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High-throughput analysis of meiotic crossover frequency and interference via flow cytometry of fluorescent pollen in Arabidopsis thaliana

Nature Protocols volume 8, pages 2119–2134 (2013)Cite this article

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Abstract

During meiosis, reciprocal exchange between homologous chromosomes occurs as a result of crossovers (COs). CO frequency varies within genomes and is subject to genetic, epigenetic and environmental control. As robust measurement of COs is limited by their low numbers, typically 1–2 per chromosome, we adapted flow cytometry for use with Arabidopsis transgenic fluorescent protein–tagged lines (FTLs) that express eCFP, dsRed or eYFP fluorescent proteins in pollen. Segregation of genetically linked transgenes encoding fluorescent proteins of distinct colors can be used to detect COs. The fluorescence of up to 80,000 pollen grains per individual plant can be measured in 10–15 min using this protocol. A key element of CO control is interference, which inhibits closely spaced COs. We describe a three-color assay for the measurement of CO frequency in adjacent intervals and calculation of CO interference. We show that this protocol can be used to detect changes in CO frequency and interference in the fancm zip4 double mutant. By enabling high-throughput measurement of CO frequency and interference, these methods will facilitate genetic dissection of meiotic recombination control.

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Figure 1: Measuring crossovers (COs) using meiotic segregation of FTL T-DNAs.
Figure 2: Micrographs of fluorescent pollen segregating for I1bc FTL T-DNAs.
Figure 3: CO frequency and interference measurements obtained by FTL tetrad counting and flow cytometry.
Figure 4: Flow cytometry acquisition plots for two-color analysis of pollen.
Figure 5: Flow cytometry acquisition plots for three-color analysis of pollen.
Figure 6: Flow cytometry histograms for three-color analysis of I1bc/+++ pollen.

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Acknowledgements

Work in the Henderson laboratory is supported by grants from the Royal Society, the Gatsby Charitable Foundation, the Isaac Newton Trust and Biotechnology and Biological Sciences Research Council grant no. BB/K007882/1. Work in the G.P.C. laboratory is supported by grant no. MCB-1121563 from the National Science Foundation. P.A.Z. is supported by grant no. 605/MOB/2011/0 from the Polish Ministry of Science and Higher Education.

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Author notes

  1. Nataliya E Yelina and Piotr A Ziolkowski: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Plant Sciences, University of Cambridge, Cambridge, UK

    Nataliya E Yelina, Piotr A Ziolkowski, Xiaohui Zhao, Krystyna A Kelly & Ian R Henderson

  2. Department of Biotechnology, Adam Mickiewicz University, Poznan, Poland

    Piotr A Ziolkowski

  3. Department of Pathology, University of Cambridge, Cambridge, UK

    Nigel Miller

  4. Department of Biology and The Carolina Center for Genome Sciences, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Daniela F Muñoz & Gregory P Copenhaver

  5. Lineberger Comprehensive Cancer Center, The University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA

    Daniela F Muñoz & Gregory P Copenhaver

  6. Department of Life Sciences, Imperial College, London, UK

    David J Mann

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  1. Nataliya E Yelina
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Contributions

N.E.Y., P.A.Z., N.M. and D.F.M. performed experiments. N.E.Y., P.A.Z., N.M., D.F.M., D.J.M., G.P.C. and I.R.H. designed experiments. N.E.Y., P.A.Z., N.M., X.Z., K.A.K., D.F.M., D.J.M., G.P.C. and I.R.H. analyzed data, performed statistical analysis and wrote the paper.

Corresponding authors

Correspondence to Gregory P Copenhaver or Ian R Henderson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Crossing schemes to obtain FTLs that can be scored by flow cytometry.

a) Crossing a homozygous FTL (ABC/ABC qrt1-2/qrt1-2) to wild type produces 100% F1 progeny that can be scored. b) Crossing a heterozygous FTL (ABC/+++ qrt1-2/qrt1-2) to wild type produces 50% - r F1 progeny that can be scored, where r corresponds to the proportion of CO recombinants between the FTL T-DNAs. COs between the FTL T-DNAs generate plants that cannot be scored, as they possess 0 or 2 copies of T-DNAs for each colour. c) To test the effect of a mutation of interest (moi) on COs using flow cytometry the mutant should first be crossed to a homozygous or heterozygous FTL of interest as shown in a) and b). Crossing to a homozygous FTL is shown in c). 100% of the F1 from this cross can be scored, but will appear like wild type if moi is recessive. Self-fertilization of these F1 plants will generate 9.375% (6.25% + 3.125%) - r mutant individuals that can be scored in the F2 generation. These schemes assume moi, QRT1 and the FTL intervals are unlinked.

Supplementary Figure 2 Micrographs of pollen sorted by flow cytometry.

a) Hydrated pollen purified by flow cytometry (see Fig. 5c). b) Close-up of hydrated pollen. c) Non-hydrated pollen purified by flow cytometry (see Fig. 5c). d) Close-up of non-hydrated pollen. All scale bars are 50 μM.

Supplementary Figure 3 Flow cytometry acquisition plots showing the effects of gating hydrated and non-hydrated I1bc/+++ pollen.

a) Forward scatter (FSC)/side scatter (SSC) plot of eYFPdsRed/++ pollen, with the R1 gate indicated. The percentage of events in gate R1 is listed. b) Pulse width/side scatter (SSC) plot of eYFPdsRed/++ R1 pollen. Gate R2 contains both hydrated and non-hydrated pollen and represents the population analysed by a FACScan machine during two-colour analysis. Gate R2' is enriched for hydrated pollen. The percentage of events in gates R2 and R2' are listed. c) eYFP (FL1)/dsRed (FL2) plot of eYFPdsRed/++ of R2' (hydrated only) pollen. The percentage of events in each quadrant is indicated in the inset cross diagrams. The R7 quadrant analysed in d) and e) is also shown and the percentage of R7 events listed. d) eYFP (FL1)/dsRed (FL2) plot of eYFPdsRed/++ R2 (non-hydrated and hydrated) pollen. The percentage of events in each quadrant is indicated by the inset cross diagrams. The percentage of events in R7 is listed and is greater than that in plot c). e) R7 pollen from plot d) back-gated to a pulse width/side scatter (SSC) plot. This demonstrates that the majority of events observed in R7 in d) are the non-hydrated pollen that is present in R2 and excluded from R2'.

Supplementary information

Supplementary Figure 1

Crossing schemes to obtain FTLs that can be scored by flow cytometry. (PDF 326 kb)

Supplementary Figure 2

Micrographs of pollen sorted by flow cytometry. (PDF 25276 kb)

Supplementary Figure 3

Flow cytometry acquisition plots showing the effects of gating hydrated and non-hydrated I1bc/+++ pollen. (PDF 1148 kb)

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Yelina, N., Ziolkowski, P., Miller, N. et al. High-throughput analysis of meiotic crossover frequency and interference via flow cytometry of fluorescent pollen in Arabidopsis thaliana. Nat Protoc 8, 2119–2134 (2013). https://doi.org/10.1038/nprot.2013.131

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