Abstract
BackgroundThe 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.ResultsHere 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.ConclusionsMeioSeed 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.
Highlights
The formation of crossovers during meiosis is pivotal for the redistribution of traits among the progeny of sexually reproducing organisms
Composition and operation of MeioSeed A batch-wise image processing pipeline was created in the program CellProfiler, making use of a machinelearning based classifier to distinguish individual seeds in microscope images and to separate them from the background
To illustrate the use of MeioSeed, salt stress was investigated as a novel trigger for changes in crossover frequency, through which we found that salt stress can increase crossover frequency between the fluorescence marker genes in Col3-4/20, and can induce genome-wide fluctuations in crossover frequency
Summary
The formation of crossovers during meiosis is pivotal for the redistribution of traits among the progeny of sexually reproducing organisms. 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. The research on crossover frequency in the model species Arabidopsis has been greatly facilitated by the availability of fluorescence reporter lines for all five chromosomes [16,17,18] These lines contain in cis linked fluorescence reporter constructs under control of seed or pollen specific promoters and are separated by known genetic distances, thereby enabling the visualization of crossover events between the two fluorescence marker constructs in the F2 seeds or in the pollen of F 1 plants, respectively. The main advantage of this approach is that it in principle only relies on the availability of a fluorescence microscope
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