Abstract
The number of crossovers during meiosis is relatively low, so multiple meioses need to be analyzed to accurately measure crossover frequency. In Arabidopsis, systems based on the segregation of fluorescent T-DNA reporters that are expressed in seeds (fluorescent-tagged lines, FTLs) allow for an accurate measurement of crossover frequency in specific chromosome regions. A major advantage of FTL-based experiments is the ability to analyze thousands of seeds for each biological replicate, which requires the use of automatic seed scoring. Here, we describe a protocol to computationally count the proportion of seeds that experienced a crossover event within the tested FTL interval and so measure the recombination frequency within that interval. We describe SeedScoring, a CellProfiler pipeline where the total time needed to measure crossover frequency in a single FTL line is approximately 5 min using a series of three images taken under a fluorescent stereomicroscope (3 min) and passing these images through the SeedScoring pipeline described in this protocol (2 min).
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Acknowledgments
This work was supported by Polish National Science Centre grants 2019/35/N/NZ2/02933 to N.K., 2020/37/N/NZ2/01226 to J.D., 2016/22/E/NZ2/00455 to P.A.Z., a Foundation for Polish Science grant POIR.04.04.00-00-5C0F/17-00 to P.A.Z, and European Research Council Consolidator Award ERC-2015-CoG-681987 “SynthHotSpot” to I.R.H. J.D. is the holder of Foundation for Polish Science START stipend (START 10.2021) and AMU Foundation scholarship for Ph.D students.
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Kbiri, N., Dluzewska, J., Henderson, I.R., Ziolkowski, P.A. (2022). Quantifying Meiotic Crossover Recombination in Arabidopsis Lines Expressing Fluorescent Reporters in Seeds Using SeedScoring Pipeline for CellProfiler. In: Lambing, C. (eds) Plant Gametogenesis. Methods in Molecular Biology, vol 2484. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2253-7_10
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DOI: https://doi.org/10.1007/978-1-0716-2253-7_10
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