The NLRs were extracted from the nucleotide genomic sequences using NLR-Annotator (Steuernagel et al. 2018) and from proteomes using hmmalign with reference HMM of the grass NB-ARC (Bailey et al. 2018). Additionally, NLRs and NLR-IDs were characterized in the Brachypodium (Gordon et al. 2017) and Maize annotations using the plant_rgenes pipeline (Sarris et al. 2016) (e-value cutoff 1e-3). The number of NB-ARC containing proteins was compared to those previously identified in Arabidopsis (Van et al. 2019) and plotted using R package ggplot2 (Wickham 2016). The NB-ARC domain alignment was manually curated for the presence of NB-ARC domain functional motifs including Walker A, WALKER-B, RNBS-C, GLPL and RNBS-D. The NLR phylogeny was determined using RAxML MPI (v8.2.9, -f a, -x 12345, -p 12345, -# 100, -m PROTCATJTT). The phylogeny was visualized and re-rooted on the longest internal branch in iTOL (Letunic and Bork 2016).

For the ITOL scripts you need to append to start the output the formating that corresponds to the iTOL annotation pages which can be found here: https://itol.embl.de/help.cgi Link to trees shared: https://itol.embl.de/shared/xCJbI9ndshEK