Project : USDA ARS

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Research Project: Gene Discovery and Crop Design for Current and New Rice Management Practices and Market Opportunities

Location: Dale Bumpers National Rice Research Center

2019 Annual Report

Objectives
1. Conserve, regenerate, characterize and expand rice germplasm and blast fungal collections to provide new genetic resources for rice research. 1A. Conserve and characterize the current NSGC rice collection through phenotypic and genotypic analysis to provide true to type and viable genetic resources for distribution to the research community. 1B. Determine the allelic value of Tropical japonica germplasm for improving US rice cultivars. 1C. Identify new sources of germplasm and associated alleles that result in increased grain yield under AWD management system and growing conditions of the southern U.S. 1D. Characterization of agronomic and physiological performance of weedy (red) rice germplasm biotypes in AWD management systems. 1E. Expand the NSGC collection with the development and characterization of an O. glaberrima/barthii and an O. rufipogon rice wild relative diversity panels and evaluate for agronomic and biotic stress tolerance traits. 1F. Characterize the rice blast fungus M. oryzae (Mo) collection for AVR genes and their response to changing climate and production practices. 2. Discover genomic regions and candidate genes/alleles associated with high yield, reduced environmental impacts, resistance to biotic and abiotic stresses, beneficial microbial interactions, and novel/superior grain qualities that are expressed across environments and management systems (GxExM) by developing and utilizing bioinformatics tools, high throughput phenotyping, and omics-driven analyses. 2A. Map QTLs for root architecture at the seedling stage… 2B. Identify QTL associated with quality production under AWD management… 2C. Evaluate three Chromosome Segment Substitution Line (CSSL) libraries… 2D. Fine map yield enhancing loci derived from O. rufipogon introgressions… 2E. Identify genomic regions associated with increased quality production in response to extremes in temperature. 2F. Identify genomic regions associated with increased quality production in response to biotic stress derived from O. sativa and its wild relatives. 2G. Fine-map and conduct candidate gene analyses for kernel fissure resistance (FR)… 2H. Identify genomic regions… 3. Identify optimum gene combinations using modeling of interactions between agronomic traits, reduced environmental impacts, biotic and abiotic stress tolerance, the plant-microbiome, and rice grain quality parameters that are expressed across environments and management systems (GxExM). 3A. Determine impact of reduced input systems on weed suppression. 3B. Identification and validation of effective QTLs for disease resistance… 3C. Determine the impact of AWD irrigation management… 3D. Determine the impact of abiotic stress… 4. Develop and deploy improved rice germplasm for production under existing and new management systems and for new market opportunities. 4A. Develop improved cultivars and germplasm for production in the southern U.S… 4B. Deploy yield enhancing loci derived from O. rufipogon introgressions… 4C. Determine if yield penalty is associated with disease resistance… 4D. Facilitate the development of new rice cultivars… Please see Project Plan for all listed Sub-objectives.

Approach
Rice is one of the most important cereal grains and it is important to sustain USA rice production for both domestic and global food security. A major challenge in rice production is diminishing irrigation resources. One approach being adopted uses alternate wetting and drying (AWD) irrigation that reduces water use by 20%. Yet there are many questions as to how to optimize quality production under this management system. In addition, record high temperatures during grainfill have resulted in yield and quality losses. This project will use genetic resources, genomic sequence data, and high throughput phenotyping to understand the genes and physiological processes that impact rice yield and quality under changing cultural practices and climates. The approach includes 1) exploring diverse genetic resources for novel traits and genes for developing improved rice cultivars that are resilient to changing climates and production practices, 2) identifying genes through mapping of quantitative trait loci (QTL), 3) identifying combinations of genes that result in increased yield and grain quality, and 4) developing and deploying unique combinations of genes in germplasm that will benefit the US rice industry. Central to the program is curation of the USDA’s world rice collection of over 19,000 cultivars. Subsets of this collection are used to create diversity panels that explore specific gene pools (e.g. O. glaberrima, O. rufipogon, Aus, etc.) for response to biotic and abiotic stresses. In addition, a collection of US rice blast (Magnaporthea oryzea) pathotypes will be evaluated for their ability to causes disease in response to different rice genotypes, management systems, and climatic environments (G x M x E). Segregating mapping populations developed from bi-parental matings and chromosome segment substitution lines (CSSLs) developed using wild species will be used to map QTL for response AWD and heat stress, yield production, disease resistance, kernel fissure resistance, and grain nutritional quality. Recombinant inbred lines (RILs) and CSSLs that possess QTLs for these traits will be used to determine which combinations of genes/QTLs provide the most robust response to production under systems using reduced inputs and having high disease and weed pressure as well as abiotic stresses such as drought and high temperatures. Although most of our previous research has focused on above ground traits, this project will include evaluation of root architecture traits and plant-soil-soil microbiome interactions that may be driving above ground phenotypes and responses. Outcomes from this research will include identification of unique germplasm, location of important QTLs, new methods for accurate and efficient phenotyping, and genetic markers linked to QTLs that can be used in marker assisted breeding. Our goal is to deploy improved germplasm that can be used directly as cultivars or as parental stocks in breeding programs that possess unique combinations of genes that provide high yield, superior milling and processing quality, are resilient to pest pressures and abiotic stress, and have unique nutritional quality that will result in high crop value.

Progress Report
The Geneticist position that has been vacant for several years and is responsible for maintaining the National Small Grains Collection (NSGC) of rice was filled in June 2019. The winter nursery conducted in collaboration with the University of Puerto Rico allowed successful rejuvenation of 245 NSGC germplasm accessions. Currently, some 400 additional accessions have been planted in Stuttgart, Arkansas, for seed production. A new study was launched to determine if 211 accessions that have the same name but different PI numbers are redundancies or are mis-named based upon phenotypic and genotypic data. Currently, there are 38,383 accessions in the Genetic Stocks Oryza collection (GSOR) and 7806 accessions were distributed to researchers this year. We imported 46 Aus accessions from the Philippines for the abiotic stress tolerance project; they have been successfully germinated at APHIS in Maryland. A database and software tools were developed for identifying the sub-population origin of rice chromosome segments. Chromosome segments from the indica subpopulation were identified in U.S. rice varieties that correspond to the semi-dwarf gene and the blast resistance gene Pita, and previously unknown introgressions were identified which may harbor novel genes introduced into the U.S. rice gene pool. To prepare for fine mapping of yield component gene regions from O. rufipogon introgressions, the 7K Infinium Rice Array (C7AIR) genomic results from chromosome segment substitution lines (CSSLs) revealed both target and background introgressions. This enabled selection of molecular markers to facilitate recombinant screening to fine map while controlling for gene interactions with background. A field trial of 50 Aus accessions and 30 other O. sativa cultivars was conducted under rain-fed water stress conditions and 10 Aus varieties with superior tolerance to drought stress were found. The study is being repeated in 2019 to collect a second year’s data. A mapping population of 192 recombinant inbred lines (RILs) was grown under two water stress levels and the more severe stress level (-30 kPa) was selected for future studies as being more effective for mapping quantitative trait loci (QTL) for tolerance to water-stress. Due to lack of funding and the government shutdown the establishment of a greenhouse screening system for heat stress is now delayed until FY 20. Research on weed suppression included evaluation of RILs from the PI312777/Katy mapping population for crop productivity under alternate wetting and drying (AWD) and flood irrigation revealing marked differences in tolerance to AWD. Some 20 RILs performed as well as the weed suppressive parent, PI312777, under AWD and will be followed up on in a second experiment. Differences in seedling root traits of 40 weed suppressive RILs grown in agar plates were analyzed with an imaging system. A diverse set of 28 red rice accessions was evaluated under AWD and flood irrigation. Increasing levels of AWD stress during the reproductive stage tended to reduce the yields of red rice and cultivated rice and altered the potential for outcrossing between them. Some experiments planned for 2019 were not conducted due to two vacancies in support positions and these studies will be delayed until the positions are filled. As a part of a USDA-NIFA (National Institute of Food and Agriculture) grant with Kansas State University, 300 rice blast disease isolates were purified from tissue of cultivars grown in the southern USA and Puerto Rico (2015 to 2018), 200 isolates were stored but 100 were lost during the furlough. Currently, 50 isolates are being evaluated with gene specific DNA markers to verify the presence of avirulence genes. High-density genotypes were determined for two mapping populations using C7AIR to identify genes affecting root size and architecture. A set of CSSLs and RILs are being evaluated for root architecture at the 2-week stage using an imaging system to quantify root numbers and sizes. A set of 122 CSSLs were evaluated for root and shoot biomass traits and QTL mapping is in process. These CSSLs were also evaluated for grain inorganic arsenic (iAs) and seven QTLs were identified. Root and shoot data collected in FY 18 are undergoing QTL analysis and a subset of RILs is being used to study genotype x soil microbial interaction and the impact on methane emissions. To identify and map new blast resistance genes, 150 markers were tested in 270 RILs derived from a 'Minghui63 (has resistance but no known resistance genes)/M202 (susceptible)' cross. Indel markers are being developed to fill in 20 genomic gaps. The RILs were evaluated with seven blast isolates with three more to be completed in FY 19. To determine the stability of blast resistance genes in newly developed genetic stocks/germplasm under different field conditions, a panel of 150 rice germplasm lines with different combinations of blast resistance genes were grown in field trials under upland and flood conditions in Crowley, Louisiana under natural blast infestations and were found to have consistent disease resistance reactions. A study is currently underway at Crowley, Louisiana, and Stuttgart, Arkansas, using varying irrigation systems to validate these findings. A backcross population has been developed to fine map the Pita/Pitr blast resistance gene region. A line segregating for the two closely linked genes was identified and 31 (17R:14S) progeny were selected for seed production and future mapping studies. To determine if the Ptr gene has a negative effect on yield potential, three out of 1,000 RILs having only the Ptr gene are being evaluated in a 2019 yield trial. The plan for fine-mapping QTLs for kernel fissure resistance using KASP markers has been redirected to use indels which will be faster and less costly. 1,700 mutant lines in the variety Katy were screened using hyperspectral imaging and 43 were identified having unique spectra that will undergo seed increase and evaluation for grain nutritional quality. With USDA-NIFA funding through Marquette University, four mapping populations are being developed that will segregate for cold tolerance and are currently at the F3 generation. These populations will be used to identify cold tolerance mechanisms which are subpopulation or subspecies (Indica, Japonica) specific. To identify QTLs linked with the concentrations of pigmented flavonoids, 800 progeny lines with red and purple bran phenotypes from the IR36m/‘242’ cross are being used. A subset of lines having varying hues of colored bran was selected and planted for seed increase in May 2019. This cross is also segregating for resistant starch (RS) and four genotypic classes differing in two starch genes were identified. Progeny from one of these classes that differed significantly in RS compared with the parent were planted weekly over 5 weeks to evaluate the effects of growing conditions during grain fill on RS; RS analyses were completed for these. A second mapping population from the double-mutant cross Katy mutant/IR36m was used to select the same four genotypic classes and F3 progeny were generated. Phenotypes and genotypes from the F3s were used to tag eight of the 20 known starch-synthesis genes. Results suggest that the mutation resides in a region of chromosome 8 known to contain two starch-synthesis genes. This region will be more finely mapped using additional recombinant progeny. As part of a USDA-NIFA grant with Louisiana State University, seed from more than 5,000 F3 plants were produced and will be evaluated for grain mineral content. These results will be combined with genotypes already determined from leaf DNA to identify QTLs via marker-trait linkages. Two field trials evaluating the impact of AWD water management on post-harvest grain quality were conducted. Data for the traits milling yield, grain chalkiness, grain shape, kernel weight, and grain functional traits were collected. Data on protein content is pending. Research is being conducted to understand the relative impact of multiple factors on storage stability of brown rice using 19 genotypes, each possessing at least one factor hypothesized to improve storage stability. Five traits including human sensory evaluation have been completed on samples taken every 3 months. Analysis of two more traits are pending. A wide range in concentrations of the lipid breakdown products was found. A subset of KBNT 1pa/ZHE733 RILs were selected based on genotypic and phenotypic data to understand the impact of genotype x environment on heat-induced chalk formation. In collaboration with ARS-Beltsville, two sets of experiments in temperature and CO2-controlled environments are complete and grain element analysis is being conducted. Yield trials are underway for potential new germplasm or variety releases that have improved blast disease resistance and improved grain market qualities.

Accomplishments
1. Identification of important plant traits for rice production that uses less water. Rice, which is usually grown in flooded paddies, is one of the biggest users of irrigation water and all USA rice varieties have been developed for production under this cultural management system. Identification of breeding materials and traits that are better suited for reduced irrigation management will help sustain USA rice production as water resources become more limited. ARS scientists at Stuttgart, Arkansas, in collaboration with the University of Arkansas and Arkansas State University have identified six plant traits that explain over 35% of plant yield when grown using a subsurface drip irrigation system that delivers uniform water stress in the field. Rice varieties that differ in response mechanisms to water stress were identified that will be useful for future gene discovery research that will ultimately aid breeders in developing improved rice varieties that can be grown with less water.

2. DNA markers developed for traits related to yield and quality. Rice variety development could be accelerated if SNP (single nucleotide polymorphism) markers were available to select for desired panicle architecture, plant maturity, translucent grains, and seed dimensions mandated by rice market classes, i.e. short grain, medium grain or long grain. Previously, ARS scientists at Stuttgart, Arkansas, identified DNA markers near genes controlling these traits but SNP markers in or tightly linked to these genes are needed for high throughput genotyping to be effectively utilized. The objective of this study was to develop SNP markers to assist rice breeders in incorporating genes controlling these traits as part of variety development programs. To this end, 18 SNP markers were developed, of which 14 markers were shown to be useful in selecting for the genes controlling desired panicle architecture, plant maturity, translucent grain, and grain dimensions. These SNP markers are now available to rice researchers for breeding and further research.

3. A mutant line with 2-fold greater embryo size increases vitamin E content and other lipophilic antioxidants in whole grain rice. Tocopherols, including vitamin E, tocotrienols, and gamma-oryzanol are lipophilic antioxidants with potential health beneficial properties for humans and functional properties for industrial use. ARS scientists at Stuttgart, Arkansas, and New Orleans, Louisiana, and with researchers at the University of Nevada, Las Vegas, evaluated concentrations of these lipophilic antioxidants and oil content in the embryo, bran and whole grain rice of a giant embryo rice grain mutant. The giant embryo mutant improves the health benefits of whole grain rice through higher contents of vitamin E, total tocopherols, tocotrienols and gamma-oryzanol as compared to the non-mutated rice variety. Bran and its products are used by the food and cosmetic industries, and the giant embryo mutant yields more bran, oil, total tocopherols and gamma-oryzanol than the control. These results demonstrate that mutation breeding can result in value-added rice varieties that have increased health beneficial compounds.

4. Nondestructive high-throughput hyperspectral imaging of rice grains can determine genetic subpopulation and grain chalkiness. For the first time, it is now possible to classify a rice grain according to its genetic subpopulation membership using high throughput non-destructive phenotyping. Previously, subpopulation classification required the use of multiple genetic markers in a destructive and expensive assay. A diverse panel of some 200 global rice genotypes was evaluated by ARS scientists at Stuttgart, Arkansas, using Vis/NIR spectroscopy which uses the visible and invisible regions of the spectrum. Results indicated that differences due to subpopulation and growing environment could be discerned. This technology will be valuable in rice germplasm curation by allowing accessions to be indexed according to genetic origin. The Vis/NIR phenotyping was also able to accurately measure the chalkiness of rice grains, which reduces crop value. This hyperspectral method will accelerate breeding progress for new high-quality rice varieties by replacing previously used evaluation methods that required intensive labor to quantify grain chalkiness.

5. Plant traits that reduce methane emissions in rice fields. Methane is an important greenhouse gas that is 25 times more potent than carbon dioxide and is the primary greenhouse gas emitted from flooded rice fields, which have ideal conditions for methanogens, the anaerobic bacteria that produce methane. Because of the extensive global rice acreage, reducing methane emissions due to rice production would have a significant impact on global warming. Research has shown that the amount of methane emitted from paddy grown rice can vary by variety, indicating that genetics has the potential for mitigating the effects of methane emissions. This study by ARS scientists at Stuttgart, Arkansas, documented that there is genetic variation for methane emissions that is related to shoot and root biomass. These findings will help breeders refine their method for identifying progeny having low methane emissions that will result in new rice varieties that will reduce global warming.

6. Rice varieties and genetic markers for developing salt tolerance in rice at the seedling stage. Salt stress reduces rice seedling growth and development and is an increasing problem for U.S. and global rice production due to saltwater intrusion into irrigation resources. Developing salt stress tolerance in rice is a challenge for U.S. rice breeders because few sources of salt tolerance are known, and knowledge of its genetic basis is limited. ARS scientists at Stuttgart, Arkansas, in collaboration with rice scientists from Cuu Long Delta Rice Research Institute in Vietnam screened 162 global rice varieties in a hydroponic system with high salt concentration and identified 12 that had tolerance to salt stress. Further, the team also discovered 12 novel molecular markers which were associated with salt tolerant traits. The newly identified varieties can serve as novel sources of salt stress tolerance in rice breeding. The genetic markers can be used in marker-assisted selection to accelerate development of new salt tolerant varieties and serve as a starting point for discovery of genes conferring enhanced salt tolerance in rice.

7. A rice tropical japonica mapping population is developed to identify genes controlling grain yield and quality. It is crucial to understand the genetic processes that control rice grain yield and quality to sustain production and marketability of USA rice. However, tropical japonica rice that is common in the USA is quite different genetically and phenotypically from the indica rice which is grown in most of Asia. Very few tropical japonica genetic populations are publicly available for gene mapping of these important traits. The Estrela/NSFTV199 mapping population that has been developed by ARS scientists at Stuttgart, Arkansas, is segregating for yield and grain quality traits and has been genotyped with 151 DNA markers to determine the approximate location of genes controlling number of panicles per plant, number of grains per panicle, grain size, days to maturity, and flag leaf dimensions. The population is being made publicly available for research groups to identify the underlying mechanisms controlling these yield and quality traits, evaluate the population for additional traits of interest, and for breeders to make crosses with their elite breeding materials to incorporate the desired trait(s) as part of variety development programs.

8. Establishment of a reference panel of blast disease fungal isolates for validating the presence of resistance genes in new rice cultivars. Rice blast is one of the most lethal rice diseases and it threatens stable rice production worldwide. Major blast resistance genes Pii, Pi-ta, Ptr, Pib, Pi-z, Pi9, and Piks/m/h are effective in preventing the infection of blast pathogen strains containing the corresponding avirulence (AVR) genes and are rapidly being incorporated into new rice varieties worldwide through a marker-assisted breeding approach. While marker-assisted selection is quite reliable, it is not 100% effective except for functional nucleotide polymorphic markers (FNP). However, the FNP is not available for many resistance genes and can be complicated to use. Before new cultivars are released for commercialization, validation of the presence by these major blast resistance genes is important to ensure resistance to this disease. ARS scientists at Stuttgart, Arkansas, in collaboration with scientists at Kansas State University, the University of Arkansas, and Louisiana State University have identified a diverse set of 30 blast fungal isolates from 1,000 that were collected from 1960 to 2015 in the USA and have verified the presence of AVR genes through gene-specific DNA markers and observing symptom development in comparison with an international rice blast differential system. These blast isolates can be used by breeders to verify the resistance in new cultivars as well as understand resistance mechanism that underlie these blast resistance genes.

9. Selecting for changes in root size using a rapid, non-destructive assessment of above-ground tiller number in rice breeding. Breeding for improved rice root systems could improve crop yield by increasing water and nutrient uptake, and smaller root systems have been shown to reduce methane emissions. Direct observation of roots under field conditions generally requires labor-intensive destructive sampling, which limits the ability to breed for optimized root size or architecture. This study by ARS scientists at Stuttgart, Arkansas, documented that genes affecting tiller (stem) number also have a large effect on root biomass. This indicates that tiller number can serve as a rapid, visual, non-destructive proxy for selecting for changes in root biomass. Further analysis of roots in one of the three study populations indicated, however, fine tuning of root biomass will require continued research to identify additional genetic loci that are independent of tiller number. These results provide a more efficient method for selecting for changes in root biomass in rice breeding programs that aim to develop varieties that utilize water and nutrients more effectively to produce higher grain yields.

10. Root traits can be used to select for reduced methane emissions in new rice varieties. Aerenchyma is a plant tissue that forms air spaces, generally in underground roots, which acts as a conduit for gases, i.e., oxygen, methane, and nitric oxide, to move through the plant. In flooded rice fields, wherein plant roots-soil-microbes interact, methane gas is formed in the rhizosphere and it is emitted into the atmosphere primarily by passage through the aerenchyma. Methane is a greenhouse gas that is known to have 25 times the global warming potential as carbon dioxide and flooded rice paddies are a major contributor. This study evaluated 39 rice varieties and demonstrated the existence of genetic variation in percent aerenchyma found in the cross section of rice roots. However, percent aerenchyma was not significantly different among varieties known to be high- or low-methane emitters indicating that it is not a major factor affecting methane emissions. When root biomass was considered together with percent aerenchyma, a positive correlation with methane emissions was observed. This study by ARS scientists at Stuttgart, Arkansas, demonstrated that varieties with bigger roots also developed larger total aerenchyma area, and as a result, methane emissions were increased. This information will be useful to breeders as they attempt to optimize root biomass for nutrient and water uptake while minimizing methane emissions from rice fields.

11. Establishment of a non-destructive single kernel method using near-infrared for classifying chalky rice kernels. Translucent whole milled rice grain is critical in domestically produced long grain rice that is sold in U.S. and foreign markets. A non-destructive, repeatable, high-throughput instrumental method that can be used in grain inspection and research associated with cultivar development is needed as well as understanding genetic and environmental factors that impact the formation of opaque chalky areas in the grain. ARS scientists at Stuttgart, Arkansas, in collaboration with scientists at Manhattan, Kansas, established a single kernel near-infrared method (SKNIR) that classifies chalky kernels with accuracy levels above 82% as compared with traditional, more labor-intensive methods. The wavelengths selected that were the most effective for discriminating grain chalk were partially based on the absorption bands for starch, protein and water. This method will accelerate improvement of U.S. cultivars for milled rice translucency which will increase crop value.

12. Establishment of digital imaging methods for predicting visual appearance traits of milled rice. Traits associated with milled rice appearance, including grain chalk, determine its grade, price and marketability. These appearance traits are evaluated visually (VI) in the USA by officially trained grain inspectors whereas a digital imaging system (IS) might provide objective and quantifiable measures for these traits. ARS scientists at Stuttgart, Arkansas, in collaboration with USA rice breeders in the Southern states and California and with commercial grain inspectors sought to identify quality parameters from IS for predicting five appearance quality traits commonly evaluated through VI. For chalkiness of the milled rice, of the three IS tested, two provided better correlation with the chalk values determined by VI. Statistical analyses identified quality parameters from the IS that partially interpreted and predicted the milled rice appearance traits - bran streaks, chalk, kernel color, uniformity of grain length and overall appearance as rated by VI. The newly established IS models for milled rice appearance traits provide increased speed and repeatability and are objective and quantitative which will benefit breeding programs developing U.S. cultivars with superior milled rice quality.

13. Identification of optimal hyperspectral wavebands for detection of discolored, diseased rice seeds. The inspection of rice grain infected by seedborne disease is important for ensuring uniform plant stands in production fields as well as preventing proliferation of some seedborne diseases. A hyperspectral imaging (HSI) technique was used by ARS scientists at Stuttgart, Arkansas, to find optimal wavelengths and develop a model for detecting discolored, diseased rice seed infected by bacterial panicle blight (Burkholderia glumae), a seedborne pathogen. Hyperspectral images of a total of 500 seeds, 250 non-diseased and 250 diseased, were evaluated, and found that the violet and red regions of the visible spectrum were key wavelengths reflecting the characteristics of the discolored rice seeds, and further showed that only two to three wavelengths are needed to differentiate between discolored, diseased and sound rice, instead of using the entire HSI wavelength regions. This research demonstrated the feasibility of developing a low-cost multispectral imaging technology based on these selected wavelengths for non-destructive and high-throughput screening of diseased rice seed.

U.S. DEPARTMENT OF AGRICULTURE

Dale Bumpers National Rice Research Center: Stuttgart, AR