Long-read sequencing reveals widespread intragenic structural variants in a recent allopolyploid crop plant.

Genome structural variation (SV) contributes strongly to trait variation in eukaryotic species and may have an even higher functional significance than single-nucleotide polymorphism (SNP). In recent years, there have been a number of studies associating large chromosomal scale SV ranging from hundreds of kilobases all the way up to a few megabases to key agronomic traits in plant genomes. However, there have been little or no efforts towards cataloguing small- (30-10 000 bp) to mid-scale (10 000-30 000 bp) SV and their impact on evolution and adaptation-related traits in plants. This might be attributed to complex and highly duplicated nature of plant genomes, which makes them difficult to assess using high-throughput genome screening methods. Here, we describe how long-read sequencing technologies can overcome this problem, revealing a surprisingly high level of widespread, small- to mid-scale SV in a major allopolyploid crop species, Brassica napus. We found that up to 10% of all genes were affected by small- to mid-scale SV events. Nearly half of these SV events ranged between 100 bp and 1000 bp, which makes them challenging to detect using short-read Illumina sequencing. Examples demonstrating the contribution of such SV towards eco-geographical adaptation and disease resistance in oilseed rape suggest that revisiting complex plant genomes using medium-coverage long-read sequencing might reveal unexpected levels of functional gene variation, with major implications for trait regulation and crop improvement.

Barley stripe mosaic virus-mediated somatic and heritable gene editing in barley (Hordeum vulgare L.).

Genome editing strategies in barley (Hordeum vulgare L.) typically rely on Agrobacterium-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants that constitutively express Cas9. Here, we explored virus-induced genome editing (VIGE) based on barley stripe mosaic virus (BSMV) in Cas9-transgenic barley. Somatic and heritable editing in the ALBOSTRIANS gene (CMF7) resulting in albino/variegated chloroplast-defective barley mutants is shown. In addition, somatic editing in meiosis-related candidate genes in barley encoding ASY1 (an axis-localized HORMA domain protein), MUS81 (a DNA structure-selective endonuclease), and ZYP1 (a transverse filament protein of the synaptonemal complex) was achieved. Hence, the presented VIGE approach using BSMV enables rapid somatic and also heritable targeted gene editing in barley.