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Research Project: Broadening and Strengthening the Genetic Base of Rice for Adaptation to a Changing Climate, Crop Production Systems, and Markets

Location: Dale Bumpers National Rice Research Center

2024 Annual Report

Objectives
1. Develop new climate-resilient rice with stable and high-quality yield, reduced irrigation needs, and lower methane emissions for sustainable U.S. agricultural production, and safer food supplies using new technologies. 2. Characterize and strengthen the rice germplasm collection and the blast fungal collection for genetic diversity to foster adaptation to climate change and to reduce vulnerability to biotic stress. 3. Identify the genes and gene networks that underlie beneficial rice traits to produce rice with increased economic productivity, resistance to biotic and abiotic stress, and enhanced value for human nutrition and processing. Identify the genes and gene networks that underlie beneficial rice traits to produce rice with increased economic productivity, resistance to biotic and abiotic stress, and enhanced value for human nutrition and processing. 4. Identify for critical environments the optimum gene combinations for climate-resilient, stable agronomic performance, and biotic/abiotic stress tolerance using artificial intelligence/machine learning approaches to analyze genomic information and high-throughput phenotyping data. Please see copy of upload Project Plan for all subobjectives; due to character limit they will not fit in this field.

Approach
The approach includes 1) developing germplasm and tools for breeding climate-resilient rice for sustainable U.S. agricultural production, 2) exploring diverse genetic resources for novel traits and genes to foster adaptation to climate change and to reduce vulnerability to biotic stress, 3) identifying the genes and gene networks that underlie beneficial traits to produce rice with increased economic productivity, resistance to biotic and abiotic stress, and enhanced value for human nutrition and processing, and 4) identifying optimum gene combinations for climate-resilient rice with stable agronomic performance and biotic/abiotic stress tolerance using artificial intelligence (AI) /machine learning to analyze genomic information and high-throughput phenotyping data. Marker assisted selection and rapid generation advance will be used to develop conventional and specialty breeding materials for release to US breeding programs. Efficient new methods will be developed for phenotyping traits associated with climate resilience and grain quality. Interactions between soil microbes, greenhouse gas emissions, grain quality and quantity under different irrigation systems will be evaluated. Mapping populations will be developed and evaluated to discover novel alleles from rice wild relatives that provide adaptation to abiotic and biotic stress. The USDA’s world rice collection of over 19,000 cultivars will be mined to discover useful novel alleles through the characterization of sub collections including a Tropical japonica Core (TRJC) collection, an aus subpopulation collection, weedy rice, and rice wild relative collections of O. glaberrima, O. barthii and O. australiensis. In addition, the US rice blast (Magnaporthea oryzea) field isolates will be characterized to guide the deployment of blast resistance genes. Several mapping populations will be used to identify genes and gene networks including a Japonica Multi-parent Advanced Generation Inter-Cross (MAGIC) population for yield components, an aus nested association mapping (NAM) population for water deficit and high temperature stress tolerance, and biparental populations for grain mineral accumulation. Genes and gene interactions involving biotic stress will be investigated using yeast two hybrid (Y2H) screening, and tradeoffs between biotic and abiotic stress responses will be investigated by gene expression analysis of varieties with differential blast resistance genes. Machine learning and artificial intelligence methods will be developed and deployed for high-throughput phenotyping using UAV or ground-based imaging systems. A database will be developed for genomic selection and a rapid cycle recurrent selection (RCRS) breeding pipeline will be established for fast rice breeding. The RCRS pipeline will be used to breed germplasm with increased quantitative resistance to sheath blight and cold tolerance. Outcomes from this research will include new public germplasm, new methods for accurate and efficient rice breeding, and genetic markers linked to traits that can be used in marker assisted breeding.

Progress Report
Objective 1: To develop improved climate resilient rice germplasm with high yield and quality, complementary crosses and backcrosses were completed for elite US germplasm and donors of novel traits. To optimize speed breeding, 10 diverse varieties were subjected to growth under differing wavelengths and pot sizes. The plants struggled to reach heading under LED lighting and were stunted and heat stressed under high-intensity discharge lamp (HID) lighting, and further refinements are needed. Leaf wax content was extracted from 134 diverse varieties under flooded conditions and alternate wetting and drying (AWD), however no significant differences were found between varieties. Seed from the tropical japonica core (TRJC) were imaged using a flatbed scanner and seed traits were determined using the SeedExtractor software. Samples and data on greenhouse gases, yield, and grain milling, processing, and nutritional quality collected from 2022 and 2023 field plots were analyzed, and soil microbial samples were collected for later analysis. Of the 69 chromosome segment substitution lines (CSSL) comprising the IR64_OrC library, 23 did not germinate or had very poor germination due to an extended heat treatment. Using alternate seed sources, 21 CSSLs were regrown, genotyped, and produced adequate seed. Of the original 69 IR64_OrC CSSLs, 67 CSSLs were prepared for submission to GSOR and public distribution. To improve mapping resolution, the Presidio x O. rufipogon (Wild-5) advanced backcross inbred line (BIL) population was genotyped with 20 additional markers for a total of 249 DNA markers. The parents and 240 BILs were evaluated for reaction to rice leaf blast disease using three different blast races (IA1, IB33, IB49). The ideal combination of alleles at three genes for maximizing the content of resistant starch (a beneficial health trait) in rice grains was determined to be Wxa+sbe3+SSIIIa, while previous study showed that Pb+Rc maximizes antioxidant content. Molecular markers were used to identify cross-progeny fixed for the desired resistant starch and antioxidant genotypes and progeny lines were selected in the field for desired agronomic traits. To investigate effects of contrasting root architecture, the TeQing into Lemont (TIL) population data on root traits, grain ionomics, and field agronomics were analyzed using Bayesian Network analysis, a machine learning technique, clarifying relationships between the traits and revealing marker-trait associations. Objective 2: In FY24, approximately 192 rice National Small Grains Collection (NSGC) accessions and 96 Genetic Stocks Oryza (GSOR) accessions are being rejuvenated using summer nurseries and greenhouses. To reduce NSGC redundancy, 616 accessions, grouped by sets that are redundant by name (RBN) were evaluated phenotypically and genotypically. Approximately 700 accessions were requested and received from Fort Collins to be genotyped and evaluated in the greenhouse and field. Thirty-five Oryza australiensis accessions are being grown in the greenhouse for evaluation and seed increase. NSGC received seed of 250 rejuvenated accessions from the 2023 growing season. Seed, data, plant, and panicle images were made public through GRIN-Global. The NSGC and GSOR shipped 2,587 and 4,373 seed packets, respectively, during 2023. The GSOR collection provided 4,356 seed packets to domestic researchers, and 16 were sent to international researchers. The TRJC panel of nearly 500 accessions were grown for seed increase. A subset of the TRJC, a micro core, was evaluated under standard flood conditions and AWD to identify drought tolerant accessions. The plots were imaged with an in-house built light-weight ground-based imaging system, hyperspectral data were collected using a spectrophotometer, and canopy temperature was measured. Joint allele calling and imputation was completed for the TRJC and Genome Wide Association Studies (GWAS) were performed on the agronomic and blast data. Accessions with acceptable agronomic characteristics were chosen from NSGC for sheath blight testing. The accessions were grown in the field during 2023 and 300 accessions were selected for sheath blight resistance evaluation. In collaboration researchers at Purdue University, West Lafayette, Indiana and the International Rice Research Institute (IRRI) in the Philippines, a set of 32 tropical japonica accessions selected for early growth stage drought tolerance were evaluated under AWD and irrigated conditions in field study at Stuttgart, Arkansas, in a high-throughput phenotyping facility at Purdue, and in a rainout shelter at IRRI using measurements of photosynthesis, biomass production and yield related traits. In collaboration with a researcher at Marquette University, Milwaukee, Wisconsin, four japonica specific recombinant inbred line populations were developed for mapping seedling stage cold tolerance. To identify superior alleles for drought tolerance from the aus rice genetic subpopulation, a panel of 100 accessions including 84 non-red pericarp aus and 16 checks are being phenotyped under water stress conditions. To identify adapted rice germplasm with new sources of sheath blight resistance, a total of 450 rice accessions were evaluated under field conditions and 280 were selected for greenhouse and field examinations. To characterize the bacterial pathogen Pantoea ananatis and help to identify resistant resources, bacterial samples were purified from a susceptible rice plant and were positively identified using polymerase chain reaction using rDNA and a gene specific marker. Objective 3: The L202 x Trembese tropical japonica recombinant inbred line (RIL) population comprised of 211 RILs and evaluated for yield and yield components was prepared for submission to GSOR and distribution to the public. A set of 200 accessions that were part of the aus-NAM population were selected randomly and advanced to F7 generation, and the F8 generation is currently in the field. All the planned agronomic and phenotyping data were collected under AWD irrigation management. Genotyping was delayed till F8 generation for genotyping platform cost savings by bundling multiple genotyping projects. To investigate the impact of reduced irrigation on grain concentration of desired (e.g. potassium) as well as undesired (e.g. arsenic) elements, grains from a prior yield study of 9 varieties x 4 irrigations x 3 years were analyzed commercially for grain element content and data analysis is in process. To evaluate identified quantitative trait loci (QTL) as targets for marker assisted selection to improve nutritional value, cross-progeny F5 plants containing residual heterozygosity were identified and their progeny were planted in single-plant plots, but long-term illness of essential personnel prevented timely in-house molecular analyses to identify the subset of progeny that were fixed at each targeted QTL, however grains were harvested for future genotyping and grain analysis. Different domains and the full-length protein of blast resistance protein Ptr from Katy and weedy rice BHA RR20 were cloned into a yeast two hybrid vector, and a previously constructed library obtained from Nipponbare inoculated with compatible and incompatible blast races is being used to identify interacting genes to study plant and pathogen genes involved in Ptr mediated disease resistance. To determine the role of Ptr in blast resistance and abiotic stress tolerance, differential rice varieties and lines Katy, M2354, Cybonnet, Saber, SC272, SC353, SC324 are being examined using real time PCR. RNA samples from the second youngest leaf at 0,6,16,24,48 and 72 hrs after inoculation with blast were used for analysis. Objective 4: To facilitate imaging of rice plants suitable for feature segmentation via artificial intelligence models, a mobile ground-based plant imaging system was developed that is lightweight and customized for rice field plots. This system was used to image diverse rice accessions under AWD. Additionally, an image-based method was developed to predict rice biomass non-destructively by measuring leaf area as a proxy. The non-destructive biomass imaging method uses a deep learning convolutional neural network architecture that was trained to segment the regions of an image corresponding to rice plants. This method was found to predict rice dry shoot biomass with high accuracy (r-squared greater than 0.75). Molecular markers and genome regions associated with cold tolerance and sheath blight resistance were curated from GWAS results along with candidate genes and overlapping quantitative trait loci (QTLs) for other traits. Inconsistencies between published GWAS studies in the way that significant associations are reported presented challenges in the curation process. A relational database schema has been developed and is being tested to store and query GWAS data for the purpose of identifying optimum rice accessions to use as breeding parents. Breedbase, an open-source comprehensive breeding management software system was modified to run as an instance on SCINet high performance computing nodes for rice breeding data and seed tracking. However, changes in SCINet authentication requirements have eliminated the previous method used to view the Breedbase web interface, and workarounds are being investigated. Useful data for optimizing genomic selection was obtained through a collaboration with researchers at the University of California-Davis, where genomic prediction models were evaluated for yield component traits, and prediction accuracies were estimated using previous multi-location phenotyping data on a representative panel of US rice lines. Greenhouse issues in FY2024 including a non-functioning heating and cooling system prevented completion of first year milestones for Sub-objectives 1E, 1F, and 2C, and negatively impacted Sub-objective 3E experiments.

Accomplishments
1. Release of Three Long Grain Rice Germplasm Lines with Improved Blast Resistance with the Ptr gene, Low Chalk, Excellent Milling Quality, and Good Yield. Rice Blast disease caused by the fungus Magnaporthe oryzae B.C. Couch is the most threatening disease of rice worldwide. ARS researchers at Stuttgart, Arkansas, in collaboration with researchers at the University of Arkansas Agricultural Experiment Station and Louisiana State University, Crowley, Louisiana, released these Cybonnet–Saber (CS) germplasm lines. Three rice (Oryza sativa L.) germplasm lines, designated as CS272, CS324, and CS353 were selected from among nine hundred recombinant inbred lines (RILs) derived from a cross of the U.S.-adapted cultivars ‘Cybonnet’ (PI 636726) and ‘Saber’ (PI 633624). These three germplasm lines have resistance to blast as determined under greenhouse conditions, and uninoculated field trials conducted in Puerto Rico in 2017 and Crowley, Louisiana in 2018 and 2019. The germplasm lines were resistant to most blast races as determined in inoculated greenhouse tests, resistant to leaf blast when tested in an upland blast nursery in Crowley, Louisiana, and resistant to panicle blast under flood conditions in Puerto Rico and Crowley. These CS germplasm lines contain the major blast resistance gene Ptr identified on chromosome 12 originating from the Cybonnet parent. These three CS germplasm lines are superior in panicle size and have many agronomic and grain-quality characteristics comparable to or better than those of both parents and can be used in rice-breeding programs for improving blast resistance, quality and yield and grown for premium long grain markets.

2. Identified the 3-gene combination that optimizes resistant starch content in rice grains. Being a carbohydrate-dense food with a high glycemic index (GI) limits the consumption and sales of rice. Rice GI can be reduced by increasing its content of resistant starch (RS), a starch-type so named because it resists digestion in the gastrointestinal tract. RS does not contribute to calorie count, blood glucose, or GI, and the undigested starch is a dietary fiber. Dietary fibers feed beneficial gut microbes, stabilize blood sugar levels, and reduce colon and other cancers, inflammation, and cardiovascular disease. To identify novel genes and gene combinations useful for developing high-RS rice varieties, ARS researchers at Stuttgart, Arkansas, identified a novel starch mutant, mapped it to the soluble starch synthase IIIa (ssIIIa) locus, and evaluated its impact on RS, individually, and in combination with two additional genes known to affect RS production: Wx and BEIIb. The Wx gene regulates amylose production, and high amylose wheat, corn, and rice have higher RS than standard versions of these crops. Of the three genes, beIIb mutation increased RS the most. The newly mapped ssIIIa mutant increased RS to a lesser degree and only when genetically combined with the high-amylose Wx-a allele and the non-mutant BEIIb allele. When the ssIIIa mutation was stacked onto the beIIb mutation it decreased rather than increased RS, indicating the two genes impact RS in antagonistic ways. When starch structures were investigated in detail, increased RS from beIIb mutation was found associated with an increase in long unbranched amylopectin chains, while ssIIIa mutation reduces chain elongation. Because of this antagonism, the 3-gene genotype that optimized RS was Wx-a+beIIb+SSIIIa. By identifying the target genotype and providing breeders with validated molecular markers for marker assisted selection, these results will speed development of high RS (low GI) rice.

3. Polyphenol-mediated covalent bonds on glutelin structural changes in rice with different bran colors. Varieties of rice with pigmented bran have emerged as potential nutraceutical and functional food ingredients with health-promoting benefits due to the presence of different classes of polyphenols in the bran. Long-term consumption of polyphenols may offer preventive and therapeutic effects for cardiovascular disease, cancer, neurodegenerative diseases, type-2 diabetes, osteoporosis, and obesity. In addition, rice protein has high digestibility, biological availability, and hypo allergenicity, indicating it is an ideal functional ingredient in food and beverage products. Glutelin represents the predominant protein fraction in both brown and milled rice. The concentration and type of polyphenol and protein molecules present in the grain can affect their structural and nutritional properties. An ARS researchers at Stuttgart, Arkansas, in collaboration with researchers at the University of Arkansas, explored how the polyphenols in purple and red bran rice may affect the functional and nutritional properties of rice glutelin. Milled, whole grain, and rice bran of brown, red, and purple bran varieties were evaluated for changes in protein structure. Phenolic acids inherently present in brown bran rice did not alter glutelin structure, whereas high concentrations of anthocyanins and flavan-3-ols/proanthocyanidins in purple bran and red bran rice, respectively, formed intra-molecular covalent bonds with glutelin making the protein more compact. These changes in glutelin structure in pigmented bran rice may improve rheological and nutritional properties and result in novel health beneficial food products using natural ingredients from rice.

4. Rice varieties with grain attributes beneficial to malting for beer brewing. There is increasing market demand for gluten-free beers, inciting interest in using malted rice in place of malted barley, and there are cost benefits from substituting even a portion of barley with rice. Cooked milled rice is presently used as an adjunct starch in some beers, which provides a distinctive light crisp flavor, this study by ARS researchers at Stuttgart, Arkansas, in collaboration with scientists at the University of Arkansas, Fayetteville, investigated the possibility of producing beer using malted rice, as needed for rice to replace all or a portion of the traditional barley. Grains from 19 genetically and chemically diverse rice varieties were malted using a small-sample pilot malting process. After mashing the malted rices, their worts were collected and analyzed chemically to compare their malting qualities with barley. All 19 rice varieties provided enough amylase enzymes for their starches to be well converted to sugars using standard malting conditions. Rice is known for containing less protein than wheat or barley grains, causing the widespread belief in the brewing community that beer cannot be produced entirely from rice without adding exogenous proteins or nitrogen. This study broke this paradigm by identifying some rice varieties that produced wort with sufficiently high protein and nitrogen contents. It was also discovered that some rice varieties with purple-pigmented brans imparted a unique and desirable color to the wort. While further study will be required to determine the flavor and color of the resultant rice beers, this chemical study of malted-rice worts provides direction and motivation for further investigation into the use of malted rice for beer production, and identifies traits and germplasm needed for the development of rice varieties containing the attributes that can enhance production of gluten-free malted-rice beers.

5. Identification of genes associated with rice seedling cold resilience. Due to global climate change resulting in extreme temperature fluctuations, it becomes increasingly necessary to explore the natural genetic variation for chilling tolerance at the seedling stage in rice. Discovering new sources of seedling tolerance will facilitate the development of new climate resilient, chilling tolerant rice varieties. Often rice seedlings struggle to survive when the seedlings experience cold temperatures because they lack chilling tolerance which limits how early rice can be planted in many rice growing regions including the USA. ARS researchers at Stuttgart, Arkansas, in collaboration with Marquette University researchers, ascertained the temperate japonica accessions, Krasnodarskij 3352 from the Krasnodar region of Western Russia, and WIR 911 from the Primorsky Krai region of Eastern Russia, were cold tolerant. This same study identified the aus accession, Carolino 164 from Chad, as cold susceptible. To discover genes associated with seedling stage tolerance, progeny from the crosses Krasnodarskij 3352 x Carolino 164, and WIR 911 x Carolino 164, were genotyped and evaluated for seedling stage cold tolerance. Analysis of this data revealed 25 potential genes for seedling stage chilling tolerance across 16 different chromosome regions. Future studies will determine which gene(s) would be the most effective for improving rice seedling chilling tolerance and transferring the gene(s) into rice varieties adapted to the United States growing environment. These new varieties should be more resilient to climate change because they would withstand cold more effectively during their early growth stages and could be planted earlier in the growing season, potentially having superior grain quality because the grains would mature before the intense heat of late summer. Also, it might be possible to harvest a second (ratoon) crop in the United States Mid-South due to earlier planting.

6. Mapping yield related traits utilizing three cultivated rice varieties by ancestral rice introgression libraries. The wild species that crop plants were domesticated from are a largely untapped reservoir of genetic variation available to plant breeders as they confront the challenges of a changing climate and feeding the increasing global population. The ancestral species of cultivated rice are Oryza rufipogon which is found throughout Asia, and Oryza nivara which is limited to South and Southeast Asia. When considered together, these species are identified as the Oryza rufipogon species complex (ORSC). To enhance efforts to incorporate the genetic variation hidden in the ORSC, three genetically and phenotypically diverse accessions originating from China, Laos and Indonesia were crossed with Cybonnet, an elite rice variety developed by the University of Arkansas. From these crosses, three populations were developed which had small segments of the ORSC DNA in the background of Cybonnet. The 212 progeny lines comprising these three populations and the Cybonnet parent were characterized by ARS researchers at Stuttgart, Arkansas, for 20 yield-related traits over two field seasons. Traits evaluated included six agronomic traits, six panicle architecture traits and eight seed (grain) size and/or weight traits. From this evaluation, 62 progeny lines were significantly different from Cybonnet for one or more yield related traits. Of these, 27 CSSLs were significantly different for seed size and/or weight traits, which directly relates to yield because increases would result in heavier seed, thus higher yield. Examination of the chromosomal regions with the wild ORSC DNA in these 62 progeny lines for genes controlling these yield-related traits, revealed 28 different genes. Sixteen of these genes are already targeted for rice improvement but this study identified twelve other genes which potentially could enhance yield and should be explored in future studies.

7. Identification of genetic loci for grain traits and disease resistance reveals a potential trade off in rice. Plants are sessile and thus are permanently attached to their site of germination and growth. To circumvent that immobility plants have evolved a sophisticated multifaceted mechanism to cope with external biotic stressors. Plants often redirect the resource for growth and reproduction when under pathogen attack. This growth–defense is commonly known as the tradeoff that allows plants to alter growth and development to adapt to external biotic stressors. The genetic mechanism of tradeoffs between grain traits and disease resistance is poorly understood in rice. ARS researchers at Stuttgart, Arkansas, mapped the genes for grain traits, including main panicle weight, grain length, width and thousand grain weight, using a genetic linkage map of a recombinant inbred line population MHM (Minghui63 x M-202). The MHM population along with the parents Minghui63 and M-202 were grown in replicated field plots. Grain length and width were measured by WinSEEDLE. Grain weight was measured using a balance. A total of 7 loci for grain length, 4 for grain weight, and 5 for thousand grain weight were mapped on 9 chromosomes. One thousand grain weight locus qTGW2 and one grain width locus qWIG2.2 were mapped at the known blast resistance gene Pi-b on rice chromosome 2. Another thousand grain weight locus qTGW3.3 and grain length locus qLNG3.3 were mapped at qBLAST3 on rice chromosome 3. Further studies of genes at these loci will reveal potential tradeoffs between disease resistance and productivity. This knowledge is important for breeders to develop high yielding and disease resistant rice varieties.

8. Water conserving management practices in rice production can be safely used without negatively impacting elements that are important in human nutrition. Reduced irrigation management practices such as alternate wetting-drying (AWD) or furrow irrigation (row rice) have been shown to save water resources, reduce methane emissions, and reduce potential arsenic concentrations in rice grains but little was known if the aerated soil conditions have any impact on arsenic, cadmium, and micronutrients (e.g., zinc, manganese) solubility in soil pore water and accumulation in rice grain. ARS researchers at Stuttgart, Arkansas, along with researchers at Cornell University, conducted a study on five varieties, two of which had previously been reported by ARS to be excluders for arsenic uptake and three that were accumulators of grain arsenic. It was found that continuously flooded (saturated) soils resulted in higher solubility of all the studied trace elements (arsenic, iron, molybdenum, cadmium, manganese, copper), except zinc which was not affected by soil moisture content. The arsenic excluder rice varieties had relatively low levels of total grain arsenic regardless of irrigation strategy. Interestingly, the rice varieties that were accumulators, had higher levels under saturated field conditions, but under the AWD, the total arsenic was greatly diminished, to a level found in the excluder varieties. For cadmium, which is considered a toxic hazard for humans, there was a trend for cadmium to increase under AWD conditions, but this was statistically significant only for the varieties that were also arsenic accumulators. However, none of the varieties accumulated cadmium at levels that met the criteria for a human health concern. In contrast, for the other elements, there was no significant difference in grain content in response to flood or AWD management. The results indicated that arsenic and cadmium in rice can be effectively controlled under AWD.

9. Water conserving management practices in rice production can reduce outcrossing potential between cultivated and weedy red rice. Outcrossing between cultivated and weedy red rice is a serious problem in the Mid-south United States rice growing areas because it leads to herbicide resistant red rice genotypes that severely limit the efficacy of herbicide use for controlling red rice. Recently the United States rice producers are adopting water conserving management practices such as alternate wetting and drying water management (AWD) which allows the soil to dry to a predetermined level before re-irrigating the field while maintain grain yield. Thus far, there was a significant knowledge gap on outcrossing potential between rice and weedy red rice under such irrigation management systems. ARS researchers at Stuttgart, Arkansas and Beltsville, Maryland, conducted a two-year replicated randomized field study. Outcrossing frequencies between two hybrid rice rice varieties (a long grain cv. CL142AR and medium grain cv. CL261), and two major weedy rice genotypes (blackhull and strawhull) were measured after growing together in plots irrigated with either conventional season-long flood or AWD irrigation. Results demonstrated that overall, AWD reduced outcrossing rates and the number of cross-hybrid seeds produced in weedy red rice. Specifically, outcrossing rates were significantly lower in medium grain than for the long grain rice cultivar, and the AWD irrigation management reduced outcrossing rates with the strawhull red rice, but not with the blackhull genotype. This study is the first of its kind to demonstrate that water conservation irrigation systems such as AWD have the potential to provide the additional benefit of reducing outcrossing between rice and weedy rice. The knowledge gained from this study will help in finding better solutions for long-term weedy rice control measures in commercial rice production by adding cultural management practices along with genetic improvement.

10. Regional Diversity of Magnaporthe oryzae in Arkansas, Louisiana and Puerto Rico from 2017 to 2022. Major resistance (R) gene mediated resistance to rice blast fungus Magnaporthe oryzae is often overcome by occurrences of new races with altered corresponding avirulence genes (AVR). ARS researchers at Stuttgart, Arkansas, in collaboration with researcher at University of Arkansas, analyzed blast diseased samples from experimental stations and commercial rice fields from Arkansas, Louisiana and Puerto Rico from 2017-2019 to determine the efficacy of major R genes, Pi-ta, Pik, Piz, Pi9, and Pi33 in these regions. A total of 185 blast isolates was purified to examine the existence of AVR genes AVR-Pita1, AVR-Pib, AVR-Pik, AVR-Piz, AVR-Pi9 and ACE1 and their genotypes were examined with 10 simple sequence repeat (SSR) markers. AVR-Pizt, AVR-Pita1 and AVR-Pi9 was found in all isolates suggesting that Piz, Pi-ta and Pi-9 is effective to prevent infections by these isolates. Among them, 117 of 185 contain all 6 AVR genes and three other group contains 3-5 AVR genes suggesting that there exhibits different degree of race shift in these isolates. SSR data revealed that isolates from each of these three sites were different suggesting that there was no migration among these three sites. Structure analysis of SSR data suggest that there are two major clusters with 46 combinations. The isolates from Arkansas showed high genetic diversity dominated by one or few genotypes. The isolates from Louisiana were more genetically diversified without any obvious predominant group. The isolates from Puerto Rico have the lowest heterozygosity. These data reveal contemporary genetic changes in rice blast fungus populations and is useful for guiding the deployment of major R genes in these regions.

11. Acquiring new germplasm to explore grain quality and heat tolerance. To access heat tolerance in rice germplasm ARS researchers in Stuttgart, Arkansas, in collaboration with the International Rice Research Institute (IRRI), Mississippi State University (MSU) and USDA APHIS, imported a Heat-MAGIC population developed by IRRI with 400 individuals. The population was developed by crossing between eight parents that possess excellent grain quality and yield, heat tolerance and excellent agronomic traits. The parents represent the indica, aus, and japonica subpopulations of rice.

12. Characterization of the genetic diversity in a collection of Oryza barthii, O. glaberrima, O. longistaminata, O. sativa ssp. indica and O. sativa ssp. japonica accessions for rice breeding. The genetic diversity conserved in rice genebanks is important to improve rice yield, grain quality, disease resistance, abiotic stress and global food security. The Africa Rice genebank holds the world’s largest collection of Oryza species collected from Africa, with about 20,681 rice accessions. About 83% of the accessions were collected within Africa, 14% (3,130) are O. glaberrima and 64% (14,480) are African O. sativa. A set of 9,013 accessions believed to be O. barthii, O. glaberrima, O. longistaminata, O. sativa ssp. indica or O. sativa ssp. japonica, were genotyped to provide insight into the genetic diversity, species and ecology. A set of 27,718 genetic markers were identified across the four Oryza species. ARS researchers at Stuttgart, Arkansas, contributed to the interpretation of the genetic diversity identified among the 9,013 rice accessions. The genetic markers were able to group the accessions into six groups, O. barthii, O. glaberrima, O. longistaminata, O. sativa ssp. japonica, O. sativa ssp. indica improved/advance cultivars and O. sativa ssp. indica traditional/landraces. The accessions proved to be highly diverse with genetic distances between 0.001 – 0.469 with 45% of pairs being highly distant. These 9,013 rice accessions are an important resource for pre-breeding, breeding and further genetic applications. The 686 African O. sativa ssp. japonica can be screened for important traits of interest that are not present in the US rice germplasm.

13. An efficient method for screening rice breeding lines against races of Magnaporthe oryzae. Blast disease of rice caused by the fungus Magnaporthe oryzae is one of the most lethal diseases of rice worldwide. Traditionally the 0-5 scale rating has reached limited success to evaluate disease reactions of breeding lines and rice varieties for breeding and genetic studies. ARS researchers at Stuttgart, Arkansas, in collaboration with researchers at Louisiana State University, developed a 0-6 scale for blast disease that allowed assignment of rice breeding lines and varieties into six resistance levels (highly resistant, resistant, moderate resistant, moderate susceptible, susceptible, and highly susceptible) by using 40 common rice varieties with known disease reactions under field conditions and tested them against four major blast races (IB1, IB17, IB49, and IE1-K) under greenhouse conditions. Disease reactions using 0-6 rating system verified field observations of rice varieties with blast resistance genes. Varieties carrying the Pi-ta gene were either highly resistant, resistant, or moderate resistant to IB17. The IE1-K race was able to break Pi-ta-mediate resistance of the rice varieties. The Piz gene conferred resistance to the IB17 and IE1-K races. The varieties M201, Cheniere, and Frontier were highly susceptible (score 6; 100% disease) to the race IE1-K. Moreover, varieties that were resistant or susceptible to all four blast races also showed similar levels of resistance/susceptibility to blast disease in the field. Taken together, our data proved that the 0-6 blast scale can efficiently determine the resistance levels of rice varieties against major blast races. This new method will assist rice breeding programs to incorporate durable resistance against major and emerging blast races.

14. Understanding the evolution of barbs (thorns, prickles) facilitates their elimination for crop improvement. In the agriculturally important genus Solanum, crops like eggplants and the desert raisin (forage) have “prickles” which are short, slender, sharp-pointed epidermal outgrowths conferring critical fitness advantages like defense against herbivores, improved plant competition, a climbing growth habit and improved water retention. Prickles are called “barbs” on the awns of rice and barley, “thorns” on roses and citrus trees, or “stipular spines” on Chinese date and sour jujube. In rice and barley, these barbs are undesirable because they damage harvesting equipment, causing it to wear-out faster and in the case of hand-harvesting, injure the person harvesting. Similar problems are noted in other crops, thus understanding genetic control of prickle development will aid in designing genetic studies to develop crops without or if desired, with prickles. There are about 1,500 Solanum species worldwide, with about 450 species clustering into subgroups which have prickles. Nine species with prickles and five prickleless species were selected from three of the subgroups for an in-depth study of the mutations in the DNA sequence of the LONELY GUY (LOG) gene which activates cell division by affecting cytokinin biosynthesis and results in the presence or absence of prickles. Cold Spring Harbor Laboratory, New York in collaboration with ARS researchers at Stuttgart, Arkansas, determined mutations in the same LOG gene were associated with loss of barbs on awns in rice and barley, thornless roses, loss of stipular spines on Chinese date trees and its fruit, and a prickleless desert raisin. This study demonstrates how knowledge of the DNA sequence can be used to easily eliminate prickles across a broad range of plant species and serves as a model for designing studies to eliminate undesirable traits (or incorporate desirable traits) utilizing available DNA sequence data.

15. Machine learning models to predict rice yields under forecasted future climates. To support the production of food for a growing global population, agricultural systems must be able to adapt to a changing climate. One way to do this is to evaluate how weather has affected yields in the past and then predict how crops will respond in the future under forecasted climate scenarios. ARS researchers at Stuttgart, Arkansas, in collaboration with Purdue University and Cornell University, used information on genetic variation, crop productivity, and past weather data in the U.S. Mid-south rice-growing area to model and predict rice yields. Based on county-level data from 1970 to 2015 that includes yields, acreage by variety, and weather data, along with molecular marker information for the varieties grown in each year for each county, artificial intelligence and machine learning models were developed to predict the historic yields. Results indicated that the models developed from this approach were highly correlated with the actual observed historic yields. These models were then used to predict yields under forecasted future climates. One significant discovery is that the more modern rice varieties developed through public breeding programs, which incorporate genetic material from diverse sources, exhibit greater resilience to predicted future climate conditions. This insight is particularly relevant for developing strategies to enhance crop resilience across various agricultural sectors.

16. Machine learning models for rice grain yield prediction using remote sensing imagery. Unoccupied aircraft systems (UASs) are increasingly utilized in agriculture to predict plant traits from remotely-sensed imagery. These UAS datasets are collected at multiple times and across multiple field locations, which presents challenges in developing prediction models that generalize well across different years and environments. The poor transferability of trained models from one agricultural context to another hampers the widespread adoption of UAS technology. In collaboration with Arkansas State University, ARS researchers at Stuttgart, Arkansas, conducted a two-year field study with five experiments involving two nitrogen rates, two seeding rates, hybrid and inbred varieties, and a genetic diversity study. UAS imagery was collected at multiple timepoints during the 2021 and 2022 growing seasons. Upon harvest, grain yield was determined for each plot. This study revealed dimension reduction approaches for machine learning could enhance yield prediction and improve generalization to new contexts and growing seasons. Only models capable of learning higher-order interactions yielded robust results for yield prediction across different growing seasons, while simpler models performed well when trained and tested on the same season and cultivars. These findings extend the toolkit for UAS image analysis, offering promising avenues for refining predictive capabilities using remote-sensing data.

U.S. DEPARTMENT OF AGRICULTURE

Dale Bumpers National Rice Research Center: Stuttgart, AR