A variety of approaches are required for the de novo assemblies to improve draft genomes, and include a method developed by Dovetail Genomics for genome scaffolding using long‐range genomic information obtained by Chicago method (Putnam et al., 2016). In this study, we evaluated genome mapping on nanochannel arrays employing BioNano Irys® system (www.bionanogenomics.com) to validate the initial draft assembly, anchor additional unplaced contigs and to resolve misassemblies, with the objective of improving the overall assembly coverage. BioNano genome mapping is a technique of optical mapping in which specific sequence motifs in single DNA molecules are fluorescently labelled. The labelled DNA molecules are loaded onto the IrysChip where they are electrophoretically linearized in thousands of silicon channels. Fluorescence imaging allows the construction of maps of the physical distances between occurrences of the sequence motifs (Lam et al., 2012). The aim was to advance the pseudomolecule assembly of the eight chromosomes of subterranean clover, based on the direct visualizations of sequence motifs on long single DNA molecules. Gene annotations for the de novo draft genome assembly of the subterranean clover TSUd_r1.1 were conducted using in silico automated Augustus and Maker pipelines only. This was compared with direct transcriptome analysis using whole genome RNA sequencing technology across five different tissues of subterranean clover to generate a valuable resource for the identification and characterization of genes and pathways underlying plant growth and development. This also provided a basis to investigate specific processes, biological functions and gene interactions for key agronomic traits.