APPLYING ADVANCES IN GENOMICS TO ACCELERATE RICE IMPROVEMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1009299
• Project No.: TEX09629
• Project Start Date: Feb 11, 2016
• Project End Date: Feb 10, 2021

Project Director
Thomson, MI, .

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Soil & Crop Sciences

Non Technical Summary
In the U.S., rice is grown on an average of 2.6M acres producing approximately 10M tons of grain per year, equivalent to $3.6B in market value, largely in the southern states of Arkansas, Louisiana, Missouri, Mississippi and Texas. Rice is an important component of a healthy diet, and it is a key to maintaining global food security. A number of challenges, however, face rice production both in the U.S. and around the world, including constraints in water supplies, emerging pests and diseases, increasing costs of inputs, and plateauing yields. At the same time, environmentally sustainable ways need to be found to produce enough food for 9.6 billion people by 2050 without converting additional land to rice production. There are also several areas where the nutritional content and healthiness of rice can be further improved. Thankfully, recent scientific advances in rice research and new plant breeding technologies now provide faster and more powerful ways to develop higher yielding, stress tolerant and more nutritious rice varieties.This project will take advantage of the latest advances in rice genetics, whole genome sequencing, and information technology to better explore the wealth of biodiversity available in rice and its wild relatives and identify new genes for rice improvement. It will leverage recent investments in plant genomics and work closely with rice breeders to bridge the gaps between genetic resources and plant breeding, between genomic discovery and application, and between the US and global rice research communities. The approaches will include testing exotic rice accessions, mapping novel genes, analyzing DNA sequence data, developing new pre-breeding materials, and helping rice breeders use the latest molecular marker technologies to speed up the process to develop improved rice varieties. The ultimate goals of the project will be to increase the competitiveness of US rice on the global market through lower production costs and superior grain quality, to improve the nutritional quality of rice for improved healthiness of rice consumers, to work with international partners to help secure a stable supply of rice around the world in the face of future constraints, and to support more environmentally sustainable methods of rice production.

Animal Health Component

0%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1530	1080	30%
202	1530	1080	20%
203	1530	1080	10%
203	1530	1081	10%
204	1530	1080	10%
204	1530	1081	10%
212	1530	1080	10%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 204 - Plant Product Quality and Utility (Preharvest); 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms; 202 - Plant Genetic Resources;

Subject Of Investigation
1530 - Rice;

Field Of Science
1081 - Breeding; 1080 - Genetics;

Keywords

plant breeding

crop variety improvement

genomics

molecular genetics

marker-assisted selection

dna sequencing

stress tolerance

disease resistance

yield improvement

human health and nutrition

rice (oryza sativa)

Goals / Objectives
The goals of the project are to: (A) to increase the competitiveness of US rice on the global market through lower production costs and superior grain quality; (B) to improve the nutritional quality of rice for improved healthiness of rice consumers; (C) to secure a stable supply of rice around the world in the face of future constraints, and (D) to support more environmentally sustainable methods of rice production.The specific objectives are to:1. Assist US rice producers by lowering production costs through improved varieties: The first objective is to help breeders develop improved rice varieties that perform well with reduced inputs (less fertilizer, pesticide, and water use) by using marker-assisted backcrossing to transfer alleles from exotic germplasm that provide nitrogen-use efficiency, disease resistance, and higher yields under water-saving conditions.2. Improve the grain quality of inbred and hybrid US rice germplasm. The second objective is to identify and characterize genes controlling grain quality traits, including milling quality (proportion of whole grains after milling), grain shape, and the taste and texture after cooking, and to develop improved molecular breeding strategies for developing varieties with superior grain quality.3. Develop improved brown rice for better nutrition and food safety: Although brown (unmillled) rice has higher levels of fiber and essential minerals and fatty acids than white (milled) rice, the vast majority of rice consumers still prefer white rice. The third objective will be to improve the various aspects of brown rice by investigating the genetic components underlying taste, texture, cooking time, shelf life, nutrition, and arsenic levels of unmilled rice, which should support future efforts at improving consumer acceptance of brown rice for better human health and nutrition.4. Improve global rice production through higher yields and stress tolerance: The fourth objective will be to increase rice yields by identifying genes controlling yield components and by integrating crop modeling and genomic selection to increase the rate of genetic gain in breeding programs, while using molecular markers to select for improved stress tolerance to reduce crop losses in farmers' fields.5. To support environmentally sustainable methods for rice production: Future yield increases should be integrated with sustainable approaches that limit the impact of agriculture. The fifth objective will be to identify and transfer exotic alleles for improved nutrient-use efficiency, weed competitiveness and disease resistance to reduce the amount of needed inputs, while at the same time increasing the overall genetic diversity of crops to be more sustainable under future constraints.

Project Methods
Allele mining of sequence data to explore genebanks for useful variation: This project will explore new methods to identify beneficial alleles defined by patterns (also known as haplotypes) of single nucleotide polymorphism (SNP) markers at key genetic loci across thousands of sequenced rice accessions. By leveraging international efforts to sequence rice accessions, such as the 3,000 Rice Genomes Project, novel alleles will be identified and validated and the rice haplotype map (HapMap) will enable imputation-based approaches to use lower density genotyping to impute functional polymorphisms in breeding populations.Discovery of trait-enhancing alleles using backcross populations: This project will implement a large-scale backcross isogenic line strategy using backcross nested association mapping (BC-NAM) combined with high-throughput phenotyping to identify novel yield-enhancing alleles that are functional in high-yielding genetic backgrounds. While yield will be the primary target trait, the BC-NAM populations will enable powerful screens across a number of different traits to identify alleles that are normally hidden but are functional in a high-yielding genetic background.Rapid QTL and association mapping using diverse germplasm: QTL mapping and cloning will be pursued for identification of genes with beneficial alleles even if they are from a rare genetic donor, while GWAS will be used to obtain a comprehensive survey of the common alleles controlling traits of interest across larger sets of germplasm. This project will compile all published QTL and GWAS data for target traits, and work with collaborators to identify novel genetic donors for further studies where large-effect and robust genetic loci are lacking.Allele validation through SNP marker testing and genome editing: Once novel genes and alleles are discovered through the approaches described above, validation of the alleles is needed before breeders can make use of them for marker-assisted selection. SNP markers will be validated for tracking alleles of interest (i.e. SNP haplotypes); testing the effects of these SNP haplotypes in germplasm panels and breeding-relevant segregating populations; and validating specific functional nucleotide polymorphisms (i.e. causal variants) using CRISPR/Cas9 genome editing to determine the phenotypic effects of different allelic variants to enable more precise marker-assisted selection in the future.Integrating technologies to accelerate rice breeding: This project will work closely with breeders to implement these technologies: (1) provide packages of high-value alleles from novel donors in elite breeding backgrounds (i.e. trait-enhancing introgressions in isogenic lines) with their corresponding SNP markers to enable precise selection of yield components and other traits; (2) implement a genome-wide selection strategy that combines marker data on training populations with crop modeling to provide more accurate prediction-based selections across the target environments; and (3) optimize the logistics of breeding to increase the population size, selection intensity, and speed of the selection cycles, through high-throughput field-based phenotyping, improved breeding informatics tools for data collection and analysis, and rapid generation advance (accelerated single seed descent) combined with large-scale SNP genotyping and imputation for haplotype tracking and genome-wide predictions.Evaluation plan:The success of this project will be evaluated through the following key milestones and measurable indicators of success:High-throughput single-nucleotide polymorphism (SNP) genotyping will be routinely used for marker-assisted selection at known major genes by Texas public rice breeding programs within 2 years of project initiation.At least one new partnership will be formed to promote breeding of traits for improved brown rice healthiness, nutritional content, and consumer acceptance by year 3.A BC-NAM population will be developed and used to identify novel trait-enhancing alleles by year 4 of the project.Gene-edited lines for single traits will be available by end of year 2 and gene-edited pyramid lines containing multiple alleles from exotic rice in a high-yielding genetic background will be available by the end of year 4.Public hybrid rice programs in the U.S. supported with new data on heterotic groups and molecular markers for hybrid seed production traits, grain quality, and improved hybrid performance predictions by the end of the project.Near-isogenic backcross lines will be provided to breeders for key traits by the end of the project.

Progress 02/11/16 to 02/10/21

Outputs
Target Audience:Efforts were made to build relationships and provide useful outputs with several target audiences, including: (1) Texas rice breeders: I continue to be in contact with the rice breeders at the Texas A&M AgriLife Research Center in Beaumont to discuss how molecular technologies can be used for rice improvement; (2) U.S. rice scientists: I am in contact with the USDA ARS rice group at the Dale Bumpers National Rice Research Center, along with others from the U.S. rice community, to discuss the genetic control of grain quality and other traits; (3) Texas A&M plant breeding community: my team in the Crop Genome Editing Lab worked to complete several seed grant projects in gene editing supported by Texas A&M AgriLife Research in collaboration with breeders and researchers working on crop improvement; (4) the international rice community: discussions were held with scientists from the International Rice Research Institute (IRRI) in the Philippines and the National Chung Hsing University in Taiwan to discuss more details for future collaborative research projects for rice improvement; and (5) the scientific community: the scientific outputs in the form of conference abstracts and publications in journal articles provide useful results in plant genetics, gene editing, and crop improvement to the broader scientific community. Changes/Problems:The only major problem encountered was restrictions in the number of lab personnel to be working in the lab during the COVID-19 lockdowns in 2020. This resulted in some delays in completing the research objectives, but overall was able to be managed, since our lab was never shut down completely due to being designated as performing essential research activities. What opportunities for training and professional development has the project provided?This project has provided multiple opportunities for training and professional development. First, a post-doctoral research associate has been involved in the project for the past three years, and has received training in CRISPR/Cas-mediated gene editing techniques and professional development through presenting at conferences, supervising graduate students, and writing manuscripts. Next, several graduate students have received training in genetic mapping and gene editing and have successfully graduated, including a PhD student performing the rice salinity tolerance QTL mapping (graduated in Fall 2020), a PhD student performing the rice grain quality GWAS experiments (graduated in Summer 2021), and two MSc students optimizing rice genome editing protocols (one graduated in Fall 2020 and one graduated in Fall 2021). How have the results been disseminated to communities of interest?The Project PI (M. Thomson) presented results from this project to the scientific community in several forums, including oral presentations at scientific conferences (International Plant and Animal Genome Conference, San Diego, CA, January 2016; Rice Technical Working Group Meeting, Galveston, TX, March 2016; ASA/CSSA/SSSA Annual Meeting, Phoenix, AZ November 2016; Southern Seed Association Annual Convention, San Antonio, TX, January 2017; Texas Seed Trade Association 86th Annual Convention, Galveston, TX, June 2017; ASA/SSSA/CSSA Annual Meeting, Tampa, FL, October 2017; Rice Technical Working Group Meeting, Long Beach, CA, February 2018; International Rice Research Congress: 8th Rice Genetics Symposium, Singapore, October 2018; International Symposium of Rice Functional Genomics, Taiwan, November 2019; Symposium on Novel Biotechnology and Breeding, NCHU, Taiwan, November 2019; ASA/SSSA/CSSA Annual Meeting, San Antonio, TX, November 2019; USA Rice Outlook Conference, Little Rock, AR, December 2019; and the Plant and Animal Genome Conference, San Diego, CA, January 2020) and invited seminars (USDA-ARS National Rice Research Center, Stuttgart, AR, July 2017; Taiwan Agricultural Research Institute, Taiwan, July 2018; University of Arkansas, Food and Agribusiness Webinar, November 2018). Moreover, 19 peer-reviewed journal articles were published in scientific journals across the 5-year project. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research project aimed to identify and characterize key genes controlling high-value traits in rice, which will ultimately lead to the goal of accelerating the development of high-yielding, stress-tolerant, more nutritious rice. Towards this end, the project has focused primarily on two approaches: QTL and association mapping of key genetic loci, and setting up a more efficient genome editing platform for functional validation of candidate genes. This 5-year project has successfully delivered the following accomplishments for each of the proposed research objectives: 1. Assist US rice producers by lowering production costs through improved varieties: The first objective was to help breeders develop improved rice varieties that increase the profitability of rice producers, which is a long-term objective that extends beyond this 5-year project. The first step towards that objective was to implement genome-wide association studies (GWAS) using a diverse set of rice germplasm accessions grown under Texas field conditions and genotyped with a genome-wide set of single nucleotide polymorphism (SNP) markers. Through a collaboration with Cornell University and the International Rice Research Institute (IRRI), a previously deployed 6K SNP array (Thomson et al. 2017) was upgraded to a better-performing 7K SNP array and validated across a germplasm panel of diverse rice accessions (Morales et al. 2020). At the same time, approximately 300 O. sativa accessions were planted in the field in the Texas A&M AgriLife Research Center in Beaumont in the summer of 2017 for phenotyping for key traits. The following year, approximately 200 O. sativa accessions were planted in the field in the summer of 2018 for additional phenotyping, including heading date and grain quality parameters. When combined with the 7K genotype data, the GWAS results have revealed the chromosome location of important genes across a number of important traits, such as for cold tolerance in rice (Thapa et al. 2020). Other studies are currently being prepared for publication, as described below. In addition to the GWAS activities, this project aimed to build the foundation for the development of more efficient CRISPR/Cas-based gene editing technologies that can accelerate future crop improvement efforts. As a first step, we optimized Agrobacterium-mediated transformation and regeneration of the U.S. rice variety Presidio (Molina-Risco et al. 2021). We also initiated experiments to test in planta transformation in rice using carbon-nanotube delivery into germline cells, which would speed up the transformation process by avoiding the lengthy tissue culture phase. Our preliminary data was used in the successful application for funding from the USDA NIFA Agriculture and Food Research Initiative (AFRI award #2020-67013-31811), which is still in progress. Once the efficient rice gene editing pipeline is established, improved rice varieties can be developed to help growers increase their profitability from higher yields with reduced inputs. 2. Improve the grain quality of inbred and hybrid US rice germplasm. The second objective was to identify and characterize genes controlling grain quality traits, including milling quality, grain shape, the taste and texture after cooking, and resistant starch levels (a potential contributor to lowering the glycemic index of rice) to develop improved molecular breeding strategies for developing varieties with superior grain quality. Phenotype data from 180 O. sativa accessions for milled rice yield, amylose content, gelatinization temperature, grain shape and weight, chalkiness, protein content, and resistant starch content was combined with genotype data from the 7K SNP array to perform the GWAS for rice grain quality traits (Islam et al, manuscript in preparation). A number of trait-associated SNPs representing significant QTLs were identified for each trait. These SNP markers and associated germplasm accessions can be used to transfer beneficial alleles to improve rice grain quality and nutritional traits for future molecular breeding initiatives. 3. Develop improved brown rice for better nutrition and food safety: Although brown (unmilled) rice has higher levels of fiber and essential minerals and fatty acids than white (milled) rice, the vast majority of rice consumers still prefer white rice. The third objective will be to improve the various aspects of brown rice to help promote a healthier alternative to white rice. In order to analyze the micronutrient content of brown rice, a GWAS was performed using the 7K SNP data along with phenotype data from Inductively Coupled Plasma (ICP) analysis for mineral content (namely K, Mg, Zn, Fe, Mn and Cu concentration), along with an analysis of arsenic content to ensure the diverse brown rice accessions had little to no arsenic in the grain. A number of beneficial QTLs were identified, and a manuscript is in preparation for publication. 4. Improve global rice production through higher yields and stress tolerance: The fourth objective will be to increase rice yields while using molecular markers to select for improved stress tolerance to reduce crop losses in farmers' fields. One major constraint for global rice production is salinity stress. Our lab has worked to identify key genes leading to tolerance of saline conditions for improved rice production. Several investigations into the salinity tolerance of rice at the reproductive stage were completed through this project: an initial QTL study was published (Mondal et al. 2019), and two additional QTLs studies were completed in 2020 and are planned for publication in 2021 (Mondal et al., manuscript in preparation). One of the additional QTL studies employed KASP SNP markers, while the other used a skim sequencing approach (AgSeq) to create a high-density QTL map. These QTLs can be used to develop stress tolerant rice varieties for saline-prone areas around the world. 5. To support environmentally sustainable methods for rice production: Future yield increases should be integrated with sustainable approaches that limit the impact of agriculture, while at the same time increasing the overall genetic diversity of crops to be more sustainable under future constraints. This is a long-term goal to transfer alleles from diverse accessions into cultivated rice for more sustainable rice production. This project initiated activities to test CRISPR/Cas-mediated allele replacements, but challenges in transferring large insertions using homology directed repair (HDR) has prevented progress so far. New techniques to make HDR more efficient will need to be explored in the next phase of the project.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Molina-Risco M, Ibarra O, Faion-Molina M, Kim B, Septiningsih EM, Thomson MJ (2021) Optimizing Agrobacterium-mediated transformation and CRISPR-Cas9 gene editing in the tropical japonica rice variety Presidio. Int. J. Mol. Sci. 22: 10909.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Continued efforts were made to build relationships with several target audiences, including: (1) Texas rice breeders: I continue to be in contact with the rice breeders at the Texas A&M AgriLife Research Center in Beaumont to discuss how molecular technologies can be used for rice improvement; (2) U.S. rice scientists: I am in contact with the USDA ARS rice group at the Dale Bumpers National Rice Research Center, along with others from the U.S. rice community, to discuss the genetic control of grain quality and other traits; (3) Texas A&M plant breeding community: my team in the Crop Genome Editing Lab worked to complete several seed grant projects in gene editing supported by Texas A&M AgriLife Research in collaboration with breeders and researchers working on crop improvement; and (4) the international rice community: discussions were held with scientists from the International Rice Research Institute (IRRI) in the Philippines and the National Chung Hsing University in Taiwan to discuss more details for future collaborative research projects for rice improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In Fall 2020 we had one Master's and one PhD student graduate from our lab with training in plant molecular genetics and gene editing. Currently, the project involves two Master's and three PhD students working on primarily on the genome editing activities. A post-doctoral research associate has also assisted with training of graduate students in the latest plant molecular biology techniques. How have the results been disseminated to communities of interest?The Project PI (M. Thomson) presented results from this project to the scientific community in several forums: (1) an invited oral presentation at the International Symposium of Rice Functional Genomics, Taiwan (November 6, 2019); (2) an invited talk at the Symposium on Novel Biotechnology and Breeding, NCHU, Taiwan (November 7, 2019); (3) an oral presentation at the ASA, SSSA, CSSA Annual Meeting, San Antonio, TX (November 12, 2019); and (4) participating in an invited panel on "Gene editing and rice: innovation, opportunity, and pitfalls" at the USA Rice Outlook Conference, Little Rock, AR (December 9, 2019). Moreover, three papers were published in peer-reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals?There are no major changes to the activities and objectives in the proposal. In brief, the following activities will be pursued in the next reporting period: 1. Allele mining at genes underlying key traits in rice: A comprehensive list of cloned genes in rice for traits of interest will be updated with detailed information on known functional nucleotide polymorphisms and allele effects. This data will be used to design guide RNAs for CRISPR-Cas9 genome editing at selected genes. 2. Optimizing a high-through genome editing platform in rice: Protocols for high-throughput CRISPR-Cas9 genome editing will be optimized using the new Crop Genome Editing Lab. Non-transgenic and efficient delivery techniques will be tested, including RNP delivery, nanoparticle-mediated delivery, and in planta transformation. 3. Investigating the genetic basis of flowering time, stress tolerance, and grain quality and nutrition: Experiments will be performed to better understand the genetic control of flowering time (using genome-wide association mapping and gene editing), abiotic stress tolerance (QTL analysis of salinity tolerance and gene editing), and grain quality and nutrition (genome-wide association mapping and gene editing) in rice.

Impacts
What was accomplished under these goals? The backbone of this research project is the identification and characterization of key genes controlling high-value traits in rice, which will ultimately lead to the goal of accelerating the development of high-yielding, stress-tolerant, nutritious rice. Towards this end, the project has focused on two approaches this past year: (1) QTL and association mapping of key genetic loci, and (2) setting up a more efficient genome editing platform for functional validation of candidate genes. During this reporting period, the project has accomplished the following: 1. To provide a low-cost and efficient genome-wide genotyping tool for QTL and association mapping in rice, we collaborated with Cornell University and IRRI (Philippines) to validate a new 7K SNP array across a diverse set of rice germplasm. This project was successfully completed and published last year (Morales et al 2020; PLOS ONE), and has been used for GWAS for cold tolerance in rice (Thapa et al. 2020; Frontiers in Genetics). 2. Data from the 7K SNP array is also being used for GWAS for rice grain quality and nutritional content. Phenotype data from 180 O. sativa accessions for milling, cooking, and nutrition quality, including milled rice yield, amylose content, grain shape and weight, chalkiness, and ICP analysis (As, K, Mg, Zn, Fe, Mn and Cu concentration) is currently being analyzed and is planned for publication in 2021. 3. Several investigations into the salinity tolerance of rice at the reproductive stage have been completed, and a PhD student performing this work successfully graduated in the Fall 2020 semester. An initial QTL study was published (Mondal et al. 2019; Plant Breeding and Biotechnology), and two additional QTLs studies were completed in 2020 and are planned for publication in 2021. One of the additional QTL studies employed KASP SNP markers, while the other used a skim sequencing approach (AgSeq) to create a high-density QTL map. 4. During this reporting period, gene editing activities in the Crop Genome Editing Lab were further optimized with the completion of the Texas A&M X-Grant project on "CRISPR Gene Editing for Healthier Foods and Improved Crop Resilience." We have a dedicated post-doctoral associate working to test innovative new approaches to bypass tissue culture in the plant transformation process to develop a high-throughput gene editing pipeline, including delivery methods based on room temperature plasma jets and carbon nanotubes. Preliminary data from that project was used to successfully obtain a two-year USDA NIFA AFRI grant entitled "Efficient gene editing of diverse crops using in planta transformation with carbon nanotubes."

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Mondal S, Borromeo TH, Diaz MGQ, Amas J, Rahman MA, Thomson MJ, Gregorio GB (2019) Dissecting QTLs Associated with Reproductive Stage Salinity Tolerance in Rice from BRRI dhan47. Plant Breeding and Biotechnology 7: 302-312.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Thapa R, Tabien RE, Thomson MJ, Septiningsih EM (2020) Genome-wide association mapping to identify genetic loci for cold tolerance and cold recovery during germination in rice. Frontiers in Genetics 11: 22.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Morales KY, Singh N, Perez FA, Ignacio JC, Thapa R, Arbelaez J, Tabien RE, Famoso A, Wang DR, Septiningsih EM, Shi Y, Kretzschmar T, McCouch SR, Thomson MJ (2020) An improved 7K SNP array, the C7AIR, provides a wealth of validated SNP markers for rice breeding and genetics studies. PLOS ONE 15(5): e0232479.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Continued efforts were made to build relationships with several target audiences, including: (1) Texas rice breeders: I continue to be in contact with the rice breeders at the Texas A&M AgriLife Research Center in Beaumont to discuss how molecular technologies can be used for rice improvement; (2) U.S. rice scientists: I am in contact with the USDA ARS rice group at the Dale Bumpers National Rice Research Center, along with others from the U.S. rice community, to discuss the genetic control of grain quality and other traits; (3) Texas A&M plant breeding community: my team in the Crop Genome Editing Lab worked to complete several seed grant projects in gene editing supported by Texas A&M AgriLife Research in collaboration with breeders and researchers working on crop improvement, and also helped organize the 2nd Texas A&M Genome Editing Symposium (held in College Station, TX in October 2019) which reached approximately 100 breeders and researchers at Texas A&M; and (4) the international rice community: discussions were held with scientists from the International Rice Research Institute (IRRI) in the Philippines and the National Chung Hsing University in Taiwan to discuss more details for future collaborative research projects for rice improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Currently, the project involves two Masters and three PhD students working on characterizing novel genetic diversity, allele mining, gene mapping, marker development, and genome editing activities. A post-doctoral research associate was also recruited in mid-2019, and has assisted with training of graduate students in the latest molecular techniques. How have the results been disseminated to communities of interest?The Project PI (M. Thomson) presented results from this project to the scientific community in several forums: (1) an oral presentation at the International Rice Research Congress: 8th Rice Genetics Symposium (October 15-17, 2018; Singapore); (2) giving an online webinar on "New technologies for rice breeding with a focus on CRISPR gene editing" for the University of Arkansas "Food and Agribusiness Webinar" series (November 1, 2018); (3) invited talk at the National Chung Hsing University (December 2018; Taichung, Taiwan); (4) poster presentation at the In Vitro Biology Annual Meeting (June 8-12, 2019; Tampa, FL); (5) invited talk at the American Society of Animal Science (ASAS) Annual Meeting (July 9-10, 2019, Austin, TX); and (6) poster presentation at the American Society of Plant Biology 2019 Meeting (August 3-7, 2019, San Jose, CA). Moreover, four papers were published in peer-reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals?There are no major changes to the activities and objectives in the proposal. In brief, the following activities will be pursued in the next reporting period: Allele mining at genes underlying key traits in rice: A comprehensive list of cloned genes in rice for traits of interest will be updated with detailed information on known functional nucleotide polymorphisms and allele effects. This data will be used to design guide RNAs for CRISPR-Cas9 genome editing at selected genes. Optimizing a high-through genome editing platform in rice: Protocols for high-throughput CRISPR-Cas9 genome editing will be optimized using the new Crop Genome Editing Lab. Non-transgenic and efficient delivery techniques will be tested, including RNP delivery, nanoparticle-mediated delivery, and in planta transformation. Investigating the genetic basis of flowering time, stress tolerance, and grain quality and nutrition: Experiments will be performed to better understand the genetic control of flowering time (using genome-wide association mapping and gene editing), abiotic stress tolerance (QTL analysis of salinity tolerance and gene editing), and grain quality and nutrition (genome-wide association mapping and gene editing) in rice.

Impacts
What was accomplished under these goals? Major activities completed: The backbone of this research project is the identification and characterization of key genes controlling high-value traits in rice, which will ultimately lead to the goal of accelerating the development of high-yielding, stress-tolerant, nutritious rice. Towards this end, the project has three approaches: exploring diverse rice accessions, compiling information on candidate genes underlying traits of interest, and setting up a genome editing platform for functional validation of candidate genes. During this reporting period, the project has accomplished the following: During this reporting period, activities in the Crop Genome Editing Lab at Texas A&M were further scaled up, with the completion of several gene editing seed grants funded by Texas A&M AgriLife Research, along with activities funded by the Texas A&M X-Grant project on "CRISPR Gene Editing for Healthier Foods and Improved Crop Resilience." This project supports a research team within the Crop Genome Editing Lab, led by a dedicated post-doctoral associate, with the goal of testing innovative new approaches to bypass tissue culture in the plant transformation process to develop a high-throughput gene editing pipeline. Initial tests were performed using delivery methods based on room temperature plasma jets and carbon nanotubes. Approximately 180 O. sativa accessions were evaluated for on key grain quality traits for milling, cooking, and nutrition quality, including milled rice yield, amylose content, grain shape and weight, chalkiness, and ICP analysis for As, K, Mg, Zn, Fe, Mn and Cu. These data will be combined with 7K SNP data to perform a genome-wide association study (GWAS) for grain quality traits in rice. The Illumina 7K SNP chip developed at Cornell University provides over 5,200 high-quality SNP data points per sample. Data analysis is ongoing, with the target for initial manuscripts to be submitted in early 2020. An updated GWAS was also performed for flowering time in rice using phenotype data from 2017 and 2018 field seasons with 7K SNP genotype data. A significant locus on chromosome 6 contained previously-known flowering time genes Hd3a and RFT1, which are now being targeted for gene editing to determine their effect in U.S. rice germplasm.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Baltazar MD, Ignacio JC, Thomson MJ, Ismail AM, Mendioro MS, Septiningsih EM (2019) QTL mapping for tolerance to anaerobic germination in rice from IR64 and the aus landrace Kharsu 80A. Breeding Sci 69: 227-233.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Collard BCY, Gregorio GB, Thomson MJ, Islam MR, Vergara G, Laborte AG, Nissila E, Kretzschmar T, Cobb JN (2019) Transforming rice breeding: re-designing the irrigated breeding pipeline at the International Rice Research Institute (IRRI). Crop Breed. Genet. Genom. 1: e190008.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Arbelaez JD, Dwiyanti MS, Tandayu E, Llantada K, Jarana A, Ignacio JC, Platten JD, Cobb J, Rutkoski JE, Thomson MJ, Kretzschmar T (2019) 1k-RiCA (1K-Rice Custom Amplicon) a novel genotyping amplicon-based SNP assay for genetics and breeding applications in rice. Rice 12: 55.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Rahman MA, Thomson MJ, De Ocampo M, Egdane JA, Salam MA, Shah-E-Alam M, and Ismail AM (2019) Assessing trait contribution and mapping novel QTLs for salinity tolerance using the Bangladeshi rice landrace Capsule. Rice 12: 63.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:During this stage of the project, continued efforts were made to build relationships with several target audiences, including: (1) Texas rice breeders: I continue to be in contact with the rice breeders at the Texas A&M AgriLife Research Center in Beaumont to discuss how molecular technologies can be used for rice improvement; (2) the international rice community: discussions were held with scientists from the International Rice Research Institute (IRRI) in the Philippines and the National Chung Hsing University in Taiwan to discuss more details for future collaborative research projects for rice improvement; and (3) Texas A&M plant breeding community: my team in the Crop Genome Editing Lab worked to advance 13 seed grant projects in gene editing supported by Texas A&M AgriLife Research in collaboration with breeders and researchers working on crop improvement. My team also helped organize the first Texas A&M Genome Editing Symposium (held in College Station, TX in October 2018) and attracted a large portion of the plant breeding community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Currently, the project involves two Masters and three PhD students working on characterizing novel genetic diversity, allele mining, gene mapping, marker development, and genome editing activities. We are also in the process of recruiting a post-doctoral research associate who will assist with training of graduate students in the latest molecular techniques. How have the results been disseminated to communities of interest?The Project PI (M. Thomson) presented ideas from this project to the scientific community in several forums: (1) an oral presentation at the Rice Technical Working Group meeting (Feb 2018, Long Beach, CA); (2) invited seminars at the National Chung Hsing University (July and December 2018; Taichung, Taiwan); and (3) giving an online webinar on "New technologies for rice breeding with a focus on CRISPR gene editing" for the University of Arkansas "Food and Agribusiness Webinar" series (November 1, 2018) https://youtu.be/6IQTKu42Los. Moreover, two papers were accepted for publication in peer-reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals?There are no major changes to the activities and objectives in the proposal. In brief, the following activities will be pursued in the next reporting period: Allele mining at genes underlying key traits in rice: A comprehensive list of cloned genes in rice for traits of interest will be updated with detailed information on known functional nucleotide polymorphisms and allele effects. This data will be used to design guide RNAs for CRISPR-Cas9 genome editing at selected genes. Optimizing a high-through genome editing platform in rice: Protocols for high-throughput CRISPR-Cas9 genome editing will be optimized using the new Crop Genome Editing Lab. Non-transgenic and efficient delivery techniques will be tested, including RNP delivery, nanoparticle-mediated delivery, and in planta transformation. Investigating the genetic basis of flowering time, stress tolerance, and grain quality and nutrition: Experiments will be performed to better understand the genetic control of flowering time (using genome-wide association mapping and gene editing), abiotic stress tolerance (QTL analysis of salinity tolerance and gene editing), and grain quality and nutrition (genome-wide association mapping and gene editing) in rice.

Impacts
What was accomplished under these goals? Major activities completed: The backbone of this research project is the identification and efficient use of novel sources of genetic diversity for key traits in rice, which will ultimately lead to the goal of accelerating the development of high-yielding, stress-tolerant, nutritious rice. To provide a strong foundation towards this end, the project has begun with three approaches: exploring diverse rice accessions, compiling information on key genes underlying traits of interest, and setting up a genome editing platform. During this reporting period, the project has accomplished the following: Approximately 200 O. sativa accessions were planted in the field in the Beaumont Research Center in the summer of 2018 for additional phenotyping, including heading date and grain quality parameters. Molecular marker data on these accessions was previously obtained using an Illumina 7K SNP chip for rice (developed at Cornell University), providing over 6,000 SNP data points per sample as the basis for performing genome wide association mapping. Data analysis is ongoing, with the target for initial manuscripts to be submitted by late 2019. During 2018, activities in the new Crop Genome Editing Lab at Texas A&M were scaled up, with the hiring of two full time staff and optimization of the CRISPR-based gene editing protocols. Texas A&M AgriLife Research funded 13 seed grant projects across 6 crop species (rice, wheat, sorghum, cotton, potato, and melon), for which the Crop Genome Editing Lab led the target gene sequence analysis, gRNA design, transformation construct development, and validation of the edited progeny. In 2018, this project also led to the successful award of a $500,000 X-Grant project from Texas A&M University. Our proposal on "CRISPR Gene Editing for Healthier Foods and Improved Crop Resilience" was selected as one of the 8 funded projects out of 20 finalists selected from 60 pre-proposals out of an initial 276 one-page submissions. This project supports the research team within the Crop Genome Editing Lab, with the primary goal of testing innovative new approaches to bypass tissue culture in the plant transformation process to develop a high-throughput gene editing pipeline (supports a full-time postdoctoral research associate in my lab for 2 years).

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Juanillas V, Dereeper A, Beaume N, Droc G, Dizon J, Mendoza JR, Perdon JP, Mansueto L, Triplett L, Lang J, Zhou G, Ratharanjan K, Plale B, Haga J, Leach JE, Ruiz M, Thomson M, Alexandrov N, Larmande P, Kretzschmar T, Mauleon RP (2019) Rice Galaxy: an open resource for plant science. Gigascience (in press)
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Baltazar MD, Ignacio JC, Thomson MJ, Ismail AM, Mendioro MS, Septiningsih EM (2019) QTL mapping for tolerance to anaerobic germination in rice from IR64 and the aus landrace Kharsu 80A. Breeding Science (in press)

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:During this stage of the project, efforts were made to build relationships with several target audiences, including: (1) Texas rice breeders: several discussions were held with rice breeders at the Texas A&M AgriLife Research Center in Beaumont to discuss how molecular technologies can be used for rice improvement; (2) the international rice community: discussions were held with the International Rice Research Institute (IRRI) in the Philippines and the National Chung Hsing University in Taiwan to discuss more details for future collaborative research projects; and (3) graduate students: two Masters and two PhD students continued their research under this project involving the latest techniques in plant genomics for rice improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Thus far, the project has recruited two Masters and two PhD students to work on characterizing novel genetic diversity, allele mining, gene mapping, marker development, and genome editing activities. A post-doctoral research associate has also joined the project, and will assist with training of graduate students in the latest molecular techniques. How have the results been disseminated to communities of interest?The Project PI (M. Thomson) presented ideas from this project to the scientific community during an oral presentation at the ASA/CSSA/SSSA annual meeting (Nov 2016). Moreover, four papers were published in peer-reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals?There are no major changes to the activities and objectives in the proposal. In brief, the following activities will be pursued in the next reporting period: Allele mining at genes underlying key traits in rice: A comprehensive list of cloned genes in rice for traits of interest will be updated with detailed information on known functional nucleotide polymorphisms and allele effects. This data will be used to design guide RNAs for CRISPR-Cas9 genome editing at selected genes. Optimizing a high-through genome editing platform in rice: Protocols for high-throughput CRISPR-Cas9 genome editing will be optimized using the new Crop Genome Editing Lab. Delivery and transformation systems will be compared for efficiency, including Agrobacterium, biolistic, and protoplast transformation. Investigating the genetic basis of stress tolerance: Experiments will be performed to better understand the genetic control of abiotic stress tolerance in rice, including genome-wide transcriptome analysis of heat stress using RNA-Seq, and genome editing of genes underlying salinity tolerance, flowering time, and grain quality in rice.

Impacts
What was accomplished under these goals? Major activities completed: The backbone of this research project is the identification and efficient use of novel sources of genetic diversity for key traits in rice, which will ultimately lead to the goal of accelerating the development of high-yielding, stress-tolerant, nutritious rice. To provide a strong foundation towards this end, the project has begun with three approaches: exploring diverse rice accessions, compiling information on key genes underlying traits of interest, and setting up a genome editing platform. During this reporting period, the project has accomplished the following: Over 300 O. sativa accessions were planted in the field in the Beaumont Research Center in the summer of 2017 for phenotyping and genotyping. Molecular marker data on these accessions was obtained using an Illumina 7K SNP chip for rice (developed at Cornell University), providing over 6,000 SNP data points per sample. Data analysis is underway to determine if the resolution is high enough for genome wide association mapping, or if imputation to deep sequence data will be needed. During 2017, the new Crop Genome Editing Lab at Texas A&M was established. A post-doctoral research associate was hired to help initiate the CRISPR-Cas9 platform for genome editing in rice, and the facilities and equipment were set up. Pilot studies were initiated to use Agrobacterium-based delivery of the Cas9 and gRNA for genome editing in rice, using sd1 (plant height) and GS3 (grain shape) as the initial target genes.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Thomson MJ, Singh N, Dwiyanti MS, Wang DR, Wright MH, Perez FA, DeClerck G, Chin JH, Malitic-Layaoen GA, Juanillas VM, Dilla-Ermita CJ, Mauleon R, Kretzschmar T, McCouch SR (2017) Large-scale deployment of a rice 6K SNP array for genetics and breeding applications. Rice 10:40.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kadam NN, Tamilselvan A, Lawas LMF, Quinones C, Bahuguna RN, Thomson MJ, Dingkuhn M, Muthurajan R, Struik PC, Yin X, Jagadish KSV (2017) Genetic control of plasticity in root morphology and anatomy of rice in response to water-deficit. Plant Physiol. 174: 2302-2315.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Dilla-Ermita CJ, Tandayu E, Juanillas VM, Detras J, Lozada DN, Dwiyanti MS, Cruz CV, Mbanjo EG, Ardales E, Diaz MG, Mendioro M, Thomson M, Kretzschmar T (2017) Genome-wide association analysis tracks bacterial leaf blight resistance loci in rice diverse germplasm. Rice 10: 8.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Gonzaga ZJC, Carandang J, Singh A, Collard BC, Thomson MJ, Septiningsih EM (2017). Mapping QTLs for submergence tolerance in rice using a population fixed for SUB1A tolerant allele. Mol. Breeding, 37: 47.

Progress 02/11/16 to 09/30/16

Outputs
Target Audience:During this initial stage of the project, efforts were made to build relationships with several target audiences, including: (1) Southern U.S. rice breeders: several discussions were held with rice breeders and geneticists from Arkansas, Texas, Louisiana, and Mississippi on what molecular tools are needed to accelerate U. S. rice breeding programs; (2) the international rice community: visits were made to the International Rice Research Institute (IRRI) in the Philippines and to the National Chung Hsing University in Taiwan to discuss opportunities for future collaborative research projects; and (3) graduate students: two Masters and two PhD students were recruited to join this project and training was initiated for them to learn the latest techniques in plant genomics for rice improvement. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Thus far, the project has recruited two Masters and two PhD students to work on characterizing novel genetic diversity, allele mining, gene mapping, marker development, and genome editing activities. A visiting scientist has also joined the project for one year, and will assist with training of graduate students in the latest molecular techniques. How have the results been disseminated to communities of interest?The Project PI (M. Thomson) presented ideas from this project to the scientific community at several conferences, including the Rice Technical Working Group meeting, the 5th International Conference on Quantitative Genetics, and the Annual Plant Biology 2016 meeting. What do you plan to do during the next reporting period to accomplish the goals?There are no major changes to the activities and objectives in the proposal. In brief, the following activities will be pursued in the next reporting period: Allele mining at genes underlying key traits in rice: A comprehensive list of cloned genes in rice for traits of interest will be completed, with detailed information on known functional nucleotide polymorphisms and allele effects. This data will be used to design guide RNAs for CRISPR-Cas9 genome editing at selected genes. At the same time, SNP haplotype patterns at selected genes will be also be extracted from the sequence data of the 3,000 Rice Genomes Project to assist with SNP marker development. Setting up a high-through genome editing platform in rice: A new laboratory for Crop Genome Editing will be set up at Texas A&M, and protocols for high-throughput CRISPR-Cas9 genome editing will be optimized. Delivery and transformation systems will be compared for efficiency, including Agrobacterium, biolistic, and protoplast transformation. Investigating the genetic basis of stress tolerance: Experiments will be performed to better understand the genetic control of abiotic stress tolerance in rice, including genome-wide transcriptome analysis of heat stress using RNA-Seq and genome editing of transporter genes underlying salinity tolerance in rice.

Impacts
What was accomplished under these goals? Major activities completed: The backbone of this research project is the identification and efficient use of novel sources of genetic diversity for key traits in rice, which will ultimately lead to the goal of accelerating the development of high-yielding, stress-tolerant, nutritious rice. To provide a strong foundation towards this end, the project has begun with three approaches: exploring diverse rice accessions, compiling information on key genes underlying traits of interest, and setting up a genome editing platform. During this initial period, the project has accomplished the following: Seeds of 400 O. sativa and 117 O. glaberrima genebank accessions were requested from the USDA GRIN system and planted in the field in the Beaumont Research Center for seed amplification. These accessions were selected to partially overlap the USDA mini-core and core collections representing a wide range of genetic diversity, but also to include additional accessions from the main collection from geographic regions known to contain rice varieties that are tolerant to abiotic stresses, including heat, anaerobic germination, and salinity. A list of known cloned genes in rice for key traits was compiled by extracting relevant details from publications and online databases, including the original genetic donor, gene structure and function, known alleles and functional polymorphisms, and allele effects. Initial traits include grain shape, grain weight, amylose content, grain color, awns and dormancy. Detailed information on the various functional alleles will form the basis for SNP marker development and for subsequent genome editing experiments using CRISPR-Cas9 for gene knock-outs, allele replacement, and up- and down-regulation. Discussions were held on the prospect of developing a new Crop Genome Editing Lab at Texas A&M. Feedback was gathered from breeders on what traits would be conducive for genome editing in their respective crops, and plans were made on what type of lab facilities would be needed to accommodate an expanded research program. A visiting post-doctoral fellow was also recruited to help set up the CRISPR-Cas9 platform for genome editing in rice.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ereful N, Liu LY, Tsai E, Kao SM, Dixit S, Mauleon R, Malabanan K, Thomson M, Laurena A, Lee D, Mackay I, Greenland A, Powell W, Leung H (2016) Analysis of allelic imbalance in rice hybrids under water stress and association of asymmetrically expressed genes with drought-response QTLs. Rice 9: 50.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Rahman MA, Thomson MJ, Shah-E-Alam M, de Ocampo M, Egdane J, Ismail AM (2016) Exploring novel genetic sources of salinity tolerance in rice through molecular and physiological characterization. Ann. Bot.-London 117: 1083-1097.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Chin JH, Lee YJ, Jiang W, Koh HJ, Thomson MJ (2016) Characterization of indica-japonica subspecies-specific InDel loci in wild relatives of rice (Oryza sativa L. subsp. indica Kato and subsp. japonica Kato). Genet. Resour. Crop Evol. doi:10.1007/s10722-016-0368-1.4
• Type: Other Status: Published Year Published: 2016 Citation: Thomson MJ (2016) Rice SNPvisor: a new initiative to characterize and validate breeding-relevant SNP haplotypes for the US rice community. Rice Technical Working Group (RTWG) meeting program and abstract book (abstract), Galveston, Texas. March 2016.
• Type: Other Status: Published Year Published: 2016 Citation: Thomson MJ (2016) Applying quantitative genetics advances towards molecular breeding in rice. 5th International Conference on Quantitative Genetics (ICQG5) program and abstract book (abstract), Madison, Wisconsin. June 2016.
• Type: Other Status: Published Year Published: 2016 Citation: Thomson MJ (2016) SNP haplotype validation for rice improvement. Annual Plant Biology Meeting program and abstract book (abstract), Austin, Texas. July 2016.