2.3 Allele mining and design of a genotyping platform for wild barley (Hordeum vulgare ssp. spontaneum)
We collected all the available DNA sequencing data, including SSR (Hubner et al. , 2009), BOPA1 (Schmalenbach et al., 2011), Exome-Seq (Looseley et al., 2017), and Genotype-by-sequencing (Chang et al. , 2022). The workflow for designing and eventually selecting informative SNP for the genome wide association study (GWAS) analysis is depicted in Figure S2 . This harvest identified 502K unique contigs. After filtering these SNPs with MAF>0.05, no heterozygosity, a total read coverage >20, and missing marker>0.2 in at least 80% of the samples, we were left with 38K markers. We then retrieved 100 bp sequence flanking each marker and carried out BLAST search against the reference genome MorexV1 IBSCV2 barley genome to assure their identity and selectively singleton unique markers. Finally, we filtered out the redundant markers and reached approximately 30K markers sent to LGC Genomics Ltd. (GmbH, Germany) to design the probe set. During the probe design, the low specificity probes those with an off-target hit were removed, and we ended with 22.7K SNPs on the SNP chip made by LGC (Queens Road, Teddington, Middlesex, TW11 0LY, UK).