Re-calibration of flow cytometry standards for plant genome size estimation

Flow cytometry (FCM) and genome sequencing are complementary methods for estimating plant genome size (GS). However, discrepancies between the GS estimates derived from genome assemblies and FCM create ambiguity regarding the accuracy of these approaches. Approximately 12,000 plant GS measurements have been reported, with hardly any of them based on genome assemblies. Currently, FCM is the most frequently used method. Accurate GS estimation by FCM relies on internal standards with known GS values. However, previous GS calibrations, often based on incomplete reference genome assemblies, have led to significant discrepancies in GS estimates. Historically, the GS of a diploid plant species was estimated by doubling the size of a consensus genome assembly. However, consensus assemblies collapse homologous chromosomes into a single sequence, typically favouring the larger haplotype and potentially overestimating GS, especially in highly heterozygous species. Here, we applied haplotype-resolved genome assemblies to accurately recalibrate the reference standards. We utilized a recent gapless, telomere-to-telomere (T2T) consensus and the most complete phased genome assemblies of the Nipponbare rice as a primary standard to recalibrate five commonly used plant standards. Using the consensus genome as a reference revealed an overestimation of over 30% in widely used previous GS estimates for <i>Pisum sativum</i> and <i>Nicotiana benthamiana</i>, approximately 18% for <i>Arabidopsis thaliana</i>, and 5% for <i>Sorghum bicolor</i> and <i>Gossypium hirsutum</i>. The GS estimates based on phased haplotype assemblies suggested an additional 6%-7% overestimation. Haplotype-resolved genome assemblies allow the recalibration of GS estimates with the potential to yield more accurate values by capturing haplotype-specific variations previously missed in consensus assemblies.