Ploidy estimation with flow cytometry
In order to assess genome size variation and ploidy, fresh leaf tissue was collected from one accession of each unique genotype, except for fine-textured samples where only two of the 24 unique genotypes were tested. Tissue was chopped with a razor blade in 1 mL cold LB01 (Doležel, Binarová, & Lucretti, 1989) along with maize line B73 tissue, which was included as an internal standard. Nuclei were stained with propidium iodide, and samples were run on an Accuri C6 flow cytometer. Genome sizes were then calculated by taking the ratio of sample DNA to B73 DNA. These calculations were then divided by the value calculated for PI 509022 (the P. vaginatum reference genome accession, 0.259 relative to B73) to determine relative genome size of each accession compared to PI 509022.