Wheat Panache: A pangenome graph database representing presence-absence variation across sixteen bread wheat genomes.

Bread wheat (Triticum aestivum L.) is one of humanity's most important staple crops, characterized by a large and complex genome with a high level of gene presence-absence variation (PAV) between cultivars, hampering genomic approaches for crop improvement. With the growing global population and the increasing impact of climate change on crop yield, there is an urgent need to apply genomic approaches to accelerate wheat breeding. With recent advances in DNA sequencing technology, a growing number of high-quality reference genomes are becoming available, reflecting the genetic content of a diverse range of cultivars. However, information on the presence or absence of genomic regions has been hard to visualize and interrogate because of the size of these genomes and the lack of suitable bioinformatics tools. To address this limitation, we have produced a wheat pangenome graph maintained within an online database to facilitate interrogation and comparison of wheat cultivar genomes. The database allows users to visualize regions of the pangenome to assess PAV between bread wheat genomes.

Graph pangenomes represent more genomic variants than reference genomes. We present a wheat graph pangenome based on 16 public assemblies. We present Wheat Panache, an online visual representation of this graph. Wheat Panache lets users search the graph for presence-absence variants. We also distribute the graph preindexed for Giraffe utilization.

Long-read and chromosome-scale assembly of the hexaploid wheat genome achieves high resolution for research and breeding.

BACKGROUND: The sequencing of the wheat (Triticum aestivum) genome has been a methodological challenge for many years owing to its large size (15.5 Gb), repeat content, and hexaploidy. Many initiatives aiming at obtaining a reference genome of cultivar Chinese Spring have been launched in the past years and it was achieved in 2018 as the result of a huge effort to combine short-read sequencing with many other resources. Reference-quality genome assemblies were then produced for other accessions, but the rapid evolution of sequencing technologies offers opportunities to reach high-quality standards at lower cost. RESULTS: Here, we report on an optimized procedure based on long reads produced on the Oxford Nanopore Technology PromethION device to assemble the genome of the French bread wheat cultivar Renan. CONCLUSIONS: We provide the most contiguous chromosome-scale assembly of a bread wheat genome to date. Coupled with an annotation based on RNA-sequencing data, this resource will be valuable for the crop community and will facilitate the rapid selection of agronomically important traits. We also provide a framework to generate high-quality assemblies of complex genomes using ONT.

De Novo Genome Assembly of the Japanese Wheat Cultivar Norin 61 Highlights Functional Variation in Flowering Time and Fusarium-Resistant Genes in East Asian Genotypes.

Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize the key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 22 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related 13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog and the association of its A homeologous alleles with the spring/winter growth habit. Furthermore, the Norin 61 genome carries typical East Asian functional variants different from CS, ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.

The Aegilops ventricosa 2NvS segment in bread wheat: cytology, genomics and breeding.

KEY MESSAGE: The first cytological characterization of the 2NvS segment in hexaploid wheat; complete de novo assembly and annotation of 2NvS segment; 2NvS frequency is increasing 2NvS and is associated with higher yield. The Aegilops ventricosa 2NvS translocation segment has been utilized in breeding disease-resistant wheat crops since the early 1990s. This segment is known to possess several important resistance genes against multiple wheat diseases including root knot nematode, stripe rust, leaf rust and stem rust. More recently, this segment has been associated with resistance to wheat blast, an emerging and devastating wheat disease in South America and Asia. To date, full characterization of the segment including its size, gene content and its association with grain yield is lacking. Here, we present a complete cytological and physical characterization of this agronomically important translocation in bread wheat. We de novo assembled the 2NvS segment in two wheat varieties, 'Jagger' and 'CDC Stanley,' and delineated the segment to be approximately 33 Mb. A total of 535 high-confidence genes were annotated within the 2NvS region, with > 10% belonging to the nucleotide-binding leucine-rich repeat (NLR) gene families. Identification of groups of NLR genes that are potentially N genome-specific and expressed in specific tissues can fast-track testing of candidate genes playing roles in various disease resistances. We also show the increasing frequency of 2NvS among spring and winter wheat breeding programs over two and a half decades, and the positive impact of 2NvS on wheat grain yield based on historical datasets. The significance of the 2NvS segment in wheat breeding due to resistance to multiple diseases and a positive impact on yield highlights the importance of understanding and characterizing the wheat pan-genome for better insights into molecular breeding for wheat improvement.

The barley pan-genome reveals the hidden legacy of mutation breeding.

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'1). 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 conditions2. 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.

Multiple wheat genomes reveal global variation in modern 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 genome1, and the lack of genome-assembly data for multiple wheat lines2,3. 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 stresses4,5. 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 Sm16, 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.

TRITEX: chromosome-scale sequence assembly of Triticeae genomes with open-source tools.

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.

Prospects of pan-genomics in barley.

The concept of a pan-genome refers to intraspecific diversity in genome content and structure, encompassing both genes and intergenic space. Pan-genomic studies employ a combination of de novo sequence assembly and reference-based alignment to discover and genotype structural variants. The large size and complex structure of Triticeae genomes were for a long time an obstacle for genomic research in barley and its relatives. Now that a reference genome is available, computational pipelines for high-quality sequence assembly are in place, and sequence costs continue to drop, investigations into the structural diversity of the barley genome seem within reach. Here, we review the recent progress on pan-genomics in the model grass Brachypodium distachyon, and the cereal crops rice and maize, and devise a multi-tiered strategy for a pan-genome project in barley. Our design involves: (1) the construction of high-quality de novo sequence assemblies for a small core set of representative genotypes, (2) short-read sequencing of a large diversity panel of genebank accessions to medium coverage and (3) the use of complementary methods such as chromosome-conformation capture sequencing and k-mer-based association genetics. The in silico representation of the barley pan-genome may inform about the mechanisms of structural genome evolution in the Triticeae and supplement quantitative genetics models of crop performance for better accuracy and predictive ability.

The Rise and Fall of African Rice Cultivation Revealed by Analysis of 246 New Genomes.

African rice (Oryza glaberrima) was domesticated independently from Asian rice. The geographical origin of its domestication remains elusive. Using 246 new whole-genome sequences, we inferred the cradle of its domestication to be in the Inner Niger Delta. Domestication was preceded by a sharp decline of most wild populations that started more than 10,000 years ago. The wild population collapse occurred during the drying of the Sahara. This finding supports the hypothesis that depletion of wild resources in the Sahara triggered African rice domestication. African rice cultivation strongly expanded 2,000 years ago. During the last 5 centuries, a sharp decline of its cultivation coincided with the introduction of Asian rice in Africa. A gene, PROG1, associated with an erect plant architecture phenotype, showed convergent selection in two rice cultivated species, Oryza glaberrima from Africa and Oryza sativa from Asia. In contrast, a shattering gene, SH5, showed selection signature during African rice domestication, but not during Asian rice domestication. Overall, our genomic data revealed a complex history of African rice domestication influenced by important climatic changes in the Saharan area, by the expansion of African agricultural society, and by recent replacement by another domesticated species.

De Novo Assemblies of Three Oryza glaberrima Accessions Provide First Insights about Pan-Genome of African Rices.

Oryza glaberrima is one of the two cultivated species of rice, and harbors various interesting agronomic traits, especially in biotic and abiotic resistance, compared with its Asian cousin O. sativa. A previous reference genome was published but newer studies highlighted some missing parts. Moreover, global species diversity is known nowadays to be represented by more than one single individual. For that purpose, we sequenced, assembled and annotated de novo three different cultivars from O. glaberrima. After validating our assemblies, we were able to better solve complex regions than the previous assembly and to provide a first insight in pan-genomic divergence between individuals. The three assemblies shown large common regions, but almost 25% of the genome present collinearity breakpoints or are even individual specific.

LTRclassifier: A website for fast structural LTR retrotransposons classification in plants.

Automatic classification of LTR retrotransposons is a big challenge in the area of massive genomics. Many tools were developed to detect them but automatic classification is somehow challenging. Here we propose a simple approach, LTRclassifier, based on HMM recognition followed by BLAST analyses (i) to classify plant LTR retrotransposons in their respective superfamily, and (ii) to provide automatically a basic functional annotation of these elements. The method was tested on various TE databases, and shown to be robust and fast. This tool is available as a web service implemented at IRD bioinformatics facility, http://LTRclassifier.ird.fr/.

TOGGLE: toolbox for generic NGS analyses.

BACKGROUND: The explosion of NGS (Next Generation Sequencing) sequence data requires a huge effort in Bioinformatics methods and analyses. The creation of dedicated, robust and reliable pipelines able to handle dozens of samples from raw FASTQ data to relevant biological data is a time-consuming task in all projects relying on NGS. To address this, we created a generic and modular toolbox for developing such pipelines. RESULTS: TOGGLE (TOolbox for Generic nGs anaLysEs) is a suite of tools able to design pipelines that manage large sets of NGS softwares and utilities. Moreover, TOGGLE offers an easy way to manipulate the various options of the different softwares through the pipelines in using a single basic configuration file, which can be changed for each assay without having to change the code itself. We also describe one implementation of TOGGLE in a complete analysis pipeline designed for SNP discovery for large sets of genomic data, ready to use in different environments (from a single machine to HPC clusters). CONCLUSION: TOGGLE speeds up the creation of robust pipelines with reliable log tracking and data flow, for a large range of analyses. Moreover, it enables Biologists to concentrate on the biological relevance of results, and change the experimental conditions easily. The whole code and test data are available at https://github.com/SouthGreenPlatform/TOGGLE .