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

Abstract

Phone: +49 641 9937420 20 21 22 highly-duplicated nature of plant genomes, which makes them difficult to assess using high-31throughput genome screening methods. Here we describe how long-read sequencing 32 technologies can overcome this problem, revealing a surprisingly high level of widespread, 33 small to mid-scale SV in a major allopolyploid crop species, Brassica napus. We found that 34 up to 10% of all genes were affected by small to mid-scale SV events. Nearly half of these 35 SV events ranged between 100 bp to 1000 bp, which makes them challenging to detect using 36 short read Illumina sequencing. Examples demonstrating the contribution of such SV towards 37 eco-geographical adaptation and disease resistance in oilseed rape suggest that revisiting 38 complex plant genomes using medium-coverage, long-read sequencing might reveal 39 unexpected levels of functional gene variation, with major implications for trait regulation 40 and crop improvement. 41 visualization based on mRNAseq data (He et al., 2017) or deletion calling from SNP array 55 data (Gabur et al., 2018;Grandke et al., 2016). Critically, numerous examples have 56 connected genome SV in B. napus to important agronomic traits (Gabur et al., 2018; Gabur et 57 al., 2019;Liu et al., 2012;Stein et al., 2017). These studies revealed the important role of SV 58 in the creation of de novo variation for adaptation and breeding, however the methods used 59were not yet capable of resolving SV at gene scale. 60 A first example of intragenic SV impacting quantitatively inherited traits in B. napus was 61 reported by Qian et al. (2016), who demonstrated that deletion of exons 2 and 3 from a B. 62 napus orthologue of Mendel's "Green Cotyledon" gene (the Staygreen gene NON-63 YELLOWING 1; NYE1) associated with quantitative variation for chlorophyll and oil content. 64 Unfortunately, such small deletions are challenging to reliably detect using short-read 65 sequencing or low-cost marker arrays, so that their genome-wide extent could not yet be 66 investigated in detail. In this study, using B. napus as an example for a plant genome with 67 widespread structural variation, we demonstrate the power of whole-genome long-read 68 sequencing for high-resolution detection of intragenic SV. The results reveal widespread 69 functional variation on a completely unexpected scale, suggesting that small to mid-scale SV 70 may be a major driver of functional gene diversity in this recent polyploid crop. With the 71 growing accessibility, accuracy and cost-effectiveness of long-read sequencing, our results 72 suggest that there could be enormous promise in revisiting complex crop genomes to discover 73 potentially novel functional SV which has previously been overlooked. 74 75 Results and discussions 76 Long read sequencing reveal novel SV diversity in B. napus 77 We sequenced 4 B. napus accessions with long reads using the Oxford Nanopore Technology 78 (ONT) an 8 further accessions using the Pacific Biosciences (PacBio) platform (obtained 79 from Song et al. (2020)). The ...

Homoeologous chromosome exchange explains the creation of a QTL affecting soil‐borne pathogen resistance in tobacco

“…Quantitative disease resistance has previously been associated with presence/absence genetic variation (Gabur et al ., 2018, 2020) and homoeologous recombination (Zhao et al ., 2006) in Brassica napus . In addition, recent whole‐genome analyses using long‐read sequence data (Song et al ., 2020) has revealed genetic variability affecting plant disease resistance to be enriched in genomic regions impacted by structural variation, including that due to homoeologous recombination in allopolyploid species (Chawla et al ., 2021; Hurgobin et al ., 2018; Samans et al ., 2017).…”

Combined use of Oxford Nanopore and Illumina sequencing yields insights into soybean structural variation biology

“…[22][23][24][25] and benchmarked [26], typically performing better than methods using short reads. These approaches have recently been applied to provide genome-wide assessments of SVs in crops such as tomato [12], rice (Oryza sativa) [27] and rapeseed [28].…”

Positional‐based cloning ‘fail‐safe’ approach is overpowered by wheat chromosome structural variation

“…While the cost of whole‐genome sequencing in wheat is not yet feasible for individual breeding programs, long‐read sequencing and greater fold coverage (i.e., 10) has become increasingly affordable. Longer sequencing reads can detect even small‐ (30–10,000 bp) to mid‐scale (10,000–30,000 bp) structural variants, which impact trait diversity and are shown to be widespread in polyploid species (Chawla et al., 2021; Gabur et al., 2019; Mahmoud et al., 2019). For example, a recent study of Brassica napus L. found that up to 10% of all genes were affected by small‐ to mid‐scale structural variants including flowering‐time pathway genes, which can influence agronomic traits (Chawla et al., 2021).…”

“…Longer sequencing reads can detect even small‐ (30–10,000 bp) to mid‐scale (10,000–30,000 bp) structural variants, which impact trait diversity and are shown to be widespread in polyploid species (Chawla et al., 2021; Gabur et al., 2019; Mahmoud et al., 2019). For example, a recent study of Brassica napus L. found that up to 10% of all genes were affected by small‐ to mid‐scale structural variants including flowering‐time pathway genes, which can influence agronomic traits (Chawla et al., 2021). Obtaining long‐read sequencing (small‐ to mid‐scale structural variants) or 10‐fold coverage (large‐scale structural variants) of parental lines for a mapping population could become a prerequisite first‐step in positional cloning population development. Use the transcriptome to identify candidate genes .…”