Evidence for high intergenic sequence variation in heterozygous Italian ryegrass (Lolium multiflorumLam.) genome revealed by a high-quality draft diploid genome assembly

Abstract

BackgroundOver the last decade, progress in DNA sequencing technologies and assembly methods allowed plant scientists to move beyond the use of model organisms and work directly on the genomes of the major crops. Forage grass research can also benefit from this revolution, enabling progress in population genetic studies, functional biology, and genomics-assisted breeding. Due to its large genome size and high repeat content, so far only incomplete and fragmented assemblies are available for the grasses of the Lolium and Festuca species complex.FindingsHere, we report a highly contiguous draft assembly of Italian ryegrass (L. multiflorum Lam.), spanning 4.5 Gb and with a N50 of 3 Mb, containing ~70,000 gene models. Thanks to its relatedness to barley, 78% of the assembly was anchored on seven pseudomolecules. The high heterozygosity of the plant allowed obtaining a diploid assembly – i.e. across 95% of the assembly, both alleles were assembled on separate sequences. This feature allowed unraveling a very high amount of intergenic sequence variation between allelic sequences.ConclusionsWe present a nearly complete genome assembly of a genotype used in contemporary Swiss forage grass breeding programs. This work shows how genomic research has improved, allowing to decode the genetic code of large, complex, and heterozygous plants. It also allows the functional characterization of the ryegrass gene repertoire and the large-scale development of molecular markers. Furthermore, it paves the road for a reference-based characterization and exploitation of the genetic variation within the Lolium and Festuca species complex.

Identification of Candidate Genes for Self-Compatibility in Perennial Ryegrass (Lolium perenne L.)

“…In absence of a chromosome-scale assembly available for perennial ryegrass, variant calling was performed with the GBSmode pipeline (Yates and Studer, 2021), using as a reference the genome assembly of Italian ryegrass (Lolium multiflorum Lam. ), a self-incompatible species showing high synteny and relatedness to perennial ryegrass (Copetti et al, 2021).…”

“…LG numbers were assigned based on the physical position of their markers on the Italian ryegrass assembly (Copetti et al, 2021).…”

Identification of candidate genes for self-compatibility in perennial ryegrass (Lolium perenneL.)

“…Variant calling was performed with the GBSmode pipeline (https://github.com/stevenandrewyates/GBSmode) using the Italian ryegrass (Lolium multiflorum Lam.) genome assembly as a reference (Copetti et al, 2021).…”

“…This process was repeated until all singletons and double recombinations within a window of four markers were removed. Linkage group numbers were assigned based on the physical position of their markers on the Italian ryegrass assembly (Copetti et al, 2021).…”

“…To detect novel DNA sequence polymorphisms for marker development, 10 ng of genomic DNA from 50 individuals belonging to the collection of 2,056 F 2 individuals were pooled and sequenced at low coverage in 2x150 bp mode on an S4 flow cell lane of an Illumina Novaseq instrument at FGCZ. The paired-end reads were mapped to an Italian ryegrass reference assembly (Copetti et al, 2021) using bowtie2 (v3.5.1) (Langmead and Salzberg, 2012) and alignments were converted to BAM format with SAMtools (v1.10) (Li et al, 2009). A BAM file containing reads that aligned on the scaffolds delimited by the markers flanking the SC locus in Do Canto et al (2018) (G05_065 and G05_095) was imported into the Integrative Genomics Viewer software (Robinson et al 2011) and polymorphisms were visually inspected.…”