Biosprint 96 dna plant kit

For the DNA extraction, plant tissue collection was done from a single plant from each accession after growing them in greenhouse for 2–3 weeks. A small piece of young leaf from a single plant of each accession was cut and collected in 96-well tissue collection box. Plant samples were then lyophilized, followed by genomic DNA extraction using Qiagen BioSprint 96 DNA Plant Kit (QIAGEN, Hilden, Germany). Extracted DNA was quantified with Quant-iT^TM PicoGreen® dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States). After DNA quantification and normalization, the DNA samples were transferred to 96-well DNA collection plates with one random well per plate left blank for quality control and library integrity. Genotyping of the DNA samples was performed using genotyping-by-sequencing (GBS) as described [13] (link). GBS libraries were prepared in 96 plexing using two restriction enzymes—a rare cutter PstI (5′-CTGCAG-3′), and a frequent cutter MspI (5′-CCGG-3′) with a common reverse adapter ligated. GBS libraries were sequenced using Illumina HiSeq2000 (Illumina, San Diego, CA, United States) platform at the University of Missouri (UMC; Columbia, Missouri) generating 100 bp long reads.