Navigating rice seedling cold resilience: QTL mapping in two inbred line populations and the search for genes.

Due to global climate change resulting in extreme temperature fluctuations, it becomes increasingly necessary to explore the natural genetic variation in model crops such as rice to facilitate the breeding of climate-resilient cultivars. To uncover genomic regions in rice involved in managing cold stress tolerance responses and to identify associated cold tolerance genes, two inbred line populations developed from crosses between cold-tolerant and cold-sensitive parents were used for quantitative trait locus (QTL) mapping of two traits: degree of membrane damage after 1 week of cold exposure quantified as percent electrolyte leakage (EL) and percent low-temperature seedling survivability (LTSS) after 1 week of recovery growth. This revealed four EL QTL and 12 LTSS QTL, all overlapping with larger QTL regions previously uncovered by genome-wide association study (GWAS) mapping approaches. Within the QTL regions, 25 cold-tolerant candidate genes were identified based on genomic differences between the cold-tolerant and cold-sensitive parents. Of those genes, 20% coded for receptor-like kinases potentially involved in signal transduction of cold tolerance responses; 16% coded for transcription factors or factors potentially involved in regulating cold tolerance response effector genes; and 64% coded for protein chaperons or enzymes potentially serving as cold tolerance effector proteins. Most of the 25 genes were cold temperature regulated and had deleterious nucleotide variants in the cold-sensitive parent, which might contribute to its cold-sensitive phenotype.

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.

Assessment of Rice Sheath Blight Resistance Including Associations with Plant Architecture, as Revealed by Genome-Wide Association Studies.

BACKGROUND: Sheath blight (ShB) disease caused by Rhizoctonia solani Kühn, is one of the most economically damaging rice (Oryza sativa L.) diseases worldwide. There are no known major resistance genes, leaving only partial resistance from small-effect QTL to deploy for cultivar improvement. Many ShB-QTL are associated with plant architectural traits detrimental to yield, including tall plants, late maturity, or open canopy from few or procumbent tillers, which confound detection of physiological resistance. RESULTS: To identify QTL for ShB resistance, 417 accessions from the Rice Diversity Panel 1 (RDP1), developed for association mapping studies, were evaluated for ShB resistance, plant height and days to heading in inoculated field plots in Arkansas, USA (AR) and Nanning, China (NC). Inoculated greenhouse-grown plants were used to evaluate ShB using a seedling-stage method to eliminate effects from height or maturity, and tiller (TN) and panicle number (PN) per plant. Potted plants were used to evaluate the RDP1 for TN and PN. Genome-wide association (GWA) mapping with over 3.4 million SNPs identified 21 targeted SNP markers associated with ShB which tagged 18 ShB-QTL not associated with undesirable plant architecture traits. Ten SNPs were associated with ShB among accessions of the Indica subspecies, ten among Japonica subspecies accessions, and one among all RDP1 accessions. Across the 18 ShB QTL, only qShB4-1 was not previously reported in biparental mapping studies and qShB9 was not reported in the GWA ShB studies. All 14 PN QTL overlapped with TN QTL, with 15 total TN QTL identified. Allele effects at the five TN QTL co-located with ShB QTL indicated that increased TN does not inevitably increase disease development; in fact, for four ShB QTL that overlapped TN QTL, the alleles increasing resistance were associated with increased TN and PN, suggesting a desirable coupling of alleles at linked genes. CONCLUSIONS: Nineteen accessions identified as containing the most SNP alleles associated with ShB resistance for each subpopulation were resistant in both AR and NC field trials. Rice breeders can utilize these accessions and SNPs to develop cultivars with enhanced ShB resistance along with increased TN and PN for improved yield potential.

Genome-wide association mapping for grain manganese in rice (Oryza sativa L.) using a multi-experiment approach.

Manganese (Mn) is an essential trace element for plants and commonly contributes to human health; however, the understanding of the genes controlling natural variation in Mn in crop plants is limited. Here, the integration of two of genome-wide association study approaches was used to increase the identification of valuable quantitative trait loci (QTL) and candidate genes responsible for the concentration of grain Mn across 389 diverse rice cultivars grown in Arkansas and Texas, USA, in multiple years. Single-trait analysis was initially performed using three different SNP datasets. As a result, significant loci could be detected using the high-density SNP dataset. Based on the 5.2 M SNP dataset, major QTLs were located on chromosomes 3 and 7 for Mn containing six candidate genes. In addition, the phenotypic data of grain Mn concentration were combined from three flooded-field experiments from the two sites and 3 years using multi-experiment analysis based on the 5.2 M SNP dataset. Two previous QTLs on chromosome 3 were identified across experiments, whereas new Mn QTLs were identified that were not found in individual experiments, on chromosomes 3, 4, 9 and 11. OsMTP8.1 was identified in both approaches and is a good candidate gene that could be controlling grain Mn concentration. This work demonstrates the utilisation of multi-experiment analysis to identify constitutive QTLs and candidate genes associated with the grain Mn concentration. Hence, the approach should be advantageous to facilitate genomic breeding programmes in rice and other crops considering QTLs and genes associated with complex traits in natural populations.

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.].

Validation of Yield Component Traits Identified by Genome-Wide Association Mapping in a tropical japonica × tropical japonica Rice Biparental Mapping Population.

The Rice Diversity Panel 1 (RDP1) was developed for genome-wide association (GWA) studies to explore five rice ( L.) subpopulations (, , , , and ). The RDP1 was evaluated for over 30 traits, including agronomic, panicle architecture, seed, and disease traits and genotyped with 700,000 single nucleotide polymorphisms (SNPs). Most rice grown in the southern United States is and thus the diversity in this subpopulation is interesting to U.S. breeders. Among the RDP1 accessions, 'Estrela' and 'NSFTV199' are both phenotypically and genotypically diverse, thus making them excellent parents for a biparental mapping population. The objectives were to (i) ascertain the GWA QTLs from the RDP1 GWA studies that overlapped with the QTLs uncovered in an Estrela × NSFTV199 recombinant inbred line (RIL) population evaluated for 15 yield traits, and (ii) identify known or novel genes potentially controlling specific yield component traits. The 256 RILs were genotyped with 132 simple sequence repeat markers and 70 QTLs were found. Perl scripts were developed for automatic identification of the underlying candidate genes in the GWA QTL regions. Approximately 100 GWA QTLs overlapped with 41 Estrela × NSFTV199 QTL (RIL QTL) regions and 47 known genes were identified. Two seed trait RIL QTLs with overlapping GWA QTLs were not associated with a known gene. Segregating SNPs in the overlapping GWA QTLs for RIL QTLs with high values will be evaluated as potential DNA markers useful to breeding programs for the associated yield trait.

The unique epigenetic features of Pack-MULEs and their impact on chromosomal base composition and expression spectrum.

Acquisition and rearrangement of host genes by transposable elements (TEs) is an important mechanism to increase gene diversity as exemplified by the ∼3000 Pack-Mutator-like TEs in the rice genome which have acquired gene sequences (Pack-MULEs), yet remain enigmatic. To identify signatures of functioning Pack-MULEs and Pack-MULE evolution, we generated transcriptome, translatome, and epigenome datasets and compared Pack-MULEs to genes and other TE families. Approximately 40% of Pack-MULEs were transcribed with 9% having translation evidence, clearly distinguishing them from other TEs. Pack-MULEs exhibited a unique expression profile associated with specificity in reproductive tissues that may be associated with seed traits. Expressed Pack-MULEs resemble regular protein-coding genes as exhibited by a low level of DNA methylation, association with active histone marks and DNase I hypersensitive sites, and an absence of repressive histone marks, suggesting that a substantial fraction of Pack-MULEs are potentially functional in vivo. Interestingly, the expression capacity of Pack-MULEs is independent of the local genomic environment, and the insertion and expression of Pack-MULEs may have altered the local chromosomal expression pattern as well as counteracted the impact of recombination on chromosomal base composition, which has profound consequences on the evolution of chromosome structure.

Assessment of Five Chilling Tolerance Traits and GWAS Mapping in Rice Using the USDA Mini-Core Collection.

Rice (Oryza sativa L.) is often exposed to cool temperatures during spring planting in temperate climates. A better understanding of genetic pathways regulating chilling tolerance will enable breeders to develop varieties with improved tolerance during germination and young seedling stages. To dissect chilling tolerance, five assays were developed; one assay for the germination stage, one assay for the germination and seedling stage, and three for the seedling stage. Based on these assays, five chilling tolerance indices were calculated and assessed using 202 O. sativa accessions from the Rice Mini-Core (RMC) collection. Significant differences between RMC accessions made the five indices suitable for genome-wide association study (GWAS) based quantitative trait loci (QTL) mapping. For young seedling stage indices, japonica and indica subspecies clustered into chilling tolerant and chilling sensitive accessions, respectively, while both subspecies had similar low temperature germinability distributions. Indica subspecies were shown to have chilling acclimation potential. GWAS mapping uncovered 48 QTL at 39 chromosome regions distributed across all 12 rice chromosomes. Interestingly, there was no overlap between the germination and seedling stage QTL. Also, 18 QTL and 32 QTL were in regions discovered in previously reported bi-parental and GWAS based QTL mapping studies, respectively. Two novel low temperature seedling survivability (LTSS)-QTL, qLTSS3-4 and qLTSS4-1, were not in a previously reported QTL region. QTL with strong effect alleles identified in this study will be useful for marker assisted breeding efforts to improve chilling tolerance in rice cultivars and enhance gene discovery for chilling tolerance.

Genetic architecture of cold tolerance in rice (Oryza sativa) determined through high resolution genome-wide analysis.

Cold temperature is an important abiotic stress which negatively affects morphological development and seed production in rice (Oryza sativa L.). At the seedling stage, cold stress causes poor germination, seedling injury and poor stand establishment; and at the reproductive stage cold decreases seed yield. The Rice Diversity Panel 1 (RDP1) is a global collection of over 400 O. sativa accessions representing the five major subpopulations from the INDICA and JAPONICA varietal groups, with a genotypic dataset consisting of 700,000 SNP markers. The objectives of this study were to evaluate the RDP1 accessions for the complex, quantitatively inherited cold tolerance traits at the germination and reproductive stages, and to conduct genome-wide association (GWA) mapping to identify SNPs and candidate genes associated with cold stress at these stages. GWA mapping of the germination index (calculated as percent germination in cold divided by warm treatment) revealed 42 quantitative trait loci (QTLs) associated with cold tolerance at the seedling stage, including 18 in the panel as a whole, seven in temperate japonica, six in tropical japonica, 14 in JAPONICA, and nine in INDICA, with five shared across all subpopulations. Twenty-two of these QTLs co-localized with 32 previously reported cold tolerance QTLs. GWA mapping of cold tolerance at the reproductive stage detected 29 QTLs, including seven associated with percent sterility, ten with seed weight per panicle, 14 with seed weight per plant and one region overlapping for two traits. Fifteen co-localized with previously reported QTLs for cold tolerance or yield components. Candidate gene ontology searches revealed these QTLs were associated with significant enrichment for genes related to with lipid metabolism, response to stimuli, response to biotic stimuli (suggesting cross-talk between biotic and abiotic stresses), and oxygen binding. Overall the JAPONICA accessions were more tolerant to cold stress than INDICA accessions.

Genome wide association mapping of grain arsenic, copper, molybdenum and zinc in rice (Oryza sativa L.) grown at four international field sites.

The mineral concentrations in cereals are important for human health, especially for individuals who consume a cereal subsistence diet. A number of elements, such as zinc, are required within the diet, while some elements are toxic to humans, for example arsenic. In this study we carry out genome-wide association (GWA) mapping of grain concentrations of arsenic, copper, molybdenum and zinc in brown rice using an established rice diversity panel of ∼ 300 accessions and 36.9 k single nucleotide polymorphisms (SNPs). The study was performed across five environments: one field site in Bangladesh, one in China and two in the US, with one of the US sites repeated over two years. GWA mapping on the whole dataset and on separate subpopulations of rice revealed a large number of loci significantly associated with variation in grain arsenic, copper, molybdenum and zinc. Seventeen of these loci were detected in data obtained from grain cultivated in more than one field location, and six co-localise with previously identified quantitative trait loci. Additionally, a number of candidate genes for the uptake or transport of these elements were located near significantly associated SNPs (within 200 kb, the estimated global linkage disequilibrium previously employed in this rice panel). This analysis highlights a number of genomic regions and candidate genes for further analysis as well as the challenges faced when mapping environmentally-variable traits in a highly genetically structured diversity panel.

Variation in grain arsenic assessed in a diverse panel of rice (Oryza sativa) grown in multiple sites.

• Inorganic arsenic (As(i) ) in rice (Oryza sativa) grains is a possible threat to human health, with risk being strongly linked to total dietary rice consumption and consumed rice As(i) content. This study aimed to identify the range and stability of genetic variation in grain arsenic (As) in rice. • Six field trials were conducted (one each in Bangladesh and China, two in Arkansas, USA over 2 yr, and two in Texas, USA comparing flooded and nonflood treatments) on a large number of common rice cultivars (c. 300) representing genetic diversity among international rice cultivars. • Within each field there was a 3-34 fold range in grain As concentration which varied between rice subpopulations. Importantly, As(i) correlated strongly with total As among a subset of 40 cultivars harvested in Bangladesh and China. • Genetic variation at all field sites was a large determining factor for grain As concentration, indicating that cultivars low in grain As could be developed through breeding. The temperate japonicas exhibited lower grain As compared with other subpopulations. Effects for year, location and flooding management were also statistically significant, suggesting that breeding strategies must take into account environmental factors.

Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa.

Asian rice, Oryza sativa is a cultivated, inbreeding species that feeds over half of the world's population. Understanding the genetic basis of diverse physiological, developmental, and morphological traits provides the basis for improving yield, quality and sustainability of rice. Here we show the results of a genome-wide association study based on genotyping 44,100 SNP variants across 413 diverse accessions of O. sativa collected from 82 countries that were systematically phenotyped for 34 traits. Using cross-population-based mapping strategies, we identified dozens of common variants influencing numerous complex traits. Significant heterogeneity was observed in the genetic architecture associated with subpopulation structure and response to environment. This work establishes an open-source translational research platform for genome-wide association studies in rice that directly links molecular variation in genes and metabolic pathways with the germplasm resources needed to accelerate varietal development and crop improvement.