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

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.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0298-3) contains supplementary material, which is available to authorized users.

HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis

“…Crossover frequency was measured by analyzing counts of fluorescent and non-fluorescent seeds from FTL/++ hemizygote plants using a CellProfiler image analysis pipeline 97 , 98 . CellProfiler enables the quantification of green-alone fluorescent seeds (N Green ), red-alone fluorescent seeds (N Red ) and total seeds (N Total ).…”

Anthropogenic Impacts on Meiosis in Plants

“… Verde (2003) has also presented evidence that meiotic recombination increases in response to droughting in two genotypes of maize ( Zea mays ). van Tol et al (2018) detected a 70% increase in recombination frequency between markers in response to salt stress of Arabidopsis , and genotyping revealing that CO fluctuation was not limited to the region between the marker genes but occurred throughout the genome. Modliszewski et al (2018) did not detect the same effect under similar conditions in the same species.…”

User-friendly analysis of droplet array images

“…Image data can be used for various type of analysis, e.g. protein expression 21 , bacterial counting 22 , cell profiling 23 , seeds analysis 24 , single cell analysis 25 , etc. In droplet-based experiment, imaging has been used both in mono-and polydisperse droplets.…”