Chromosome‐level haplotype‐resolved genome assembly provides insights into the highly heterozygous genome of Italian ryegrass ( <i>Lolium multiflorum</i> Lam.)

Italian ryegrass (Lolium multiflorum Lam.) is an important forage grass, providing a major source of feed for ruminants in temperate regions. Due to its highly heterozygous and repeat-rich genome, high-quality chromosome-level genome assemblies are scarce for Italian ryegrass. Here, we sequenced the genome of a genotype from the Italian ryegrass cultivar 'Rabiosa' (hereafter referred to as Rabiosa), and we obtained Oxford Nanopore Technologies long reads (∼60-fold coverage), Illumina short reads (∼85-fold coverage), and high-throughput chromosome conformation capture data (∼60-fold coverage). With Rabiosa as one of the parents, we constructed an F₁ population consisting of 304 individuals, which were genotyped by reduced representation sequencing for linkage map construction and quantitative trait locus (QTL) analysis. Using whole-genome sequencing data of Rabiosa and the genetic linkage map, we first generated a chromosome-level unphased haploid assembly (scaffold N50 of 338.75 Mb, total Benchmarking Universal Single-Copy Orthologs [BUSCO] score of 94.60%). Then, based on the unphased assembly and a reference-based phasing approach, we generated a chromosome-level haplotype-resolved assembly containing both haplotypes (scaffold N50 of ∼250 Mb and total BUSCO score of ∼90% for each haplome). Between the two haplotypes of Rabiosa, we observed a highly collinear gene order at the chromosome level and a high sequence variation at the local level. With a graph-based reference built from the unphased and the haplotype-resolved assemblies of Rabiosa, we conducted a QTL analysis, and two QTL significantly associated with stem rust resistance were detected. The genome assemblies of Rabiosa will serve as invaluable genomic resources to facilitate genomic applications in forage grass research and breeding.