Wheat Genotyping-by-Sequencing Protocol

Seeds were vernalized for 2 days at 4°C and grown for two weeks at room temperature in petri dishes covered with filter paper. DNA was extracted from first leaves using the Bioline Isolate II Plant DNA Kit. In a few cases where germination did not occur DNA was extracted from pulverized seeds with the Roche HighPure PCR Product Purification Kit. DNA was quantified by a Qubit dsDNA HS assay with a Qubit 2.0 Fluorometer and DNA integrity was checked by electrophoresis in 1% agarose gels. The resulting samples had DNA concentrations between 30–100 ng μl^–1.
Genotyping-by-sequencing (Genomic Diversity Facility, Cornell University) was carried out as described by Elshire et al. [35 (link)]. Optimization was attempted with PstI and EcoT221 and the former chosen for genome complexity reduction. Unique sequence tags were identified from the FASTQ files and aligned to release 31 of the T. aestivum genome using BWA v.0.7.8-r455 [36 (link)]. SNP calling was carried out with the TASSEL-GBS pipeline [37 (link)] and the vcf files were handled with VCFtools v.0.1.13 [38 (link)] and TASSEL [39 (link)]. Different filters for coverage, missing data, biallelic SNPs, indels and minimum allele frequency were applied as described in Results. The GBS data are available at the European Nucleotide Archive, study PRJEB42105.