A pooled-sample draft genome assembly provides insights into host plant-specific transcriptional responses of a Solanaceae-specializing pest, Tupiocoris notatus (Hemiptera: Miridae).

The assembly of genomes from pooled samples of genetically heterogenous samples of conspecifics remains challenging. In this study, we show that high-quality genome assemblies can be produced from samples of multiple wild-caught individuals. We sequenced DNA extracted from a pooled sample of conspecific herbivorous insects (Hemiptera: Miridae: Tupiocoris notatus) acquired from a greenhouse infestation in Tucson, Arizona (in the range of 30-100 individuals; 0.5 mL tissue by volume) using PacBio highly accurate long reads (HiFi). The initial assembly contained multiple haplotigs (>85% BUSCOs duplicated), but duplicate contigs could be easily purged to reveal a highly complete assembly (95.6% BUSCO, 4.4% duplicated) that is highly contiguous by short-read assembly standards (N 50 = 675 kb; Largest contig = 4.3 Mb). We then used our assembly as the basis for a genome-guided differential expression study of host plant-specific transcriptional responses. We found thousands of genes (N = 4982) to be differentially expressed between our new data from individuals feeding on Datura wrightii (Solanaceae) and existing RNA-seq data from Nicotiana attenuata (Solanaceae)-fed individuals. We identified many of these genes as previously documented detoxification genes such as glutathione-S-transferases, cytochrome P450s, and UDP-glucosyltransferases. Together our results show that long-read sequencing of pooled samples can provide a cost-effective genome assembly option for small insects and can provide insights into the genetic mechanisms underlying interactions between plants and herbivorous pests.

An improved chromosome-level genome assembly of perennial ryegrass (Lolium perenne L.).

This work is an update and extension of the previously published article "Ultralong Oxford Nanopore Reads Enable the Development of a Reference-Grade Perennial Ryegrass Genome Assembly" by Frei et al. The published genome assembly of the doubled haploid perennial ryegrass (Lolium perenne L.) genotype Kyuss (Kyuss v1.0) marked a milestone for forage grass research and breeding. However, order and orientation errors may exist in the pseudo-chromosomes of Kyuss, since barley (Hordeum vulgare L.), which diverged 30 million years ago from perennial ryegrass, was used as the reference to scaffold Kyuss. To correct for structural errors possibly present in the published Kyuss assembly, we de novo assembled the genome again and generated 50-fold coverage high-throughput chromosome conformation capture (Hi-C) data to assist pseudo-chromosome construction. The resulting new chromosome-level assembly Kyuss v2.0 showed improved quality with high contiguity (contig N50 = 120 Mb), high completeness (total BUSCO score = 99%), high base-level accuracy (QV = 50), and correct pseudo-chromosome structure (validated by Hi-C contact map). This new assembly will serve as a better reference genome for Lolium spp. and greatly benefit the forage and turf grass research community.

High-resolution bulked segregant analysis enables candidate gene identification for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.).

Bacterial wilt, caused by Xanthomonas translucens pv. graminis (Xtg), is a serious disease of economically important forage grasses, including Italian ryegrass (Lolium multiflorum Lam.). A major QTL for resistance to Xtg was previously identified, but the precise location as well as the genetic factors underlying the resistance are yet to be determined. To this end, we applied a bulked segregant analysis (BSA) approach, using whole-genome deep sequencing of pools of the most resistant and most susceptible individuals of a large (n = 7484) biparental F2 population segregating for resistance to Xtg. Using chromosome-level genome assemblies as references, we were able to define a ~300 kb region highly associated with resistance on pseudo-chromosome 4. Further investigation of this region revealed multiple genes with a known role in disease resistance, including genes encoding for Pik2-like disease resistance proteins, cysteine-rich kinases, and RGA4- and RGA5-like disease resistance proteins. Investigation of allele frequencies in the pools and comparative genome analysis in the grandparents of the F2 population revealed that some of these genes contain variants with allele frequencies that correspond to the expected heterozygosity in the resistant grandparent. This study emphasizes the efficacy of combining BSA studies in very large populations with whole genome deep sequencing and high-quality genome assemblies to pinpoint regions associated with a binary trait of interest and accurately define a small set of candidate genes. Furthermore, markers identified in this region hold significant potential for marker-assisted breeding strategies to breed resistance to Xtg in Italian ryegrass cultivars more efficiently.

Pan-genome inversion index reveals evolutionary insights into the subpopulation structure of Asian rice.

Understanding and exploiting genetic diversity is a key factor for the productive and stable production of rice. Here, we utilize 73 high-quality genomes that encompass the subpopulation structure of Asian rice (Oryza sativa), plus the genomes of two wild relatives (O. rufipogon and O. punctata), to build a pan-genome inversion index of 1769 non-redundant inversions that span an average of ~29% of the O. sativa cv. Nipponbare reference genome sequence. Using this index, we estimate an inversion rate of ~700 inversions per million years in Asian rice, which is 16 to 50 times higher than previously estimated for plants. Detailed analyses of these inversions show evidence of their effects on gene expression, recombination rate, and linkage disequilibrium. Our study uncovers the prevalence and scale of large inversions (≥100 bp) across the pan-genome of Asian rice and hints at their largely unexplored role in functional biology and crop performance.

Fine-Mapping and Comparative Genomic Analysis Reveal the Gene Composition at the S and Z Self-incompatibility Loci in Grasses.

Self-incompatibility (SI) is a genetic mechanism of hermaphroditic plants to prevent inbreeding after self-pollination. Allogamous Poaceae species exhibit a unique gametophytic SI system controlled by two multi-allelic and independent loci, S and Z. Despite intense research efforts in the last decades, the genes that determine the initial recognition mechanism are yet to be identified. Here, we report the fine-mapping of the Z-locus in perennial ryegrass (Lolium perenne L.) and provide evidence that the pollen and stigma components are determined by two genes encoding DUF247 domain proteins (ZDUF247-I and ZDUF247-II) and the gene sZ, respectively. The pollen and stigma determinants are located side-by-side and were genetically linked in 10,245 individuals of two independent mapping populations segregating for Z. Moreover, they exhibited high allelic diversity as well as tissue-specific gene expression, matching the expected characteristics of SI determinants known from other systems. Revisiting the S-locus using the latest high-quality whole-genome assemblies revealed a similar gene composition and structure as found for Z, supporting the hypothesis of a duplicated origin of the two-locus SI system of grasses. Ultimately, comparative genomic analyses across a wide range of self-compatible and self-incompatible Poaceae species revealed that the absence of a functional copy of at least one of the six putative SI determinants is accompanied by a self-compatible phenotype. Our study provides new insights into the origin and evolution of the unique gametophytic SI system in one of the largest and economically most important plant families.

Low-Input High-Molecular-Weight DNA Extraction for Long-Read Sequencing From Plants of Diverse Families.

Long-read DNA sequencing technologies require high molecular weight (HMW) DNA of adequate purity and integrity, which can be difficult to isolate from plant material. Plant leaves usually contain high levels of carbohydrates and secondary metabolites that can impact DNA purity, affecting downstream applications. Several protocols and kits are available for HMW DNA extraction, but they usually require a high amount of input material and often lead to substantial DNA fragmentation, making sequencing suboptimal in terms of read length and data yield. We here describe a protocol for plant HMW DNA extraction from low input material (0.1 g) which is easy to follow and quick (2.5 h). This method successfully enabled us to extract HMW from four species from different families (Orchidaceae, Poaceae, Brassicaceae, Asteraceae). In the case of recalcitrant species, we show that an additional purification step is sufficient to deliver a clean DNA sample. We demonstrate the suitability of our protocol for long-read sequencing on the Oxford Nanopore Technologies PromethION® platform, with and without the use of a short fragment depletion kit.

Origin and Diversification of the Saguaro Cactus (Carnegiea gigantea): A Within-Species Phylogenomic Analysis.

Reconstructing accurate historical relationships within a species poses numerous challenges, not least in many plant groups in which gene flow is high enough to extend well beyond species boundaries. Nonetheless, the extent of tree-like history within a species is an empirical question on which it is now possible to bring large amounts of genome sequence to bear. We assess phylogenetic structure across the geographic range of the saguaro cactus, an emblematic member of Cactaceae, a clade known for extensive hybridization and porous species boundaries. Using 200 Gb of whole genome resequencing data from 20 individuals sampled from 10 localities, we assembled two data sets comprising 150,000 biallelic single nucleotide polymorphisms (SNPs) from protein coding sequences. From these, we inferred within-species trees and evaluated their significance and robustness using five qualitatively different inference methods. Despite the low sequence diversity, large census population sizes, and presence of wide-ranging pollen and seed dispersal agents, phylogenetic trees were well resolved and highly consistent across both data sets and all methods. We inferred that the most likely root, based on marginal likelihood comparisons, is to the east and south of the region of highest genetic diversity, which lies along the coast of the Gulf of California in Sonora, Mexico. Together with striking decreases in marginal likelihood found to the north, this supports hypotheses that saguaro's current range reflects postglacial expansion from the refugia in the south of its range. We conclude with observations about practical and theoretical issues raised by phylogenomic data sets within species, in which SNP-based methods must be used rather than gene tree methods that are widely used when sequence divergence is higher. These include computational scalability, inference of gene flow, and proper assessment of statistical support in the presence of linkage effects. [Phylogenomics; phylogeography; rooting; Sonoran Desert.].

Identification of Candidate Genes for Self-Compatibility in Perennial Ryegrass (Lolium perenne L.).

Self-incompatibility (SI) is a genetic mechanism preventing self-pollination in ~40% of plant species. Two multiallelic loci, called S and Z, control the gametophytic SI system of the grass family (Poaceae), which contains all major forage grasses. Loci independent from S and Z have been reported to disrupt SI and lead to self-compatibility (SC). A locus causing SC in perennial ryegrass (Lolium perenne L.) was previously mapped on linkage group (LG) 5 in an F2 population segregating for SC. Using a subset of the same population (n = 68), we first performed low-resolution quantitative trait locus (QTL) mapping to exclude the presence of additional, previously undetected contributors to SC. The previously reported QTL on LG 5 explained 38.4% of the phenotypic variation, and no significant contribution from other genomic regions was found. This was verified by the presence of significantly distorted markers in the region overlapping with the QTL. Second, we fine mapped the QTL to 0.26 centimorgan (cM) using additional 2,056 plants and 23 novel sequence-based markers. Using Italian ryegrass (Lolium multiflorum Lam.) genome assembly as a reference, the markers flanking SC were estimated to span a ~3 Mb region encoding for 57 predicted genes. Among these, seven genes were proposed as relevant candidate genes based on their annotation and function described in previous studies. Our study is a step forward to identify SC genes in forage grasses and provides diagnostic markers for marker-assisted introgression of SC into elite germplasm.

The genome of Shorea leprosula (Dipterocarpaceae) highlights the ecological relevance of drought in aseasonal tropical rainforests.

Hyperdiverse tropical rainforests, such as the aseasonal forests in Southeast Asia, are supported by high annual rainfall. Its canopy is dominated by the species-rich tree family of Dipterocarpaceae (Asian dipterocarps), which has both ecological (e.g., supports flora and fauna) and economical (e.g., timber production) importance. Recent ecological studies suggested that rare irregular drought events may be an environmental stress and signal for the tropical trees. We assembled the genome of a widespread but near threatened dipterocarp, Shorea leprosula, and analyzed the transcriptome sequences of ten dipterocarp species representing seven genera. Comparative genomic and molecular dating analyses suggested a whole-genome duplication close to the Cretaceous-Paleogene extinction event followed by the diversification of major dipterocarp lineages (i.e. Dipterocarpoideae). Interestingly, the retained duplicated genes were enriched for genes upregulated by no-irrigation treatment. These findings provide molecular support for the relevance of drought for tropical trees despite the lack of an annual dry season.

Multiple Wheat Genomes Reveal Novel Gli-2 Sublocus Location and Variation of Celiac Disease Epitopes in Duplicated α-Gliadin Genes.

The seed protein α-gliadin is a major component of wheat flour and causes gluten-related diseases. However, due to the complexity of this multigene family with a genome structure composed of dozens of copies derived from tandem and genome duplications, little was known about the variation between accessions, and thus little effort has been made to explicitly target α-gliadin for bread wheat breeding. Here, we analyzed genomic variation in α-gliadins across 11 recently published chromosome-scale assemblies of hexaploid wheat, with validation using long-read data. We unexpectedly found that the Gli-B2 locus is not a single contiguous locus but is composed of two subloci, suggesting the possibility of recombination between the two during breeding. We confirmed that the number of immunogenic epitopes among 11 accessions varied. The D subgenome of a European spelt line also contained epitopes, in agreement with its hybridization history. Evolutionary analysis identified amino acid sites under diversifying selection, suggesting their functional importance. The analysis opens the way for improved grain quality and safety through wheat breeding.

Ultralong Oxford Nanopore Reads Enable the Development of a Reference-Grade Perennial Ryegrass Genome Assembly.

Despite the progress made in DNA sequencing over the last decade, reconstructing telomere-to-telomere genome assemblies of large and repeat-rich eukaryotic genomes is still difficult. More accurate basecalls or longer reads could address this issue, but no current sequencing platform can provide both simultaneously. Perennial ryegrass (Lolium perenne L.) is an example of an important species for which the lack of a reference genome assembly hindered a swift adoption of genomics-based methods into breeding programs. To fill this gap, we optimized the Oxford Nanopore Technologies' sequencing protocol, obtaining sequencing reads with an N50 of 62 kb-a very high value for a plant sample. The assembly of such reads produced a highly complete (2.3 of 2.7 Gb), correct (QV 45), and contiguous (contig N50 and N90 11.74 and 3.34 Mb, respectively) genome assembly. We show how read length was key in determining the assembly contiguity. Sequence annotation revealed the dominance of transposable elements and repeated sequences (81.6% of the assembly) and identified 38,868 protein coding genes. Almost 90% of the bases could be anchored to seven pseudomolecules, providing the first high-quality haploid reference assembly for perennial ryegrass. This protocol will enable producing longer Oxford Nanopore Technology reads for more plant samples and ushering forage grasses into modern genomics-assisted breeding programs.

Reciprocal allopolyploid grasses (Festuca × Lolium) display stable patterns of genome dominance.

Allopolyploidization entailing the merger of two distinct genomes in a single hybrid organism, is an important process in plant evolution and a valuable tool in breeding programs. Newly established hybrids often experience massive genomic perturbations, including karyotype reshuffling and gene expression modifications. These phenomena may be asymmetric with respect to the two progenitors, with one of the parental genomes being "dominant." Such "genome dominance" can manifest in several ways, including biased homoeolog gene expression and expression level dominance. Here we employed a k-mer-based approach to study gene expression in reciprocal Festuca pratensis Huds. × Lolium multiflorum Lam. allopolyploid grasses. Our study revealed significantly more genes where expression mimicked that of the Lolium parent compared with the Festuca parent. This genome dominance was heritable to successive generation and its direction was only slightly modified by environmental conditions and plant age. Our results suggest that Lolium genome dominance was at least partially caused by its more efficient trans-acting gene expression regulatory factors. Unraveling the mechanisms responsible for propagation of parent-specific traits in hybrid crops contributes to our understanding of allopolyploid genome evolution and opens a way to targeted breeding strategies.

A chromosome-scale assembly of allotetraploid Brassica juncea (AABB) elucidates comparative architecture of the A and B genomes.

Brassica juncea (AABB), commonly referred to as mustard, is a natural allopolyploid of two diploid species-B. rapa (AA) and B. nigra (BB). We report a highly contiguous genome assembly of an oleiferous type of B. juncea variety Varuna, an archetypical Indian gene pool line of mustard, with ~100× PacBio single-molecule real-time (SMRT) long reads providing contigs with an N50 value of >5 Mb. Contigs were corrected for the misassemblies and scaffolded with BioNano optical mapping. We also assembled a draft genome of B. nigra (BB) variety Sangam using Illumina short-read sequencing and Oxford Nanopore long reads and used it to validate the assembly of the B genome of B. juncea. Two different linkage maps of B. juncea, containing a large number of genotyping-by-sequencing markers, were developed and used to anchor scaffolds/contigs to the 18 linkage groups of the species. The resulting chromosome-scale assembly of B. juncea Varuna is a significant improvement over the previous draft assembly of B. juncea Tumida, a vegetable type of mustard. The assembled genome was characterized for transposons, centromeric repeats, gene content and gene block associations. In comparison to the A genome, the B genome contains a significantly higher content of LTR/Gypsy retrotransposons, distinct centromeric repeats and a large number of B. nigra specific gene clusters that break the gene collinearity between the A and the B genomes. The B. juncea Varuna assembly will be of major value to the breeding work on oleiferous types of mustard that are grown extensively in south Asia and elsewhere.

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.

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.

Evolution and Diversity of the Wild Rice Oryza officinalis Complex, across Continents, Genome Types, and Ploidy Levels.

The Oryza officinalis complex is the largest species group in Oryza, with more than nine species from four continents, and is a tertiary gene pool that can be exploited in breeding programs for the improvement of cultivated rice. Most diploid and tetraploid members of this group have a C genome. Using a new reference C genome for the diploid species O. officinalis, and draft genomes for two other C genome diploid species Oryza eichingeri and Oryza rhizomatis, we examine the influence of transposable elements on genome structure and provide a detailed phylogeny and evolutionary history of the Oryza C genomes. The O. officinalis genome is 1.6 times larger than the A genome of cultivated Oryza sativa, mostly due to proliferation of Gypsy type long-terminal repeat transposable elements, but overall syntenic relationships are maintained with other Oryza genomes (A, B, and F). Draft genome assemblies of the two other C genome diploid species, Oryza eichingeri and Oryza rhizomatis, and short-read resequencing of a series of other C genome species and accessions reveal that after the divergence of the C genome progenitor, there was still a substantial degree of variation within the C genome species through proliferation and loss of both DNA and long-terminal repeat transposable elements. We provide a detailed phylogeny and evolutionary history of the Oryza C genomes and a genomic resource for the exploitation of the Oryza tertiary gene pool.

Evolution of l-DOPA 4,5-dioxygenase activity allows for recurrent specialisation to betalain pigmentation in Caryophyllales.

The evolution of l-DOPA 4,5-dioxygenase activity, encoded by the gene DODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that l-DOPA 4,5-dioxygenase activity evolved via a single Caryophyllales-specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear. To address this, we functionally characterised 23 distinct DODA proteins for l-DOPA 4,5-dioxygenase activity, from four betalain-pigmented and five anthocyanin-pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of l-DOPA 4,5-dioxygenase activity. We find that low l-DOPA 4,5-dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated l-DOPA 4,5-dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro-synteny. In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated l-DOPA 4,5-dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.

Gene duplication and transposition of mobile elements drive evolution of the Rpv3 resistance locus in grapevine.

A wild grape haplotype (Rpv3-1) confers resistance to Plasmopara viticola. We mapped the causal factor for resistance to an interval containing a TIR-NB-LRR (TNL) gene pair that originated 1.6-2.6 million years ago by a tandem segmental duplication. Transient coexpression of the TNL pair in Vitis vinifera leaves activated pathogen-induced necrosis and reduced sporulation compared with control leaves. Even though transcripts of the TNL pair from the wild haplotype appear to be partially subject to nonsense-mediated mRNA decay, mature mRNA levels in a homozygous resistant genotype were individually higher than the mRNA trace levels observed for the orthologous single-copy TNL in sensitive genotypes. Allelic expression imbalance in a resistant heterozygote confirmed that cis-acting regulatory variation promotes expression in the wild haplotype. The movement of transposable elements had a major impact on the generation of haplotype diversity, altering the DNA context around similar TNL coding sequences and the GC-content in their proximal 5'-intergenic regions. The wild and domesticated haplotypes also diverged in conserved single-copy intergenic DNA, but the highest divergence was observed in intraspecific and not in interspecific comparisons. In this case, introgression breeding did not transgress the genetic boundaries of the domesticated species, because haplotypes present in modern varieties sometimes predate speciation events between wild and cultivated species.

Genome sequence of the model rice variety KitaakeX.

BACKGROUND: The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to transform and propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species. RESULTS: Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics. CONCLUSIONS: The high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.