Chlorophyll fluorescence

Fluorescence data was collected using a MINI-PAM-II fluorometer (Walz Heinz GmbH, Effeltrich, Germany).

To conduct measurements:

• The leaf clip was attached to a leaf and measurements of photosynthetically active radiation (PAR) and the photosynthetic efficiency of photosystem II (PhiPSII) were recorded.

• This was repeated ~10 times per plot.

• Each FI plot was followed by its RI plot counterpart.

Plots of blocks 1 and 4 were measured 49 days after sowing between 14:45 and 15:45. Plots of blocks 2 and 5 were measured 48 days after sowing between 15:00 and 17:00. All blocks were measured 50 days after sowing between 10:24 and 12:49.

The fluorescence kinetics of saturation pulse analyses for measurements were inspected using WinControl-3 Software (version 3.29-rev.1112) (Walz Heinz GmbH, Effeltrich, Germany). Measurements that did not pass inspection were discarded from subsequent analyses. PhiPSII values were clustered according to genotype, treatment and PAR level using custom R scripts.

PAR was divided into four levels:

• low (<500 μmol photons m -2 s -1 ),\
• mid-low (500-1000 μmol photons m -2 s -1 ),
• mid-high (1000-1500 μmol photons m -2 s -1 ) and
• high (>1500 μmol photons m -2 s -1 ).

Agglomerative hierarchical clustering was applied on the scaled PhiPSII means for all plots using the ‘agnes’ function of the R package ‘cluster’ [1] with a cut-off of five clusters.

Reference:

[1] Maechler, M.; Rousseeuw, P.; Struyf, A.; Hubert, M.; Hornik, K.; Studer, M.; Roudier, P.; Gonzalez, J.; Kozlowski, K.; Schubert, E.; et al. “Finding Groups in Data”: Cluster Analysis Extended Rousseeuw et al. Available online: https://cran.r-project.org/web/packages/cluster/cluster.pdf (accessed on 26 February 2021).