51-mer depth distribution for homozygous parental lines. (A) 51-mer frequency distribution for W7984 (red), compared with Opata (black). W7984 was sequenced more deeply to enable de novo WGS assembly. Uptick at low depth (below 51-mer frequency of approximately 5) corresponds to sequencing error. Peak frequency (approximately 18 for W7984, approximately 11 for Opata) represents the typical number of 51-mers covering nucleotides in the non-repetitive regions of the genome. (B) Cumulative frequency distribution for W7984 and Opata as a function of estimated genomic copy count (51-mer frequency divided by peak 51-mer frequency from panel (A)). Note logarithmic scale on the horizontal axis. The two curves lie on top of each other, as expected for two accessions from the same species. Approximately 45% of the hexaploid wheat genome is found in regions that are single copy as measured by 51-mers (estimated genomic copy count ≤2), and the remainder is typically at high 51-mer copy number (approximately 40% of the genome is found in 10 or more copies). The distribution rises smoothly through estimated genome copy counts of two and three, indicating the three subgenomes of hexaploid wheat are largely differentiated at the scale of a 51-mer.