| Summary: | Plant genomes provide information on biosynthetic pathways involved in the production of industrially relevant compounds. Genome size estimates are essential for the initiation of genome projects. The genome size of rooibos (<i>Aspalathus linearis</i> species complex) was estimated using DAPI flow cytometry and k-mer analyses. For flow cytometry, a suitable nuclei isolation buffer, plant tissue and a transport medium for rooibos ecotype samples collected from distant locations were identified. When using radicles from commercial rooibos seedlings, Woody Plant Buffer and <i>Vicia faba</i> as an internal standard, the flow cytometry-estimated genome size of rooibos was 1.24 ± 0.01 Gbp. The estimates for eight wild rooibos growth types did not deviate significantly from this value. K-mer analysis was performed using Illumina paired-end sequencing data from one commercial rooibos genotype. For biocomputational estimation of the genome size, four k-mer analysis methods were investigated: A standard formula and three popular programs (BBNorm, GenomeScope, and FindGSE). GenomeScope estimates were strongly affected by parameter settings, specifically CovMax. When using the complete k-mer frequency histogram (up to 9 × 10<sup>5</sup>), the programs did not deviate significantly, estimating an average rooibos genome size of 1.03 ± 0.04 Gbp. Differences between the flow cytometry and biocomputational estimates are discussed.
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