Rooibos (Aspalathus linearis) Genome Size Estimation Using Flow Cytometry and K-Mer Analyses

Area of Science:

• Plant genomics
• Bioinformatics
• Flow cytometry

Background:

• Plant genomes are vital for understanding biosynthetic pathways of valuable compounds.
• Accurate genome size estimation is a prerequisite for initiating genome sequencing projects.
• Rooibos (Aspalathus linearis) is an economically important plant with limited genomic data.

Purpose of the Study:

• To estimate the genome size of rooibos (Aspalathus linearis) using two complementary methods.
• To optimize protocols for DAPI flow cytometry of rooibos samples.
• To evaluate different k-mer analysis tools for genome size estimation in rooibos.

Main Methods:

• DAPI flow cytometry was performed using radicles from commercial rooibos seedlings and Vicia faba as an internal standard.
• K-mer analysis was conducted on Illumina paired-end sequencing data from a commercial rooibos genotype.
• Four k-mer analysis methods (standard formula, BBNorm, GenomeScope, FindGSE) were investigated.

Main Results:

• Flow cytometry estimated the rooibos genome size at 1.24 ± 0.01 Gbp, consistent across different ecotypes.
• K-mer analyses, particularly using complete k-mer frequency histograms, estimated the genome size at 1.03 ± 0.04 Gbp.
• GenomeScope estimates were sensitive to parameter settings (CovMax), while other k-mer methods showed less deviation.

Conclusions:

• Both flow cytometry and k-mer analyses provide reliable genome size estimates for rooibos.
• Optimized protocols facilitate accurate genome size determination for rooibos and related species.
• The established genome size data will support future genomic research and breeding efforts in rooibos.