Multitrait engineering of Hassawi red rice for sustainable cultivation.

Sustainable agriculture requires locally adapted varieties that produce nutritious food with limited agricultural inputs. Genome engineering represents a viable approach to develop cultivars that fulfill these criteria. For example, the red Hassawi rice, a native landrace of Saudi Arabia, tolerates local drought and high-salinity conditions and produces grain with diverse health-promoting phytochemicals. However, Hassawi has a long growth cycle, high cultivation costs, low productivity, and susceptibility to lodging. Here, to improve these undesirable traits via genome editing, we established efficient regeneration and Agrobacterium-mediated transformation protocols for Hassawi. In addition, we generated the first high-quality reference genome and targeted the key flowering repressor gene, Hd4, thus shortening the plant's lifecycle and height. Using CRISPR/Cas9 multiplexing, we simultaneously disrupted negative regulators of flowering time (Hd2, Hd4, and Hd5), grain size (GS3), grain number (GN1a), and plant height (Sd1). The resulting homozygous mutant lines flowered extremely early (∼56 days) and had shorter stems (approximately 107 cm), longer grains (by 5.1%), and more grains per plant (by 50.2%), thereby enhancing overall productivity. Furthermore, the awns of grains were 86.4% shorter compared to unedited plants. Moreover, the modified rice grain displayed improved nutritional attributes. As a result, the modified Hassawi rice combines several desirable traits that can incentivize large-scale cultivation and reduce malnutrition.

A high-performance computational workflow to accelerate GATK SNP detection across a 25-genome dataset.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) are the most widely used form of molecular genetic variation studies. As reference genomes and resequencing data sets expand exponentially, tools must be in place to call SNPs at a similar pace. The genome analysis toolkit (GATK) is one of the most widely used SNP calling software tools publicly available, but unfortunately, high-performance computing versions of this tool have yet to become widely available and affordable. RESULTS: Here we report an open-source high-performance computing genome variant calling workflow (HPC-GVCW) for GATK that can run on multiple computing platforms from supercomputers to desktop machines. We benchmarked HPC-GVCW on multiple crop species for performance and accuracy with comparable results with previously published reports (using GATK alone). Finally, we used HPC-GVCW in production mode to call SNPs on a "subpopulation aware" 16-genome rice reference panel with ~ 3000 resequenced rice accessions. The entire process took ~ 16 weeks and resulted in the identification of an average of 27.3 M SNPs/genome and the discovery of ~ 2.3 million novel SNPs that were not present in the flagship reference genome for rice (i.e., IRGSP RefSeq). CONCLUSIONS: This study developed an open-source pipeline (HPC-GVCW) to run GATK on HPC platforms, which significantly improved the speed at which SNPs can be called. The workflow is widely applicable as demonstrated successfully for four major crop species with genomes ranging in size from 400 Mb to 2.4 Gb. Using HPC-GVCW in production mode to call SNPs on a 25 multi-crop-reference genome data set produced over 1.1 billion SNPs that were publicly released for functional and breeding studies. For rice, many novel SNPs were identified and were found to reside within genes and open chromatin regions that are predicted to have functional consequences. Combined, our results demonstrate the usefulness of combining a high-performance SNP calling architecture solution with a subpopulation-aware reference genome panel for rapid SNP discovery and public deployment.

High-Quality Genome Assemblies Reveal Evolutionary Dynamics of Repetitive DNA and Structural Rearrangements in the Drosophila virilis Subgroup.

High-quality genome assemblies across a range of nontraditional model organisms can accelerate the discovery of novel aspects of genome evolution. The Drosophila virilis group has several attributes that distinguish it from more highly studied species in the Drosophila genus, such as an unusual abundance of repetitive elements and extensive karyotype evolution, in addition to being an attractive model for speciation genetics. Here, we used long-read sequencing to assemble five genomes of three virilis group species and characterized sequence and structural divergence and repetitive DNA evolution. We find that our contiguous genome assemblies allow characterization of chromosomal arrangements with ease and can facilitate analysis of inversion breakpoints. We also leverage a small panel of resequenced strains to explore the genomic pattern of divergence and polymorphism in this species and show that known demographic histories largely predicts the extent of genome-wide segregating polymorphism. We further find that a neo-X chromosome in Drosophila americana displays X-like levels of nucleotide diversity. We also found that unusual repetitive elements were responsible for much of the divergence in genome composition among species. Helitron-derived tandem repeats tripled in abundance on the Y chromosome in D. americana compared to Drosophila novamexicana, accounting for most of the difference in repeat content between these sister species. Repeats with characteristics of both transposable elements and satellite DNAs expanded by 3-fold, mostly in euchromatin, in both D. americana and D. novamexicana compared to D. virilis. Our results represent a major advance in our understanding of genome biology in this emerging model clade.

Oryza glumaepatula: A wild relative to improve drought tolerance in cultivated rice.

Developing drought-resistant rice (Oryza sativa, L.) is essential for improving field productivity, especially in rain-fed areas affected by climate change. Wild relatives of rice are potential sources for drought-resistant traits. Therefore, we compared root growth and drought response among 22 wild Oryza species, from which Oryza glumaepatula was selected as a promising source for further exploration. A geographically diverse panel of 69 O. glumaepatula accessions was then screened for drought stress-related traits, and 6 of these accessions showed lower shoot dry weight (SDW) reduction, greater percentage of deep roots, and lower stomatal density (STO) under drought than the drought tolerant O. sativa variety, Sahbhagi dhan. Based on whole-genome resequencing of all 69 O. glumaepatula accessions and variant calling to a high-quality O. glumaepatula reference genome, we detected multiple genomic loci colocating for SDW, root dry weight at 30 to 45 cm depth, and STO in consecutive drought trials. Geo-referencing indicated that the potential drought donors originated in flood-prone locations, corroborating previous hypotheses about the coexistence of flood and drought tolerance within individual Oryza genomes. These findings present potential donor accessions, traits, and genomic loci from an AA genome wild relative of rice that, together with the recently developed reference genome, may be useful for further introgression of drought tolerance into the O. sativa backgrounds.

Genome-wide association analysis identifies natural allelic variants associated with panicle architecture variation in African rice, Oryza glaberrima Steud.

African rice (Oryza glaberrima Steud), a short-day cereal crop closely related to Asian rice (Oryza sativa L.), has been cultivated in Sub-Saharan Africa for ∼ 3,000 years. Although less cultivated globally, it is a valuable genetic resource in creating high-yielding cultivars that are better adapted to diverse biotic and abiotic stresses. While inflorescence architecture, a key trait for rice grain yield improvement, has been extensively studied in Asian rice, the morphological and genetic determinants of this complex trait are less understood in African rice. In this study, using a previously developed association panel of 162 O. glaberrima accessions and new SNP variants characterized through mapping to a new version of the O. glaberrima reference genome, we conducted a genome-wide association study of four major morphological panicle traits. We have found a total of 41 stable genomic regions that are significantly associated with these traits, of which 13 co-localized with previously identified QTLs in O. sativa populations and 28 were unique for this association panel. Additionally, we found a genomic region of interest on chromosome 3 that was associated with the number of spikelets and primary and secondary branches. Within this region was localized the O. sativa ortholog of the PHYTOCHROME B gene (Oglab_006903/OgPHYB). Haplotype analysis revealed the occurrence of natural sequence variants at the OgPHYB locus associated with panicle architecture variation through modulation of the flowering time phenotype, whereas no equivalent alleles were found in O. sativa. The identification in this study of genomic regions specific to O. glaberrima indicates panicle-related intra-specific genetic variation in this species, increasing our understanding of the underlying molecular processes governing panicle architecture. Identified candidate genes and major haplotypes may facilitate the breeding of new African rice cultivars with preferred panicle traits.

High quality genome assemblies reveal evolutionary dynamics of repetitive DNA and structural rearrangements in the Drosophila virilis sub-group.

High-quality genome assemblies across a range of non-traditional model organisms can accelerate the discovery of novel aspects of genome evolution. The Drosophila virilis group has several attributes that distinguish it from more highly studied species in the Drosophila genus, such as an unusual abundance of repetitive elements and extensive karyotype evolution, in addition to being an attractive model for speciation genetics. Here we used long-read sequencing to assemble five genomes of three virilis group species and characterized sequence and structural divergence and repetitive DNA evolution. We find that our contiguous genome assemblies allow characterization of chromosomal arrangements with ease and can facilitate analysis of inversion breakpoints. We also leverage a small panel of resequenced strains to explore the genomic pattern of divergence and polymorphism in this species and show that known demographic histories largely predicts the extent of genome-wide segregating polymorphism. We further find that a neo-X chromosome in D. americana displays X-like levels of nucleotide diversity. We also found that unusual repetitive elements were responsible for much of the divergence in genome composition among species. Helitron-derived tandem repeats tripled in abundance on the Y chromosome in D. americana compared to D. novamexicana, accounting for most of the difference in repeat content between these sister species. Repeats with characteristics of both transposable elements and satellite DNAs expanded by three-fold, mostly in euchromatin, in both D. americana and D. novamexicana compared to D. virilis. Our results represent a major advance in our understanding of genome biology in this emerging model clade.

Multi-omics resources for targeted agronomic improvement of pigmented rice.

Pigmented rice (Oryza sativa L.) is a rich source of nutrients, but pigmented lines typically have long life cycles and limited productivity. Here we generated genome assemblies of 5 pigmented rice varieties and evaluated the genetic variation among 51 pigmented rice varieties by resequencing an additional 46 varieties. Phylogenetic analyses divided the pigmented varieties into four varietal groups: Geng-japonica, Xian-indica, circum-Aus and circum-Basmati. Metabolomics and ionomics profiling revealed that black rice varieties are rich in aromatic secondary metabolites. We established a regeneration and transformation system and used CRISPR-Cas9 to knock out three flowering time repressors (Hd2, Hd4 and Hd5) in the black Indonesian rice Cempo Ireng, resulting in an early maturing variety with shorter stature. Our study thus provides a multi-omics resource for understanding and improving Asian pigmented rice.

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.

Prediction of crossover recombination using parental genomes.

Meiotic recombination is a crucial cellular process, being one of the major drivers of evolution and adaptation of species. In plant breeding, crossing is used to introduce genetic variation among individuals and populations. While different approaches to predict recombination rates for different species have been developed, they fail to estimate the outcome of crossings between two specific accessions. This paper builds on the hypothesis that chromosomal recombination correlates positively to a measure of sequence identity. It presents a model that uses sequence identity, combined with other features derived from a genome alignment (including the number of variants, inversions, absent bases, and CentO sequences) to predict local chromosomal recombination in rice. Model performance is validated in an inter-subspecific indica x japonica cross, using 212 recombinant inbred lines. Across chromosomes, an average correlation of about 0.8 between experimental and prediction rates is achieved. The proposed model, a characterization of the variation of the recombination rates along the chromosomes, can enable breeding programs to increase the chances of creating novel allele combinations and, more generally, to introduce new varieties with a collection of desirable traits. It can be part of a modern panel of tools that breeders can use to reduce costs and execution times of crossing experiments.

FrangiPANe, a tool for creating a panreference using left behind reads.

We present here FrangiPANe, a pipeline developed to build panreference using short reads through a map-then-assemble strategy. Applying it to 248 African rice genomes using an improved CG14 reference genome, we identified an average of 8 Mb of new sequences and 5290 new contigs per individual. In total, 1.4 G of new sequences, consisting of 1 306 676 contigs, were assembled. We validated 97.7% of the contigs of the TOG5681 cultivar individual assembly from short reads on a newly long reads genome assembly of the same TOG5681 cultivar. FrangiPANe also allowed the anchoring of 31.5% of the new contigs within the CG14 reference genome, with a 92.5% accuracy at 2 kb span. We annotated in addition 3252 new genes absent from the reference. FrangiPANe was developed as a modular and interactive application to simplify the construction of a panreference using the map-then-assemble approach. It is available as a Docker image containing (i) a Jupyter notebook centralizing codes, documentation and interactive visualization of results, (ii) python scripts and (iii) all the software and libraries requested for each step of the analysis. We foreseen our approach will help leverage large-scale illumina dataset for pangenome studies in GWAS or detection of selection.

The gyrfalcon (Falco rusticolus) genome.

High-quality genome assemblies are characterized by high-sequence contiguity, completeness, and a low error rate, thus providing the basis for a wide array of studies focusing on natural species ecology, conservation, evolution, and population genomics. To provide this valuable resource for conservation projects and comparative genomics studies on gyrfalcon (Falco rusticolus), we sequenced and assembled the genome of this species using third-generation sequencing strategies and optical maps. Here, we describe a highly contiguous and complete genome assembly comprising 20 scaffolds and 13 contigs with a total size of 1.193 Gbp, including 8,064 complete Benchmarking Universal Single-Copy Orthologs (BUSCOs) of the total 8,338 BUSCO groups present in the library aves_odb10. Of these BUSCO genes, 96.7% were complete, 96.1% were present as a single copy, and 0.6% were duplicated. Furthermore, 0.8% of BUSCO genes were fragmented and 2.5% (210) were missing. A de novo search for transposable elements (TEs) identified 5,716 TEs that masked 7.61% of the F. rusticolus genome assembly when combined with publicly available TE collections. Long interspersed nuclear elements, in particular, the element Chicken-repeat 1 (CR1), were the most abundant TEs in the F. rusticolus genome. A de novo first-pass gene annotation was performed using 293,349 PacBio Iso-Seq transcripts and 496,195 transcripts derived from the assembly of 42,429,525 Illumina PE RNA-seq reads. In all, 19,602 putative genes, of which 59.31% were functionally characterized and associated with Gene Ontology terms, were annotated. A comparison of the gyrfalcon genome assembly with the publicly available assemblies of the domestic chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and hummingbird (Calypte anna) revealed several genome rearrangements. In particular, nine putative chromosome fusions were identified in the gyrfalcon genome assembly compared with those in the G. gallus genome assembly. This genome assembly, its annotation for TEs and genes, and the comparative analyses presented, complement and strength the base of high-quality genome assemblies and associated resources available for comparative studies focusing on the evolution, ecology, and conservation of Aves.

The International Oryza Map Alignment Project (IOMAP): the Americas-past achievements and future directions.

The wild relatives of rice hold unexplored genetic diversity that can be employed to feed an estimated population of 10 billion by 2050. The Oryza Map Alignment Project (OMAP) initiated in 2003 has provided comprehensive genomic resources for comparative, evolutionary, and functional characterization of the wild relatives of rice, facilitating the cloning of >600 rice genes, including those for grain width (GW5) and submergence tolerance (SUB1A). Following in the footsteps of the original project, the goal of 'IOMAP: the Americas' is to investigate the present and historic genetic diversity of wild Oryza species endemic to the Americas through the sequencing of herbaria and in situ specimens. The generation of a large diversity panel describing past and current genetic status and potential erosion of genetic variation in the populations will provide useful knowledge for the conservation of the biodiversity in these species. The wild relatives of rice in the Americas present a wide range of resistance traits useful for crop improvement and neodomestication approaches. In the race against time for a sustainable food future, the neodomestication of the first cereal species recently accomplished in O. alta opens the door to the potential neodomestication of the other wild Oryza species in Americas.

Full-Length Transcriptome Sequencing Reveals Alternative Splicing and lncRNA Regulation during Nodule Development in Glycine max.

Alternative splicing (AS) is a ubiquitous phenomenon among eukaryotic intron-containing genes, which greatly contributes to transcriptome and proteome diversity. Here we performed the isoform sequencing (Iso-Seq) of soybean underground tissues inoculated and uninoculated with Rhizobium and obtained 200,681 full-length transcripts covering 26,183 gene loci. It was found that 80.78% of the multi-exon loci produced more than one splicing variant. Comprehensive analysis of these identified 7874 differentially splicing events with highly diverse splicing patterns during nodule development, especially in defense and transport-related processes. We further profiled genes with differential isoform usage and revealed that 2008 multi-isoform loci underwent stage-specific or simultaneous major isoform switches after Rhizobium inoculation, indicating that AS is a vital way to regulate nodule development. Moreover, we took the lead in identifying 1563 high-confidence long non-coding RNAs (lncRNAs) in soybean, and 157 of them are differentially expressed during nodule development. Therefore, our study uncovers the landscape of AS during the soybean-Rhizobium interaction and provides systematic transcriptomic data for future study of multiple novel directions in soybean.

Gene fusion as an important mechanism to generate new genes in the genus Oryza.

BACKGROUND: Events of gene fusion have been reported in several organisms. However, the general role of gene fusion as part of new gene origination remains unknown. RESULTS: We conduct genome-wide interrogations of four Oryza genomes by designing and implementing novel pipelines to detect fusion genes. Based on the phylogeny of ten plant species, we detect 310 fusion genes across four Oryza species. The estimated rate of origination of fusion genes in the Oryza genus is as high as 63 fusion genes per species per million years, which is fixed at 16 fusion genes per species per million years and much higher than that in flies. By RNA sequencing analysis, we find more than 44% of the fusion genes are expressed and 90% of gene pairs show strong signals of purifying selection. Further analysis of CRISPR/Cas9 knockout lines indicates that newly formed fusion genes regulate phenotype traits including seed germination, shoot length and root length, suggesting the functional significance of these genes. CONCLUSIONS: We detect new fusion genes that may drive phenotype evolution in Oryza. This study provides novel insights into the genome evolution of Oryza.

Long-read genome sequencing of bread wheat facilitates disease resistance gene cloning.

The cloning of agronomically important genes from large, complex crop genomes remains challenging. Here we generate a 14.7 gigabase chromosome-scale assembly of the South African bread wheat (Triticum aestivum) cultivar Kariega by combining high-fidelity long reads, optical mapping and chromosome conformation capture. The resulting assembly is an order of magnitude more contiguous than previous wheat assemblies. Kariega shows durable resistance to the devastating fungal stripe rust disease1. We identified the race-specific disease resistance gene Yr27, which encodes an intracellular immune receptor, to be a major contributor to this resistance. Yr27 is allelic to the leaf rust resistance gene Lr13; the Yr27 and Lr13 proteins show 97% sequence identity2,3. Our results demonstrate the feasibility of generating chromosome-scale wheat assemblies to clone genes, and exemplify that highly similar alleles of a single-copy gene can confer resistance to different pathogens, which might provide a basis for engineering Yr27 alleles with multiple recognition specificities in the future.

Genome assembly of the JD17 soybean provides a new reference genome for comparative genomics.

Cultivated soybean (Glycine max) is an important source for protein and oil. Many elite cultivars with different traits have been developed for different conditions. Each soybean strain has its own genetic diversity, and the availability of more high-quality soybean genomes can enhance comparative genomic analysis for identifying genetic underpinnings for its unique traits. In this study, we constructed a high-quality de novo assembly of an elite soybean cultivar Jidou 17 (JD17) with chromosome contiguity and high accuracy. We annotated 52,840 gene models and reconstructed 74,054 high-quality full-length transcripts. We performed a genome-wide comparative analysis based on the reference genome of JD17 with 3 published soybeans (WM82, ZH13, and W05), which identified 5 large inversions and 2 large translocations specific to JD17, 20,984-46,912 presence-absence variations spanning 13.1-46.9 Mb in size. A total of 1,695,741-3,664,629 SNPs and 446,689-800,489 Indels were identified and annotated between JD17 and them. Symbiotic nitrogen fixation genes were identified and the effects from these variants were further evaluated. It was found that the coding sequences of 9 nitrogen fixation-related genes were greatly affected. The high-quality genome assembly of JD17 can serve as a valuable reference for soybean functional genomics research.

Rice domestication.

Rice is a staple food crop for more than one-third of the global population (http://www.sustainablerice.org/), of which 90% live at or near the poverty line. Thus, rice genetic improvement is important for global food security and is critical for enhancing socioeconomic benefits and reducing the environmental impacts of agriculture. In continued efforts to address the long-standing problem of food security and sustainable agriculture, scientists are utilizing genes from diverse varieties of rice to improve the resilience of rice to pests, diseases and environmental stress. This Primer describes the history of rice domestication, the importance of wild relatives of rice for crop improvement, and the domestication of wild species of rice not previously planted by farmers - a new approach called neodomestication.

Starch Synthesis-Related Genes (SSRG) Evolution in the Genus Oryza.

Cooking quality is an important attribute in Common/Asian rice (Oryzasativa L.) varieties, being highly dependent on grain starch composition. This composition is known to be highly dependent on a cultivar's genetics, but the way in which their genes express different phenotypes is not well understood. Further analysis of variation of grain quality genes using new information obtained from the wild relatives of rice should provide important insights into the evolution and potential use of these genetic resources. All analyses were conducted using bioinformatics approaches. The analysis of the protein sequences of grain quality genes across the Oryza suggest that the deletion/mutation of amino acids in active sites result in variations that can negatively affect specific steps of starch biosynthesis in the endosperm. On the other hand, the complete deletion of some genes in the wild species may not affect the amylose content. Here we present new insights for Starch Synthesis-Related Genes (SSRGs) evolution from starch-specific rice phenotypes.

Two gap-free reference genomes and a global view of the centromere architecture in rice.

Rice (Oryza sativa), a major staple throughout the world and a model system for plant genomics and breeding, was the first crop genome sequenced almost two decades ago. However, reference genomes for all higher organisms to date contain gaps and missing sequences. Here, we report the assembly and analysis of gap-free reference genome sequences for two elite O. sativa xian/indica rice varieties, Zhenshan 97 and Minghui 63, which are being used as a model system for studying heterosis and yield. Gap-free reference genomes provide the opportunity for a global view of the structure and function of centromeres. We show that all rice centromeric regions share conserved centromere-specific satellite motifs with different copy numbers and structures. In addition, the similarity of CentO repeats in the same chromosome is higher than across chromosomes, supporting a model of local expansion and homogenization. Both genomes have over 395 non-TE genes located in centromere regions, of which ∼41% are actively transcribed. Two large structural variants at the end of chromosome 11 affect the copy number of resistance genes between the two genomes. The availability of the two gap-free genomes lays a solid foundation for further understanding genome structure and function in plants and breeding climate-resilient varieties.