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.

Loss of a Premature Stop Codon in the Rice Wall-Associated Kinase 91 (WAK91) Gene Is a Candidate for Improving Leaf Sheath Blight Disease Resistance.

Leaf sheath blight disease (SB) of rice caused by the soil-borne fungus Rhizoctonia solani results in 10-30% global yield loss annually and can reach 50% under severe outbreaks. Many disease resistance genes and receptor-like kinases (RLKs) are recruited early on by the host plant to respond to pathogens. Wall-associated receptor kinases (WAKs), a subfamily of receptor-like kinases, have been shown to play a role in fungal defense. The rice gene WAK91 (OsWAK91), co-located in the major SB resistance QTL region on chromosome 9, was identified by us as a candidate in defense against rice sheath blight. An SNP mutation T/C in the WAK91 gene was identified in the susceptible rice variety Cocodrie (CCDR) and the resistant line MCR010277 (MCR). The consequence of the resistant allele C is a stop codon loss, resulting in an open reading frame with extra 62 amino acid carrying a longer protein kinase domain and additional phosphorylation sites. Our genotype and phenotype analysis of the parents CCDR and MCR and the top 20 individuals of the double haploid SB population strongly correlate with the SNP. The susceptible allele T is present in the japonica subspecies and most tropical and temperate japonica lines. Multiple US commercial rice varieties with a japonica background carry the susceptible allele and are known for SB susceptibility. This discovery opens the possibility of introducing resistance alleles into high-yielding commercial varieties to reduce yield losses incurred by the sheath blight disease.

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.

Reference genome of the nutrition-rich orphan crop chia (Salvia hispanica) and its implications for future breeding.

Chia (Salvia hispanica L.) is one of the most popular nutrition-rich foods and pseudocereal crops of the family Lamiaceae. Chia seeds are a rich source of proteins, polyunsaturated fatty acids (PUFAs), dietary fibers, and antioxidants. In this study, we present the assembly of the chia reference genome, which spans 303.6 Mb and encodes 48,090 annotated protein-coding genes. Our analysis revealed that ~42% of the chia genome harbors repetitive content, and identified ~3 million single nucleotide polymorphisms (SNPs) and 15,380 simple sequence repeat (SSR) marker sites. By investigating the chia transcriptome, we discovered that ~44% of the genes undergo alternative splicing with a higher frequency of intron retention events. Additionally, we identified chia genes associated with important nutrient content and quality traits, such as the biosynthesis of PUFAs and seed mucilage fiber (dietary fiber) polysaccharides. Notably, this is the first report of in-silico annotation of a plant genome for protein-derived small bioactive peptides (biopeptides) associated with improving human health. To facilitate further research and translational applications of this valuable orphan crop, we have developed the Salvia genomics database (SalviaGDB), accessible at https://salviagdb.org.

Beyond gene ontology (GO): using biocuration approach to improve the gene nomenclature and functional annotation of rice S-domain kinase subfamily.

The S-domain subfamily of receptor-like kinases (SDRLKs) in plants is poorly characterized. Most members of this subfamily are currently assigned gene function based on the S-locus Receptor Kinase from Brassica that acts as the female determinant of self-incompatibility (SI). However, Brassica like SI mechanisms does not exist in most plants. Thus, automated Gene Ontology (GO) pipelines are not sufficient for functional annotation of SDRLK subfamily members and lead to erroneous association with the GO biological process of SI. Here, we show that manual bio-curation can help to correct and improve the gene annotations and association with relevant biological processes. Using publicly available genomic and transcriptome datasets, we conducted a detailed analysis of the expansion of the rice (Oryza sativa) SDRLK subfamily, the structure of individual genes and proteins, and their expression.The 144-member SDRLK family in rice consists of 82 receptor-like kinases (RLKs) (67 full-length, 15 truncated),12 receptor-like proteins, 14 SD kinases, 26 kinase-like and 10 GnK2 domain-containing kinases and RLKs. Except for nine genes, all other SDRLK family members are transcribed in rice, but they vary in their tissue-specific and stress-response expression profiles. Furthermore, 98 genes show differential expression under biotic stress and 98 genes show differential expression under abiotic stress conditions, but share 81 genes in common.Our analysis led to the identification of candidate genes likely to play important roles in plant development, pathogen resistance, and abiotic stress tolerance. We propose a nomenclature for 144 SDRLK gene family members based on gene/protein conserved structural features, gene expression profiles, and literature review. Our biocuration approach, rooted in the principles of findability, accessibility, interoperability and reusability, sets forth an example of how manual annotation of large-gene families can fill in the knowledge gap that exists due to the implementation of automated GO projections, thereby helping to improve the quality and contents of public databases.

Potential of Platinum Standard Reference Genomes to Exploit Natural Variation in the Wild Relatives of Rice.

As the world's population expands from 7.6 billion to 10 billion over the next 30 years, scientists and farmers across the globe must explore every angle necessary to provide a safe, stable and sustainable food supply for generations to come. Rice, and its wild relatives in the genus Oryza, will play a significant role in helping to solve this 10 billion people question due to its place as a staple food for billions. The genus Oryza is composed of 27 species that span 15 million years of evolutionary diversification and have been shown to contain a plethora of untapped adaptive traits, e.g., biotic and abiotic resistances, which can be used to improve cultivated rice. Such traits can be introduced into cultivated rice, in some cases by conventional crossing, and others via genetic transformation and gene editing methods. In cases where traits are too complex to easily transfer to cultivated rice [e.g., quantitative trait loci (QTL)], an alternative strategy is to domesticate the wild relative that already contains the desired adaptive traits - i.e., "neodomestication". To utilize the Oryza genus for crop improvement and neodomestication, we first need a set of genomic resources that can be used to efficiently identify, capture, and guide molecular crop improvement. Here, we introduce the concept of platinum standard reference genome sequences (PSRefSeq) - a new standard by which contiguous near-gap free reference genomes can now be produced. By having a set of PSRefSeqs for every Oryza species we set a new bar for how crop wild relatives can be integrated into crop improvement programs.

A platinum standard pan-genome resource that represents the population structure of Asian rice.

As the human population grows from 7.8 billion to 10 billion over the next 30 years, breeders must do everything possible to create crops that are highly productive and nutritious, while simultaneously having less of an environmental footprint. Rice will play a critical role in meeting this demand and thus, knowledge of the full repertoire of genetic diversity that exists in germplasm banks across the globe is required. To meet this demand, we describe the generation, validation and preliminary analyses of transposable element and long-range structural variation content of 12 near-gap-free reference genome sequences (RefSeqs) from representatives of 12 of 15 subpopulations of cultivated Asian rice. When combined with 4 existing RefSeqs, that represent the 3 remaining rice subpopulations and the largest admixed population, this collection of 16 Platinum Standard RefSeqs (PSRefSeq) can be used as a template to map resequencing data to detect virtually all standing natural variation that exists in the pan-genome of cultivated Asian rice.

Gramene 2018: unifying comparative genomics and pathway resources for plant research.

Gramene (http://www.gramene.org) is a knowledgebase for comparative functional analysis in major crops and model plant species. The current release, #54, includes over 1.7 million genes from 44 reference genomes, most of which were organized into 62,367 gene families through orthologous and paralogous gene classification, whole-genome alignments, and synteny. Additional gene annotations include ontology-based protein structure and function; genetic, epigenetic, and phenotypic diversity; and pathway associations. Gramene's Plant Reactome provides a knowledgebase of cellular-level plant pathway networks. Specifically, it uses curated rice reference pathways to derive pathway projections for an additional 66 species based on gene orthology, and facilitates display of gene expression, gene-gene interactions, and user-defined omics data in the context of these pathways. As a community portal, Gramene integrates best-of-class software and infrastructure components including the Ensembl genome browser, Reactome pathway browser, and Expression Atlas widgets, and undergoes periodic data and software upgrades. Via powerful, intuitive search interfaces, users can easily query across various portals and interactively analyze search results by clicking on diverse features such as genomic context, highly augmented gene trees, gene expression anatomograms, associated pathways, and external informatics resources. All data in Gramene are accessible through both visual and programmatic interfaces.