Grasses are considered one of the most important angiosperm groups in economic terms. The Setaria sphacelata complex is an intriguing species with considerable variation in genome size (GS). It is currently being explored from a genomic perspective especially because of its great adaptability under winter periods. Repetitive DNA sequences are known to contribute significantly to GS diversity, which in turn can play a role in impacting the ecology and evolution of a species. This study is aimed to characterize the GS and repetitive elements of different germplasm accessions of S. sphacelata. We analyzed 540 plants from 70 accessions collected worldwide. Flow cytometry was used for GS estimation, together with chromosome counting, fluorescent in situ hybridization (FISH), and bioinformatic analysis. Most (i.e., 80%) accessions were tetraploids (2n = 4x = 36) with GS ranging from 3.0 to 3.7 pg/2C. Additionally, a low number of diploids (2n = 2x = 18; ∼1.6 to 2 pg/2C), pentaploids (2n = 5x = 45: ∼4 pg/2C), hexaploids (2n = 6x = 54; ∼4.4 pg/2C), and octoploids (2n = 8x = 72; ∼6 pg/2C,) were detected. The greatest variation was found in accessions from South Africa. RepeatExplorer2 showed that 39% and 43% of the genomes of two diploid accessions comprised repetitive sequences, with Ty3/gypsy retrotransposons being the most abundant repeats. However, variation in the percentage of two Ty3/gypsy sublineages, Athila and Ogre, suggests different process of amplification and deletion of repeats during the evolution of the species complex. In contrast, FISH revealed some satellite repeats with a conserved localized distribution in pericentromeric regions between accessions. Although differences in the distribution of the FISH signals over the chromosomes were observed between the different ploidy levels, the data suggest that increases in the ploidy level were associated with the occurrence of hybridization and DNA loss.
