Barley F2 Population Genotyping

Forty-eight F₂s (24 each for wild type and stripped) and parental lines were first genotyped with a barley 50 k iSelect SNP Array [43 (link)]. Marker positions are based on the barley pseudo-molecule assembly of Morex V1 [44 (link)]. Genotype calling was performed with the de novo calling algorithm in GenomeStudio (Illumina). Clusters of polymorphic SNPs were inspected and manually adjusted if necessary. The linked SNPs were used to develop semi-thermal asymmetric reverse PCR (STARP) markers to genotype the F₂ population [45 (link)]. PCR was conducted in a 10 μl reaction volume with Taq DNA polymerase (New England Biolab) according to the manufacturer’s instructions. Sequences of priming element-adjustable primer (PEA-primer) 1 and 2 are 5′-AGCTGGTT-SP9-GCAACAGGAACCAGCTATGAC-3′ and 5′-ACTGCTCAAGAG-SP9-GACGCAAGTGAGCAGTATGAC-3′, respectively [45 (link)]. The thermal amplification program followed a touchdown protocol as described previously [45 (link), 46 (link)]. Stained with GelRed™ nucleic acid stain (MilliporeSigma), amplicons were analyzed with 6% polyacrylamide gel which was imaged using a Typhoon™ FLA 9500 variable mode laser scanner (GE Healthcare Life Sciences, Marlborough, MA). The markers used in the present study are listed in Table 1.