MeioSeed: a CellProfiler-based program to count fluorescent seeds for crossover frequency analysis in Arabidopsis thaliana

Abstract Background The formation of crossovers during meiosis is pivotal for the redistribution of traits among the progeny of sexually reproducing organisms. In plants the molecular mechanisms underlying the formation of crossovers have been well established, but relatively little is known about the factors that determine the exact location and the frequency of crossover events in the genome. In the model plant species Arabidopsis, research on these factors has been greatly facilitated by reporter lines containing linked fluorescence marker genes under control of promoters active in seeds or pollen, allowing for the visualization of crossover events by fluorescence microscopy. However, the usefulness of these reporter lines to screen for novel modulators of crossover frequency in a high throughput manner relies on the availability of programs that can accurately count fluorescent seeds. Such a program was previously not available in scientific literature. Results Here we present MeioSeed, a novel CellProfiler-based program that accurately counts GFP and RFP fluorescent Arabidopsis seeds with adjustable detection thresholds for fluorescence intensity, making use of a robust seed classifier which was trained by machine learning in Ilastik. Using the previously published reporter line Col3-4/20 as an example, we explain the use of MeioSeed and the steps taken to optimize the thresholding settings of the program to fit the published model for recombination frequency and transgene segregation. The use of MeioSeed is illustrated by investigating salt stress as a novel abiotic trigger for changes in crossover frequency in Col3-4/20 (♂) × Ler-0 (♀) F1 hybrids. Salt stress was found to trigger increases in crossover frequency between the marker genes of up to 70% compared to the control treatment without salt stress. Genotyping of control and salt treated populations revealed that the changes in crossover frequency were not limited to the region between the marker genes, but that fluctuations in crossover frequency are likely to occur genome-wide after treatment with high salt concentrations. Conclusions MeioSeed allows for the high throughput recognition and counting of fluorescent Arabidopsis seeds and can facilitate the screening for novel abiotic and biotic modulators of crossover frequency using reporter lines in Arabidopsis

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Additional file 1. Operation and processing steps of the MeioSeed Algorithm. Additional text from the results section describing how MeioSeed should be operated and which steps the CellProfiler algorithm takes to process fluorescence microscopy images into images with seed counts

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Additional file 3. Overview of rapeseeds (Brassica napus cv. Westar) counted with MeioSeed. The same settings as for Arabidopsis seeds were used, except for a seed classifier file which was trained in Ilastik to recognize rapeseeds

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Additional file 2. Genetic maps of F2 populations resulting from treatment of Col3-4/20 (♂) × Ler-0 (♀) F1 hybrids with either 0 mM or 300 mM NaCl, generated by KASP genotyping with 39 SNP marker sets (384 chromosomes per treatment)

Enhancement of Arabidopsis growth characteristics using genome interrogation with artificial transcription factors - Fig 3

<p><b>A</b>) Quantification of the relative RSA of the selected 3F-EAR lines (T2; segregating) and 3F-VP16 lines (T3; only VP16-05-014 segregating) compared to Col-0 at 25 dpg. The RSA of each plant was calculated in terms of percentage of the average of Col-0. Error bars represent SEM values (n = 182 for Col-0, n = 16–18 for the transgenic lines). Significant differences with the Col-0 are indicated by an * (<i>p</i> < 0.05). <b>B</b>) Number of true leaves with discernable petioles at 25 dpg. Error bars represent SEM values (n = 36 for Col-0, n = 16–18 for the transgenic lines). Significant increases compared to Col-0 indicated by asterisks (*) (<i>p</i> < 0.05). <b>C)</b> Overview of the rosette phenotypes of the selected 3F-ATF lines (25 dpg), and of the 9 bp DNA recognition sequences of the 3Fs that were isolated from them. The presented individuals had RSA values closest to the average value found for their genotypes.</p