A gap-free tomato genome built from complementary PacBio and Nanopore long DNA sequences reveals extensive linkage drag during breeding

Abstract

The assembly and scaffolding of plant crop genomes facilitates the characterization of genetically diverse cultivated and wild germplasm. The cultivated tomato has been improved through the introgression of genetic material from related wild species, including resistance to pandemic strains of Tobacco Mosaic virus (TMV) from Solanum peruvianum. Here we applied PacBio HiFi and ONT nanopore sequencing to develop independent, highly contiguous and complementary assemblies of an inbred TMV-resistant tomato variety. We merged the HiFi and ONT assemblies to generate a long-read-only assembly where all twelve chromosomes were represented as twelve contiguous sequences (N50=68.5 Mbp). The merged assembly was validated by chromosome conformation capture data and is highly consistent with previous tomato assemblies that made use of genetic maps and HiC for scaffolding. Our long-read-only assembly reveals that a complex series of structural variants linked to the TMV resistance gene likely contributed to linkage drag of a 64.1 Mbp region of the S. peruvianum genome during tomato breeding. We show that this minimal introgression region is present in six cultivated tomato hybrid varieties developed in three commercial breeding programs. Our results suggest that complementary long read technologies can facilitate the rapid generation of near complete genome sequences.

Structural variant-based pangenome construction has low sensitivity to variability of haplotype-resolved bovine assemblies

“…Cutting-edge sequencing and bioinformatic improvements (Baid et al, 2021; Silvestre-Ryan & Holmes, 2021), like the ONT Guppy5 basecaller, will likely assist more efficient assembly, resulting in higher QV and reduced computational load; however, currently the ONT specific requirements might be computationally prohibitive, especially when assembling many samples. Currently there are no approaches to co-assembling HiFi and ONT data that fully exploit the different advantages of the reads; however, HiFi and ONT assemblies can be merged post hoc (Alonge et al, 2021) which may improve contiguity (van Rengs et al, 2021), although we observed only minor increases (Supplementary Table 8).…”

“…These results indicate that optimal minigraph pangenomes would use high-quality, complete genomes as the backbone, like emerging T2T assemblies (Belser et al, 2021;Nurk et al, 2021;Rengs et al, 2021). Alternatively, reference-free approaches to pangenomes (Armstrong et al, 2020) may also circumvent these issues.…”

“…The quality and completeness of the pangenome backbone can have an impact, seen on the incomplete Angus chromosome 28 or the ARS-UCD1.2 chromosomes generally lacking centromeric sequence, but again we found no specific bias between CLR-or ONT-based backbones. These results indicate that optimal minigraph pangenomes would use high-quality, complete genomes as the backbone, like emerging telomere-to-telomere assemblies (Belser et al, 2021;Nurk et al, 2021;van Rengs et al, 2021). Alternatively, reference-free approaches that use base-level alignment may circumvent these issues and resolve variation down to single nucleotides (Armstrong et al, 2020;Garrison, n.d.), which may prove crucial for breakpoint resolution and related analyses.…”

The potential of integrative phenomics to harness underutilized crops for improving stress resilience

“…However, when going beyond model plants and major established crops, challenges remain. Whilst resolving homozygous genomes ( van Rengs et al., 2021 ) and pangenomes ( Bayer et al., 2020 ) of crops with a medium genome size is now achievable with the advent of novel sequencing technologies ( Mascher et al., 2021 ), challenges remain with consistent gene annotation and ontology developments. Rapid progress is being made with more mature tools, facilitating structural ( Holst et al, 2023 ) and functional genome annotation ( Bolger et al., 2018 ; Schwacke et al., 2019 ), whilst plant genome data continues to be gathered in comprehensive databases ( Vandepoele, 2017 ).…”