Developmental normalization of phenomics data generated by high throughput plant phenotyping systems.

Background: Sowing time is commonly used as the temporal reference for (Arabidopsis) experiments in high throughput plant phenotyping (HTPP) systems. This relies on the assumption that germination and seedling establishment are uniform across the population. However, individual seeds have different development trajectories even under uniform environmental conditions. This leads to increased variance in quantitative phenotyping approaches. We developed the Digital Adjustment of Plant Development (DAPD) normalization method. It normalizes time-series HTPP measurements by reference to an early developmental stage and in an automated manner. The timeline of each measurement series is shifted to a reference time. The normalization is determined by cross-correlation at multiple time points of the time-series measurements, which may include rosette area, leaf size, and number.

Results: The DAPD method improved the accuracy of phenotyping measurements by decreasing the statistical dispersion of quantitative traits across a time-series. We applied DAPD to evaluate the relative growth rate in Arabidopsis plants and demonstrated that it improves uniformity in measurements, permitting a more informative comparison between individuals. Application of DAPD decreased variance of phenotyping measurements by up to 2.5 times compared to sowing-time normalization. The DAPD method also identified more outliers than any other central tendency technique applied to the non-normalized dataset.

Conclusions: DAPD is an effective method to control for temporal differences in development within plant phenotyping datasets. In principle, it can be applied to HTPP data from any species/trait combination for which a relevant developmental scale can be defined.