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Fluorescent Arabidopsis tetrads: a visual assay for quickly developing large crossover and crossover interference data sets

Nature Protocols volume 3, pages 41–50 (2008)Cite this article

Abstract

In most organisms, one crossover (CO) event inhibits the chances of another nearby event. The term used to describe this phenomenon is 'CO interference'. Here, we describe a protocol for quickly generating large data sets that are amenable to CO interference analysis in the flowering plant, Arabidopsis thaliana. We employ a visual assay that utilizes transgenic marker constructs encoding pollen-expressed fluorescent proteins of three colors in the quartet mutant background. In this genetic background, male meiotic products—the pollen grains—remain physically attached thereby facilitating tetrad analysis. We have developed a library of mapped marker insertions that, when crossed together, create adjacent intervals that can be rapidly and simultaneously screened for COs. This assay system is capable of detecting and differentiating single COs as well as two-, three- and four-strand double COs. We also describe how to analyze the data that are produced by this method. To generate and score a double interval in a wild-type and mutant background using this protocol will take 22–27 weeks.

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Figure 1: Map of fluorescent transgenes.
Figure 2: Single locus segregation patterns in pollen tetrads.
Figure 3: Classification of tetrad fluorescent patterns.
Figure 4: Examples of multicolor fluorescent tetrads.

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Acknowledgements

We thank Corbin Jones and Frank Stahl for critical reading of this manuscript. We also thank the NSF (MCB-0618691) and DOE (DE-FGO2-05ER15651) for financial support.

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Authors and Affiliations

  1. Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Luke E Berchowitz & Gregory P Copenhaver

  2. Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Luke E Berchowitz & Gregory P Copenhaver

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Correspondence to Gregory P Copenhaver.

Supplementary information

Supplementary Fig. 1

Blank tetrad classification template (PDF 255 kb)

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Berchowitz, L., Copenhaver, G. Fluorescent Arabidopsis tetrads: a visual assay for quickly developing large crossover and crossover interference data sets. Nat Protoc 3, 41–50 (2008). https://doi.org/10.1038/nprot.2007.491

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