Candidate gene identification in Morex reference and amplification from Bowman + Rph15

Based on the hypersensitive infection type of the Rph15 resistance, we used the physical interval of 44‐57Mb reported in Martin et al. (2020a) for the introgression of the Rph15 resistance from PI 355447 into Bowman to search for predicted annotated NLR candidate resistance genes in the Morex reference assembly using the NLR annotator pipeline reported by Steuernagel et al. (2020). The sequence from 44 800 000 to 57 300 000 bp on chromosome 2H of the Morex reference genome (Mascher et al., 2017) was extracted by samtools version 1.9.0. The sequence was cut into fragments of 20 kb length with 5 kb overlap, and the NLR prediction was performed based as previously described (https://github.com/steuernb/NLR‐Annotator Steuernagel et al., 2020).

We extracted flanking sequences from the Morex reference around the candidate gene locus and design a single primer pair (RGA4 F: and RGA4 R) based on the Morex rph15 candidate gene sequence (HORVU2Hr1G019120.5 GenBank accession {"type":"entrez-nucleotide","attrs":{"text":"AY641411.1","term_id":"55792395","term_text":"AY641411.1"}}AY641411.1) to amplify a 9442 base pair genomic fragment including approximately: 1.3 kb of sequence upstream from the translation Start codon and 2.5 kb of sequence downstream of the STOP codon. Polymerase chain reaction (PCR) was performed using Phusion polymerase as per the manufacturer’s instructions (New England Biosciences). PCR was performed using 50 µL reactions including: 1 × HF buffer, 100 ng of genomic DNA, 1 µm of primers, 3% of dimethyl sulfoxide (DMSO), 1 Unit of Phusion polymerase (New England Biosciences). PCR was performed at 1 cycle of 98°C for 3 min followed by 35 cycles of denaturing at 98°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 3 min and 30 s and then incubating at 72°C for 10 min. The PCR products were separated on a 1% agarose gel, and a single band of the expected size was excised and purified. The gel‐purified PCR product was then cloned into the sub‐cloning vector TOPO XL as per manufacturer’s instructions (Invitrogen). We amplified and cloned genomic fragments from resistant donors for Rph15 (BW719) and Rph16 (HS‐680) and the universally susceptible (Gus) barley accessions. The plasmid DNA of five positive clones from each amplicon was sent for Sanger sequencing using internal primers, and the sequences were compared with the template from Morex. All primers used in this study are detailed in Table S4.