Primer design. The diagram shows the NBS domain motifs used in primer design. The motifs shown in blue are relatively conserved between TIR and non-TIR classes of NBS sequence while the domains in yellow have consistent differences. The three types of primer sets are shown with arrows to mark the location of the primers used. We used a total of 24 primer combinations that would specifically amplify TIR-NBS and non-TIR-NBS sequences, as well as combinations that would amplify all NBS sequences. All combinations were designed to amplify the kinase-2 region containing either a tryptophan (non-TIR) or aspartic acid (TIR) to aid in classification of the sequence.