Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping ...approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.
Chromosome-scale genome sequence assemblies underpin pan-genomic studies. Recent genome assembly efforts in the large-genome Triticeae crops wheat and barley have relied on the commercial ...closed-source assembly algorithm DeNovoMagic. We present TRITEX, an open-source computational workflow that combines paired-end, mate-pair, 10X Genomics linked-read with chromosome conformation capture sequencing data to construct sequence scaffolds with megabase-scale contiguity ordered into chromosomal pseudomolecules. We evaluate the performance of TRITEX on publicly available sequence data of tetraploid wild emmer and hexaploid bread wheat, and construct an improved annotated reference genome sequence assembly of the barley cultivar Morex as a community resource.
Abstract
Durable crop disease resistance is an essential component of global food security. Continuous pathogen evolution leads to a breakdown of resistance and there is a pressing need to ...characterize new resistance genes for use in plant breeding. Here we identified an accession of wild emmer wheat (
Triticum turgidum
ssp.
dicoccoides
), PI 487260, that is highly resistant to multiple stripe rust isolates. Genetic analysis revealed resistance was conferred by a single, incompletely dominant gene designated as
Yr84
. Through bulked segregant analysis sequencing (BSA-Seq) we identified a 52.7 Mb resistance-associated interval on chromosome 1BS. Detected variants were used to design genetic markers for recombinant screening, further refining the interval of
Yr84
to a 2.3–3.3 Mb in tetraploid wheat genomes. This interval contains 34 candidate genes encoding for protein domains involved in disease resistance responses. Furthermore, KASP markers closely-linked to
Yr84
were developed to facilitate marker-assisted selection for rust resistance breeding.
Seed mutagenesis is one strategy to create a population with thousands of useful mutations for the direct selection of desirable traits, to introduce diversity into varietal improvement programs, or ...to generate a mutant collection to support gene functional analysis. However, phenotyping such large collections, where each individual may carry many mutations, is a bottleneck for downstream analysis. Targeting Induced Local Lesions in Genomes (TILLinG), when coupled with next-generation sequencing allows high-throughput mutation discovery and selection by genotyping. We mutagenized an advanced durum breeding line, UAD0951096_F2:5 and performed short-read (2x125 bp) Illumina sequencing of the exome of 100 lines using an available exome capture platform. To improve variant calling, we generated a consolidated exome reference using the recently available genome sequences of the cultivars Svevo and Kronos to facilitate the alignment of reads from the UAD0951096_F2:5 derived mutants. The resulting exome reference was 484.4 Mbp. We also developed a user-friendly, searchable database and bioinformatic analysis pipeline that allowed us to predict zygosity of the mutations discovered and extracts flanking sequences for rapid marker development. Here, we present these tools with the aim of allowing researchers fast and accurate downstream selection of mutations discovered by TILLinG by sequencing to support functional annotation of the durum wheat genome.
An annotated reference sequence representing the hexaploid bread wheat genome in 21 pseudomolecules has been analyzed to identify the distribution and genomic context of coding and noncoding elements ...across the A, B, and D subgenomes. With an estimated coverage of 94% of the genome and containing 107,891 high-confidence gene models, this assembly enabled the discovery of tissue- and developmental stage-related coexpression networks by providing a transcriptome atlas representing major stages of wheat development. Dynamics of complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. This community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding.
Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the ...size and complexity of the wheat genome
, and the lack of genome-assembly data for multiple wheat lines
. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses
. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm1
, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.
Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop ...species (known as the 'pan-genome'
). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions
. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.
SUMMARY
Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for the improvement of wheat. To understand ...their diversity and advance their utilization, we produced whole‐genome assemblies of Aegilops longissima and Aegilops speltoides. Whole‐genome comparative analysis, along with the recently sequenced Aegilops sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and are most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides genome is closest to the wheat B subgenome, and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome‐wide analysis of nucleotide‐binding leucine‐rich repeat (NLR) genes revealed species‐specific and lineage‐specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.
Significance Statement
Whole‐genome comparative analysis using de novo assemblies of Aegilops longissima and Aegilops speltoides, along with the recently sequenced Aegilops sharonensis genome, strengthen evolutionary evidence that Ae. longissima and Ae. sharonensis genomes are highly similar and are most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. A detailed NLR annotation and haplotype block analysis aims to help further research using these species to improve wheat.
The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their ...manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance.
PDF
