and 8C refer to the copy numbers of chromosomes in the nuclei analyzed and they were assigned by comparing to a known diploid WT control.
Extended Data Fig. 5 Representative gating of nuclei using a combination of light scatter and fluorescence properties.
(a) Scatter plot comparing the relative size of nuclei from debris using forward scatter (FSC-A) and DNA fluorescence (y-axis). (b) Scatter plot comparing the relative granularity/complexity of nuclei from debris using side scatter (SSC-A) and DNA fluorescence (x-axis). Gates drawn (in a and b) exemplify the selected nuclei based on their light scatter and DNA fluorescence properties. c) Count of nuclei with different relative DNA content resulting from the gating described (in a and b).
Supplementary Table 1.
R script used for statistical analysis.
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Aboobucker, S.I., Zhou, L. & Lübberstedt, T. Haploid male fertility is restored by parallel spindle genes in Arabidopsis thaliana. Nat. Plants 9, 214–218 (2023). https://doi.org/10.1038/s41477-022-01332-6