Neutrality test (dN/dS and H
statistic)
To identify the loci under positive selection, we performed dN/dS
analysis. For each cluster obtained from ClusterPicker, we used the most
similar homolog in snapdragon (Antirrhimum majus L.; Li, Zhang,
et al. (2019)) as ancestral NLR ortholog sequence for the sequences in
the cluster and conducted multiple sequence alignment using MAFFT,
estimated phylogenetic tree with FastTree and reverse-transcribed the
alignment of the sequences. We calculated the dN/dS ratios (ratio
between non-synonymous mutations and synonymous mutations) using PAML
software (Yang, 2007) within each of the clusters and used ratio
>1 as an indicator of putative positive selection. We also
calculated per base dN/dS ratios since, due to functional constraints on
conserved protein domains, it is much more likely that certain regions
in a gene are under selective pressure rather than the whole gene.
We carried out the Fay & Wu H statistic analysis (Fay & Wu, 2000) on
NLR transcripts by mapping the Plantago reads to the reference
transcriptome assembly using ANGSD software (Korneliussen, Albrechtsen,
& Nielsen, 2014). To identify loci under selective pressure, we
calculated the Fay & Wu H (H) values within a sliding window of nine
nucleotides. We chose H values less than -3.5 to signify selection.
Functional domains within the transcripts were identified with
InterProScan (Jones et al., 2014) search with the online InterPro
service of the EMBL-EBI website (Mitchell et al., 2019). The significant
H value loci were plotted against the transcript domain structure in R.