Genetic loci regulating the concentrations of anthocyanins and proanthocyanidins in the pericarps of purple and red rice.

The pigmented flavonoids, anthocyanins and proanthocyanidins, have health promoting properties. Previous work determined that the genes Pb and Rc turn on and off the biosynthesis of anthocyanins (purple) and proanthocyanidins (red), respectively. Not yet known is how the concentrations of these pigmented flavonoids are regulated in grain pericarps. Quantitative trait locus (QTL) analysis in a population of rice (Oryza sativa L.) F5 recombinant inbred lines from white pericarp "IR36ae" x red+purple pericarp "242" revealed three QTLs associated with grain concentrations of anthocyanins (TAC) or proanthocyanidins (PA). Both TAC and PA independently mapped to a 1.5 Mb QTL region on chromosome 3 between RM3400 (at 15.8 Mb) and RM15123 (17.3 Mb), named qPR3. Across 2 years, qPR3 explained 36.3% of variance in TAC and 35.8% in PA variance not attributable to Pb or Rc. The qPR3 region encompasses Kala3, a MYB transcription factor previously known to regulate purple grain characteristics. Study of PbPbRcrc progeny showed that TAC of RcRc near isogenic lines (NILs) was 2.1-4.5x that of rcrc. Similarly, study of PbPbRcRc NILs, which had 70% higher PA than pbpbRcRc NILs, revealed a mutual enhancement, not a trade-off between these compounds that share precursors. This suggests that Pb and Rc upregulate genes in a shared pathway as they activate TAC and PA synthesis, respectively. This study provides molecular markers for facilitating marker-assisted selection of qPR3, qPR5, and qPR7 to enhance grain concentrations of pigmented flavonoids and documented that stacking Rc and Pb genes further increases both flavonoid compounds.

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.

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.

Relationships Among Arsenic-Related Traits, Including Rice Grain Arsenic Concentration and Straighthead Resistance, as Revealed by Genome-Wide Association.

There is global concern that rice grains and foods can contain harmful amounts of arsenic (As), motivating breeders to produce cultivars that restrict As accumulation in grains to protect human health. Arsenic is also toxic to plants, with straighthead disorder (StHD), causing panicle sterility, being observed in rice. The genetic variation in StHD resistance suggests that plants have evolved mechanisms that reduce As toxicity, possibly via regulation of As uptake, transport, or detoxification/sequestration. Because these mechanisms could also underlie the wide (3- to 100-fold) differences in grain As concentration (grain-As) observed among diverse rice genotypes, it was hypothesized that some genes reduce both grain-As content and StHD susceptibility and may be detectable as co-located StDH and As quantitative trait loci (QTL). We used a machine-learning Bayesian network approach plus high-resolution genome-wide association study (GWAS) to identify QTL for grain-As and StHD resistance within the USDA Rice Minicore Collection (RMC). Arsenic enters roots through phosphorus (P) and silica (Si) transporters, As detoxification involves sulfur (S), and cell signaling to activate stress tolerance mechanisms is impacted by Si, calcium (Ca), and copper (Cu). Therefore, concentrations of Si, P, S, Ca, and Cu were included in this study to elucidate physiological mechanisms underlying grain-As and StHD QTL. Multiple QTL (from 9 to 33) were identified for each of the investigated As-associated traits. Although the QTL for StHD, Si, and grain-As did not overlap as heavily as our hypothesis predicted (4/33 StHD and 4/15 As QTL co-located), they do provide useful guidance to future research. Furthermore, these are the first StHD and Si QTL to be identified using high-density mapping, resulting in their being mapped to shorter, more precise genomic regions than previously reported QTL. The candidate genes identified provide guidance for future research, such as gene editing or mutation studies to further investigate the role of antioxidants and ROS scavenging to StHD resistance, as indicated by candidate genes around the commonly reported qStHD8-2 QTL. Other genes indicated for future study for improving grain-As and StHD include several multidrug and toxic compound extrusion (MATE) genes, F-box genes, and NIPs not documented to date to transport As.

Identification of Superior Alleles for Seedling Stage Salt Tolerance in the USDA Rice Mini-Core Collection.

Salt stress is a major constraint to rice acreage and production worldwide. The purpose of this study was to evaluate the natural genetic variation available in the United States Department of Agriculture (USDA) rice mini-core collection (URMC) for early vigor traits under salt stress and identify quantitative trait loci (QTLs) for seedling-stage salt tolerance via a genome-wide association study (GWAS). Using a hydroponic system, the seedlings of 162 accessions were subjected to electrical conductivity (EC) 6.0 dS m-1 salt stress at the three-to-four leaf stage. After completion of the study, 59.4% of the accessions were identified as sensitive, 23.9% were identified as moderately tolerant, and 16.7% were identified as highly tolerant. Pokkali was the most tolerant variety, while Nerica-6 was the most sensitive. Adapting standard International Rice Research Institute (IRRI) protocols, eight variables associated with salt tolerance were determined. The GWAS of the URMC, using over three million single-nucleotide polymorphisms (SNPs), identified nine genomic regions associated with salt tolerance that were mapped to five different chromosomes. Of these, none were in the known Saltol QTL region, suggesting different probable genes and mechanisms responsible for salt tolerance in the URMC. The study uncovered genetic loci that explained a large portion of the variation in salt tolerance at the seedling stage. Fourteen highly salt-tolerant accessions, six novel loci, and 16 candidate genes in their vicinity were identified that may be useful in breeding for salt stress tolerance. Identified QTLs can be targeted for fine mapping, candidate gene verification, and marker-assisted breeding in future studies.

Association Analysis of Three Diverse Rice (Oryza sativa L.) Germplasm Collections for Loci Regulating Grain Quality Traits.

Rice ( L.) end-use cooking quality is vital for producers and billions of consumers worldwide. Grain quality is a complex trait with interacting genetic and environmental factors. Deciphering the complex genetic architecture associated with grain quality provides essential information for improved breeding strategies to enhance desirable traits that are stable across variable climatic and environmental conditions. In this study, genome-wide association (GWA) analysis of three rice diversity panels, the USDA rice core subset (1364 accessions), the minicore (MC) (173 accessions after removing non-), and the high density rice array-MC (HDMC) (383 accessions), with simple sequence repeats, single nucleotide polymorphic markers, or both, revealed large- and small-effect loci associated with known genes and previously uncharacterized genomic regions. Clustering of the significant regions in the GWA results suggests that multiple grain quality traits are inherited together. The 11 novel candidate loci for grain quality traits and the seven candidates for grain chalk identified are involved in the starch biosynthesis pathway. This study highlights the intricate pleiotropic relationships that exist in complex genotype-phenotypic associations and gives a greater insight into effective breeding strategies for grain quality improvement.

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.

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.

Recent and projected increases in atmospheric CO2 concentration can enhance gene flow between wild and genetically altered rice (Oryza sativa).

Although recent and projected increases in atmospheric carbon dioxide can alter plant phenological development, these changes have not been quantified in terms of floral outcrossing rates or gene transfer. Could differential phenological development in response to rising CO(2) between genetically modified crops and wild, weedy relatives increase the spread of novel genes, potentially altering evolutionary fitness? Here we show that increasing CO(2) from an early 20(th) century concentration (300 µmol mol(-1)) to current (400 µmol mol(-1)) and projected, mid-21(st) century (600 µmol mol(-1)) values, enhanced the flow of genes from wild, weedy rice to the genetically altered, herbicide resistant, cultivated population, with outcrossing increasing from 0.22% to 0.71% from 300 to 600 µmol mol(-1). The increase in outcrossing and gene transfer was associated with differential increases in plant height, as well as greater tiller and panicle production in the wild, relative to the cultivated population. In addition, increasing CO(2) also resulted in a greater synchronicity in flowering times between the two populations. The observed changes reported here resulted in a subsequent increase in rice dedomestication and a greater number of weedy, herbicide-resistant hybrid progeny. Overall, these data suggest that differential phenological responses to rising atmospheric CO(2) could result in enhanced flow of novel genes and greater success of feral plant species in agroecosystems.

High-resolution mapping of Rsn1, a locus controlling sensitivity of rice to a necrosis-inducing phytotoxin from Rhizoctonia solani AG1-IA.

Rhizoctonia solani is a necrotrophic fungal pathogen that causes disease on many crop-plant species. Anastomosis group 1-IA is the causal agent of sheath blight of rice (Oryza sativa L.), one of the most important rice diseases worldwide. R. solani AG1-IA produces a necrosis-inducing phytotoxin and rice cultivar's sensitivity to the toxin correlates with disease susceptibility. Unlike genetic analyses of sheath blight resistance where resistance loci have been reported as quantitative trait loci, phytotoxin sensitivity is inherited as a Mendelian trait that permits high-resolution mapping of the sensitivity genes. An F(2) mapping population derived from parent cultivars 'Cypress' (toxin sensitive) and 'Jasmine 85' (toxin insensitive) was used to map Rsn1, the necrosis-inducing locus. Initial mapping based on 176 F(2) progeny and 69 simple sequence repeat (SSR) markers located Rsn1 on the long arm of chromosome 7, with tight linkage to SSR marker RM418. A high-resolution genetic map of the region was subsequently developed using a total of 1,043 F(2) progeny, and Rsn1 was mapped to a 0.7 cM interval flanked by markers NM590 and RM418. Analysis of the corresponding 29 Kb genomic sequences from reference cultivars 'Nipponbare' and '93-11' revealed the presence of four putative genes within the interval. Two are expressed cytokinin-O-glucosyltransferases, which fit an apoptotic pathway model of toxin activity, and are individually being investigated further as potential candidates for Rsn1.

A natural mutation in rc reverts white-rice-pericarp to red and results in a new, dominant, wild-type allele: Rc-g.

The Rc locus regulates pigmentation of the rice bran layer, and selection for the rc allele (white pericarp) occurred during domestication of the crop. White bran is now ubiquitous among cultivated varieties throughout rice growing regions of the world. We identified a new allele that arose by natural mutation within the rc pseudogene of the cultivar 'Wells'. The mutation restored the reading frame of the gene, and reverted the bran layer pigmentation to red (wild-type). By sequencing the Rc locus in plants derived from red seeds, and linkage analysis in a segregating population, we were able to demonstrate that mutation within rc resulted in the new, dominant, wild-type allele Rc-g.