Plant material, DNA extraction, and sequencing

A total of 75 sorghum WGS data sets were published with sufficient sequencing depth for analysis at the time of the study [1,22,25], but a subset of only 53 of these accessions were available through the United States Department of Agriculture U.S National Plant Germplasm System Grin-Global [59] for validation of the new SSRs. Seedlings of these 53 accessions were grown in Pro-Mix® Biofungicide™ growing medium (Premier Tech Horticulture, Rivière-du-Loup, Qc) until they were large enough to provide leaf samples. Leaf discs were harvested from immature tissues using the LGC BioArk Leaf kits according to the manufacturer’s instructions. The DNA was extracted by LGC genomics using LGC sbeadex™ chemistry and libraries were prepared using the LGC genomics SeqSNP pipeline. Sequencing was performed by LGC genomics using Illumina® [60] sequencing-by-synthesis technology on an Illumina® NextSeq 550 with paired-end 150-base pair reads. All sequencing data is publicly available through the submission to NCBI (https://www.ncbi.nlm.nih.gov/) as BioProject (PRJNA610844), with 53 BioSample accession numbers (SAMN14318439—SAMN14318491), and 53 Sequence Read Archive (SRA) submission accession numbers (SRR11252447—SRR11252499).