Identifying genes associated with abiotic stress tolerance suitable for CRISPR/Cas9 editing in upland rice cultivars adapted to acid soils.

Five genes of large phenotypic effect known to confer abiotic stress tolerance in rice were selected to characterize allelic variation in commercial Colombian tropical japonica upland rice cultivars adapted to drought-prone acid soil environments (cv. Llanura11 and Porvenir12). Allelic variants of the genes ART1, DRO1, SUB1A, PSTOL1, and SPDT were characterized by PCR and/or Sanger sequencing in the two upland cultivars and compared with the Nipponbare and other reference genomes. Two genes were identified as possible targets for gene editing: SUB1A (Submergence 1A), to improve tolerance to flooding, and SPDT (SULTR3;4) (SULTR-like Phosphorus Distribution Transporter), to improve phosphorus utilization efficiency and grain quality. Based on technical and regulatory considerations, SPDT was targeted for editing. The two upland cultivars were shown to carry the SPDT wild-type (nondesirable) allele based on sequencing, RNA expression, and phenotypic evaluations under hydroponic and greenhouse conditions. A gene deletion was designed using the CRISPR/Cas9 system, and specialized reagents were developed for SPDT editing, including vectors targeting the gene and a protoplast transfection transient assay. The desired edits were confirmed in protoplasts and serve as the basis for ongoing plant transformation experiments aiming to improve the P-use efficiency of upland rice grown in acidic soils.

Phenotypic Variation and the Impact of Admixture in the Oryza rufipogon Species Complex (ORSC).

Crop wild relatives represent valuable reservoirs of variation for breeding, but their populations are threatened in natural habitats, are sparsely represented in genebanks, and most are poorly characterized. The focus of this study is the Oryza rufipogon species complex (ORSC), wild progenitor of Asian rice (Oryza sativa L.). The ORSC comprises perennial, annual and intermediate forms which were historically designated as O. rufipogon, O. nivara, and O. sativa f. spontanea (or Oryza spp., an annual form of mixed O. rufipogon/O. nivara and O. sativa ancestry), respectively, based on non-standardized morphological, geographical, and/or ecologically-based species definitions and boundaries. Here, a collection of 240 diverse ORSC accessions, characterized by genotyping-by-sequencing (113,739 SNPs), was phenotyped for 44 traits associated with plant, panicle, and seed morphology in the screenhouse at the International Rice Research Institute, Philippines. These traits included heritable phenotypes often recorded as characterization data by genebanks. Over 100 of these ORSC accessions were also phenotyped in the greenhouse for 18 traits in Stuttgart, Arkansas, and 16 traits in Ithaca, New York, United States. We implemented a Bayesian Gaussian mixture model to infer accession groups from a subset of these phenotypic data and ascertained three phenotype-based group assignments. We used concordance between the genotypic subpopulations and these phenotype-based groups to identify a suite of phenotypic traits that could reliably differentiate the ORSC populations, whether measured in tropical or temperate regions. The traits provide insight into plant morphology, life history (perenniality versus annuality) and mating habit (self- versus cross-pollinated), and are largely consistent with genebank species designations. One phenotypic group contains predominantly O. rufipogon accessions characterized as perennial and largely out-crossing and one contains predominantly O. nivara accessions characterized as annual and largely inbreeding. From these groups, 42 "core" O. rufipogon and 25 "core" O. nivara accessions were identified for domestication studies. The third group, comprising 20% of our collection, has the most accessions identified as Oryza spp. (51.2%) and levels of O. sativa admixture accounting for more than 50% of the genome. This third group is potentially useful as a "pre-breeding" pool for breeders attempting to incorporate novel variation into elite breeding lines.

A Coordinated Suite of Wild-Introgression Lines in Indica and Japonica Elite Backgrounds.

Rice, Oryza sativa L., is a cultivated, inbreeding species that serves as the staple food for the largest number of people on earth. It has two strongly diverged varietal groups, Indica and Japonica, which result from a combination of natural and human selection. The genetic divergence of these groups reflects the underlying population structure of their wild ancestors, and suggests that a pre-breeding strategy designed to take advantage of existing genetic, geographic and ecological substructure may provide a rational approach to the utilization of crop wild ancestors in plant improvement. Here we describe the coordinated development of six introgression libraries (n = 63 to 81 lines per library) in both Indica (cv. IR64) and Japonica (cv. Cybonnet) backgrounds using three bio-geographically diverse wild donors representing the Oryza rufipogon Species Complex from China, Laos and Indonesia. The final libraries were genotyped using an Infinium 7K rice SNP array (C7AIR) and analyzed under greenhouse conditions for several simply inherited (Mendelian) traits. These six interspecific populations can be used as individual Chromosome Segment Substitution Line libraries and, when considered together, serve as a powerful genetic resource for systematic genetic dissection of agronomic, physiological and developmental traits in rice.

Corrigendum: A Coordinated Suite of Wild-Introgression Lines in Indica and Japonica Elite Backgrounds.

[This corrects the article DOI: 10.3389/fpls.2020.564824.].

Open access resources for genome-wide association mapping in rice.

Increasing food production is essential to meet the demands of a growing human population, with its rising income levels and nutritional expectations. To address the demand, plant breeders seek new sources of genetic variation to enhance the productivity, sustainability and resilience of crop varieties. Here we launch a high-resolution, open-access research platform to facilitate genome-wide association mapping in rice, a staple food crop. The platform provides an immortal collection of diverse germplasm, a high-density single-nucleotide polymorphism data set tailored for gene discovery, well-documented analytical strategies, and a suite of bioinformatics resources to facilitate biological interpretation. Using grain length, we demonstrate the power and resolution of our new high-density rice array, the accompanying genotypic data set, and an expanded diversity panel for detecting major and minor effect QTLs and subpopulation-specific alleles, with immediate implications for rice improvement.

Substitution mapping of dth1.1, a flowering-time quantitative trait locus (QTL) associated with transgressive variation in rice, reveals multiple sub-QTL.

A quantitative trait locus (QTL), dth1.1, was associated with transgressive variation for days to heading in an advanced backcross population derived from the Oryza sativa variety Jefferson and an accession of the wild rice relative Oryza rufipogon. A series of near-isogenic lines (NILs) containing different O. rufipogon introgressions across the target region were constructed to dissect dth1.1 using substitution mapping. In contrast to the late-flowering O. rufipogon parent, O. rufipogon alleles in the substitution lines caused early flowering under both short- and long-day lengths and provided evidence for at least two distinct sub-QTL: dth1.1a and dth1.1b. Potential candidate genes underlying these sub-QTL include genes with sequence similarity to Arabidopsis GI, FT, SOC1, and EMF1, and Pharbitis nil PNZIP. Evidence from families with nontarget O. rufipogon introgressions in combination with dth1.1 alleles also detected an early flowering QTL on chromosome 4 and a late-flowering QTL on chromosome 6 and provided evidence for additional sub-QTL in the dth1.1 region. The availability of a series of near-isogenic lines with alleles introgressed from a wild relative of rice provides an opportunity to better understand the molecular basis of transgressive variation in a quantitative trait.