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).