Goldengate genotyping assay for veracode manual protocol

DNA was extracted from leaf tissue using a CTAB method as described by Rogers and Bendich [65 ]. DNA was quantified with the Quant-iT™ PicoGreen® Assay (Invitrogen, Carlsbad, USA), using the Appliskan multiplate reader (Thermo Scientific, Courtaboeuf, France). DNA concentrations were adjusted to 50 ng/μL for each sample. For each assay, five plates of 96 samples containing 50 μL of genomic DNA normalized to 50 ng/μL were provided for genotyping using the “GoldenGate Genotyping Assay for VeraCode Manual Protocol” (Illumina Inc., San Diego, USA) [66 ]. The automatic allele calling for each locus was accomplished using the Genome Studio software (Illumina Inc., San Diego, USA). The homozygous and heterozygous clusters were checked visually and they were manually edited when necessary. Technical replicates and signal intensities were verified; only the most reliable calls were retained. A quality mark was then given to each SNP as follows: (A) Excellent genotyping; (B) Polymorphism detected but low fluorescence; (C) Polymorphism detected but low cluster separation; (D) Polymorphism detected but some accessions (> 10%) were not genotyped; and (E) Failed or No polymorphism detected (Additional file 9: Table S5).