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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

2023 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
This will be the final report this project and will be replaced with pending project entitled “Broadening and Strengthening the Genetic Base of Rice for Adaptation to a Changing Climate, Crop Production Systems, and Markets”. ARS in-house and grant supported research have progressed and ended with 4 scientific vacancies. Most subobjectives of Objectives 1 and 2 were completed. A total of 1800 rice accessions from 2019-2022 were rejuvenated for the National Small Grains Collection (NSGC), and several mapping populations were characterized including recombinant inbred lines (RIL) derived from the crosses of Minghui63 by M202, Cybonnet (CYBT) by Saber (SABR), a Multiparent Advanced Generation Intercross (MAGIC) population, a Tropical japonica (TRJ) core diversity panel, and TeQing-into-Lemont backcross introgression lines (TILs), resulting in the release of two rice varieties and one germplasm line. Quantitative trait loci (QTL) and genetic resources were identified for agronomically important traits including tiller number, biomass, disease resistance, and tolerance to soil moisture extremes and straighthead. Methods were developed to scan single kernels and disinfect seed. In FY23, approximately an additional 1000 rice accessions of NSGC are being rejuvenated using summer nurseries and greenhouses. Seed, data, plant and panicle images will be made public through the Germplasm Research Information Network (GRIN)-Global. The NSGC accessions that had three accessions with the same name in GRIN-Global were evaluated phenotypically and genotypically to reduce redundancy in the collection. About 300 accessions requested from Fort Collins were evaluated for the correct phenotype in the field, per, and will be genotyped later. Also, another set of 300 accessions from Fort Collins will be grown in the greenhouse this summer to evaluate for correctness. During the 2022 field season, 198 rice accessions from the NSGC rejuvenations were identified as daylength sensitive in the Arkansas environment. The NSGC and the Genetic Stocks Oryza (GSOR) shipped 1,093 and 27,569 rice accessions, respectively, during 2022. GSOR provided 4,595 accessions to domestic researchers of which 478 were to Arkansas researchers, and 22,974 were sent to international researchers. The TRJ core diversity panel (over 500 accessions) was established, extensively genotyped, and phenotyped. Of these, 398 TRJ accessions and two non-TRJ controls were genotyped via skim sequencing with an average of 3X genome coverage. SNP calling was accomplished using Google Deep Variant and Glnexus on the USDA-ARS SCINet High-Performance Computer system and imputation was performed resulting in a set of 5,231,433 single nucleotide polymorphisms (SNPs). After filtering, 436,022 biallelic SNPs were used in a genome-wide association study to identify genetic loci affecting agronomic and morphological traits. This will serve as an important resource for the new project plan and enable mining genes/alleles to benefit US rice breeding efforts. For the MAGIC population, about two-thirds of the year 2 yield component data collection was completed. Once completed, QTL and Bayesian Network analyses will be conducted to identify genes underlying yield related traits. Of the 150 accessions included in the Africa Rice Panel, 87 were skim sequenced. Once the genotypic data is analyzed and combined with published genotypes for the remaining accessions, the genome-wide association mapping will be conducted utilizing previously collected phenotypic data. A total of 200 blast isolates from the southern USA and Puerto Rico were characterized for the existence of five avirulence genes and 11 single sequence repeat markers, and for pathogenicity toward selected differential rice varieties to identify effective blast resistance genes for deployment. A total of 30 blast races was assembled for breeders to identify and verify blast resistance genes. The root architecture of 2-week-old seedlings for about 120 TILs (3 reps each) was defined using more than 100 data points per sample acquired using winRhizo analysis of root images. Data collected in FY22 was analyzed for outliers, BLUPs for future trait-by-trait and QTL analyses are now being calculated. Preliminary data from three mapping populations resulted in one (CYBT x SABR) being selected for field evaluation under alternative wetting and drying (AWD) management in FY23. Six Chromosome Segment Substitution Line (CSSL) libraries were characterized for six agronomic traits and seed size. To determine whether introgressions from the wild donors enhanced yield and yield components, the three Cybonnet CSSL libraries were evaluated over two field seasons and characterized for 17 yield related traits. Current analyses suggest the wild introgressions in the Cybonnet CSSLs did not enhance grain yield but rather decreased it. Analysis of the genotypes for the Presidio/O. rufipogon (Wild-5) advanced backcross population revealed 14 monomorphic regions, thus the population is currently being genotyped with 49 additional markers in these regions. The population is being evaluated for reaction to rice leaf blast disease using two methods and three blast races. The second year of testing 144 diverse rice lines along with checks is in progress for identifying heat stress tolerant TRJ rice germplasm. Increasing dietary intake of resistant starch (RS), a dietary fiber, can stabilize blood sugar levels and decrease cancer risk, making it desirable to increase RS in rice grains. Three QTL for RS discovered in FY22 were validated in FY23 using F6:7 progeny lines of IR36m x ‘242’. Grains from F4 plants of a cross between the novel starch mutant line (KatyM) and IR36m were provided to collaborator who verified the novel KatyM mutation alters starch structure. The individual and combined effects of the KatyM mutation with two genes previously known to affect the grain RS content were studied. The KatyM mutation was found to not be functionally compatible with the sbe3 mutation, and the gene combination that provided the highest grain RS was Wxa combined with sbe3. Two QTL for reduced grain arsenic content on chromosome (chr) 2 and chr7, and one that increased phosphorus, magnesium, and potassium altogether on chr2 were validated in FY23 using grains from F3:4 plants. Thirty TILs selected for having extreme differences in tiller number and root biomass, were grown a second year under AWD versus flooded management and collected plant samples were analyzed. Data validated FY22 findings that some of these lines tolerate the severe dry conditions associated with AWD better by maintaining a high number of tillers per plant, while other lines retained yield under AWD by maintaining higher panicle size and fertility. A QTL for maintained tiller number initially mapped to chromosome 4 using F2 data (FY21) was validated using both F2:3 progeny (FY22) and F3:4 progeny (FY23). Similarly, using progeny from a second cross, a QTL for high fertility and panicle weight under AWD was mapped and validated to chr5. The two TILs were crossed, and progeny containing both AWD-tolerance QTLs selected using molecular markers in FY23 are currently being evaluated for AWD-tolerance in field plots. Due to 4 critical vacancies, significant portions of the Objectives 3 and 4 were dropped. For the Subobjective 3B, to evaluate and validate yield potentials of rice lines with blast resistant QTL, 12 rice germplasm with different combinations of blast resistance genes were grown for yield, and yield components under continuous flood and AWD for 3 years. In FY23, two rice resistant rice germplasm lines with enhanced yield potential were selected and are being grown in uniform regional rice nursery for potential germplasm release. For the Subobjective 3C, to determine the impact of AWD irrigation management on soil-microbe- root interactions in rice production, two rice varieties Koshihikari and Saber were grown under three irrigation managements (3 reps each), AWD, mid-summer drainage and continuous flood. A total of 72 soil samples were collected from 4 plant growth stages and will be analyzed to determine if there is a correlation between differences in soil microbes and yield components. For the Subobjective 4A, analysis of the segregating progeny of the three Jefferson/O. rufipogon introgression lines for yield-related traits revealed the previously reported yield advantage was not a result of the single targeted O. rufipogon introgression on chr2, chr4 or chr8, but rather multiple loci (introgressions), thus these loci were not fine-mapped (Subobjective 2D) or pyramided, and subsequently evaluated in the field for yield enhancing traits. For the Subobjective 4C, in FY23, 45 BC6F8 (Katy into M202) rice breeding lines selected based on panicle size and blast resistance genes Pi-ta/Ptr are being grown using replicated yield plots to determine if they exhibit a yield penalty. For the Subobjective 4D, to facilitate the development of new rice varieties, an unoccupied aerial vehicle (UAV) was used to predict plant nitrogen status and days to heading, facilitating the use of this technology for phenotyping biotic and abiotic stress under field conditions.

Accomplishments
1. Release of USDA-Tiara purple bran rice: Delicious and nutritious. Consumers desire to have both flavorful and nutritious food. Most rice is consumed in the milled form where the outer bran layer is removed, but this is where most of the protein, vitamins, fiber, and antioxidants are in the grain. ARS researchers in Stuttgart, Arkansas, conducted research over 16 years to develop a long grain rice variety, USDA-Tiara, having purple bran that is enriched with anthocyanins, a potent antioxidant also found in blueberries. In addition, it is an aromatic rice having a nutty, popcorn flavor when cooked. Such specialty cultivars offer an economic opportunity for small independent growers who can maintain identity preservation versus the co-mingling of rice varieties that is performed at large rice mills. Thus, the release of USDA-Tiara, which is now in commercial production in the southern U.S., will bring greater value to rural communities as well as enhanced nutrition for consumers.

2. Release of Santee Gold rice: An heirloom rice with superior culinary properties. Rice production dates to the 18th century on the southeast coast of the USA with the variety Carolina Gold. It was an extremely valuable export because of its superior cooking quality, serving as the foundation for the US rice industry, and it is still grown today for gourmet chefs. After a 14-year breeding process, Santee Gold was released by the ARS researchers in Stuttgart, Arkansas, a variety which has greater crop productivity and longer grain shape as compared to its parent Carolina Gold while maintaining its renown culinary properties. Santee Gold is currently in commercial production in the southern USA and provides a new avenue for increased economic value for independent farmers and millers interested in capturing value added markets.

3. Genetic loci for three key weedy traits identified. Weedy rice is a non-domestic relative of cultivated rice that poses a significant threat to crop production by competing with crops and causes considerable yield loss. The annual yield loss in the United States alone due to weedy rice infestations could feed approximately 12 million people. Weedy rice has evolved independently from various cultivated rice groups through a process of de-domestication, during which it has evolved a suite of traits collectively known as the "agricultural weed syndrome." These traits include rapid growth, high nutrient use efficiency, seed dormancy, seed dispersal, and herbicide resistance. However, the genetic basis underlying these weediness traits remains to be fully elucidated. To address this knowledge gap, an ARS researcher in Stuttgart, Arkansas, with researchers at the University of Massachusetts, Amherst, Massachusetts, developed a novel mapping population through a cross between BHA weedy rice and aus cultivars to explore the genetic mechanisms underpinning three key weedy traits: flowering time, plant height, and seed shattering. Genetic loci were mapped using a combination of bulked segregant analysis and high-throughput whole-genome re-sequencing, resulting in a more time-efficient and accurate method. The loci and candidate genes identified not only provide insights into the genetic basis of evolutionary mechanisms of these traits but also have the potential to inform weed management strategies and help identify rice varieties that are more likely to produce weedy descendants.

4. Acquiring new germplasm to explore heat and drought tolerance. The United States rice collection which is part of the National Small Grains Collection (NSGC) contains only 205 accessions that are not Oryza sativa, the cultivated rice grown in the USA. New Rice for Africa (NERICA) germplasm has O. glaberrima DNA incorporated into O. sativa to improve the drought tolerance of O. sativa. Additionally, O. australiensis is a source of heat tolerance. To expand the sources of drought and heat tolerance for U.S. rice improvement, ARS researchers in Stuttgart, Arkansas, in collaboration with USDA-APHIS imported eighteen NERICA germplasm lines, from AfricaRice, Ivory Coast and 35 O. australiensis accessions from the International Rice Research institute (IRRI), Philippines. In addition to drought tolerance, the NERICA lines reportedly have high yields. Besides heat tolerance, the O. australiensis accessions possess salt tolerance. Seed is currently being increased for distribution and the accessions are being phenotypically characterized in the field (NERICA lines) and greenhouse (O. australiensis). These new accessions are a potential source of beneficial alleles for improving drought and heat tolerance in US cultivars as a response to climate change.

5. Exploring the genetic diversity in rice cultivars conserved in the AfricaRice Gene bank for rice improvement. The crop diversity conserved in gene banks is important to global food security. Rice (Oryza sativa L.) is grown in 40 of the 54 countries in Africa. The AfricaRice gene bank conserves 14,480 rice cultivars with 75% of these cultivars originating from African countries and includes both traditional farmer’s cultivars (landraces) and modern rice cultivars. Genotyping these cultivars (or accessions) provides insight into the genetic diversity among the accessions and enhances use of the collection. ARS researchers in Stuttgart, Arkansas, and Pullman, Washington, contributed to interpretation of the genetic diversity identified among 5,738 rice accessions from the AfricaRice Center, Abidjan, Ivory Coast. The DNA marker variation among the genotyped accessions grouped them into the Indica and Japonica subspecies, traditional (landraces) and modern cultivars, and lowland, upland and mangrove forest (swamp) ecologies for growing rice. Based on the genetic diversity, a subset of 600 accessions was selected and identified as the AfricaRice Oryza sativa Core Collection (AROSCC). The AROSCC captured more than 95% of the genetic variation observed among all the genotyped accessions and is an important resource for rice breeders and rice improvement programs around the world. It can be screened for important traits of interest, and the likelihood of finding these useful traits in one or more of the 600 accessions is nearly as high as if all 5,783 accessions were screened, thus saving considerable time and resources.

6. Identification of genes for panicle architecture and seed traits that enhance yield. Rice is the staple food for over half of the world’s 7.6 billion people and population growth is predicted to reach 8.6 billion in 2030 and 9.8 billion in 2050. To meet this growing demand for food, it is essential to understand the plant processes that control grain yield, thus expediting breeding efforts to produce more pounds of rice per acre. Research conducted by ARS researchers in Stuttgart, Arkansas, in collaboration with the University of Arkansas focused on identifying genes that control traits associated with improved rice yield, including number of panicles per plant, number of panicle branches per panicle, number of seeds per panicle, seed size (length and width,) and seed weight. To better understand the biological processes controlling these traits, a global collection of 400 diverse rice varieties was phenotyped for these yield-related traits and mapped with DNA markers to identify the genes controlling these traits. To validate these results, two diverse rice varieties in this collection, L-202, which is short statured with a long grain originating from California, USA and Trembese which is tall, with a medium grain length originating from Indonesia, were selected as parents for population development. The population was advanced eight generations and evaluated for the same yield-related traits. Using DNA markers, we identified 37 chromosome regions across both studies where genes controlling yield-related traits are located. Most significant was a genomic region on chromosome 7 which affects the number of seeds per panicle, panicle branching, seed length, and seed weight. These results will be used to develop user friendly DNA markers associated with these yield-related traits that can be used by rice breeders to accelerate selection for the desired panicle architecture, seed size (length and width) and seed weight. Ultimately, these “tools” will be used to improve rice yield.

7. Quantification of trade-offs between rice yield and grain quality under heat stress. Increased heat stress during cropping season poses significant challenges to rice production, yet the complex stoichiometry between rice yield, grain quality, and high daytime (HDT) and nighttime temperature (HNT) presents significant gaps in scientific knowledge. ARS researchers in Stuttgart, Arkansas, in collaboration with Clemson University performed meta-analyses and synthesized the state of knowledge. The comparison of various components of yield and grain quality against HDT and HNT demonstrated that HDT- and HNT-stresses have their unique effects. The study also suggested that grain quality should be considered for selection and breeding of heat stress tolerant rice varieties in response to rising ambient temperatures during cropping season. These meta-analyses will serve as a powerful resource tool to give directions and guide in future planning of heat stress tolerance research in rice crop.

8. Saving water and preserving rice grain nutrition. New methods of rice production are being adopted that save water resources and reduce methane emissions. Methods which allow the soil to go through drying periods during the season can affect the availability of certain minerals for plant uptake. Research was conducted by ARS researchers in Stuttgart, Arkansas, in collaboration with Cornell University to evaluate the effect of water saving practices as compared to conventional flooded fields on the availability of several trace elements in the soil. Reduced irrigation can be safely used without negatively impacting elements that are important for plant growth. Both arsenic and cadmium, which are concerns for human health, can also be effectively controlled under these systems. Thus, rice can be grown under a management system that will save water resources, have a reduced environmental effect, and will not compromise the nutrients needed for plant growth and human consumption.

9. Discovery of genetic loci that increase rice pericarp concentrations of anthocyanins and proanthocyanidins. Anthocyanins and proanthocyanidins are the antioxidant pigmented flavonoids that give blueberries, cranberries, and red wine their colour and health-beneficial attributes. Rice grains with purple coloured bran also contain anthocyanins, and red-bran rice contains proanthocyanidins. ARS researchers in Stuttgart, Arkansas, identified new genes and gene combinations that increase the concentrations of these desired compounds in rice grains. At the outset of this study, it was known that the biosynthesis of anthocyanins (purple) and proanthocyanidins (red) in rice bran are turned on and off by the Pb and Rc genes, respectively. Study of the two compounds individually revealed two QTLs, one each on chromosomes 3 and 7, that increased anthocyanin content, and identified the same region of chromosome 3 plus a QTL region on chromosome 5 as enhancing proanthocyanidin content. Anthocyanins and proanthocyanidins share a large portion of their biosynthetic pathways. The shared QTL on chromosome 3 appears to enhance activity at one or more of the shared biosynthetic steps, while the other QTLs impact downstream steps that are unique to anthocyanin or proanthocyanidin synthesis. Because of shared precursors between anthocyanins and proanthocyanidins, it was anticipated that the synthesis of one compound would divert limited precursors from the synthesis of the other compound, causing a trade-off between the two compounds. What was observed was mutual enhancement, suggesting that Pb and Rc not only turn on synthesis of anthocyanins and proanthocyanidins, respectively, but also increase flux through shared biosynthetic pathway such that concentrations of both antioxidant pigments was maximized by combining Pb and Rc along with the QTLs identified on chromosome 3, 5, and 7. Rice breeders can use this gene-stacking information, and the molecular markers used to tag the 3 identified QTLs to breed rice varieties with increased content of beneficial antioxidant compounds.

10. Grain arsenic content found reduced by sequestration of arsenic in flag leaves. Arsenic in rice poses a concern for some consumers and baby food producers. Arsenic is also toxic to plants and causes straighthead disorder in rice. ARS researchers in Stuttgart, Arkansas, in collaboration with Louisiana State University determined that one method used by rice plants to reduce accumulation of arsenic in grains is to trap it by sequestration in flag leaves. Retaining arsenic in leaves during the grain filling period effectively prevents it from being transported to grains. Because sequestration of arsenic in leaf cells involves several sulphur containing compounds, the researchers also investigated if increasing plant sulphur content through foliar fertilization could further reduce grain arsenic concentrations or decrease the severity of arsenic-induced straighthead disorder. While an increase in grain sulphur concentration verified uptake of sulphur fertilizer through leaves, foliar application of sulphur did not prove to be a useful method for limiting the accumulation of arsenic in US produced rice, nor did it reduce straighthead severity. The connection discovered between leaf sequestration and grain concentration of arsenic will direct future research aimed at identifying genes and production methods to reduce rice grain arsenic concentrations.

U.S. DEPARTMENT OF AGRICULTURE

Dale Bumpers National Rice Research Center: Stuttgart, AR