Source: MARQUETTE UNIVERSITY submitted to
MECHANISMS OF COLD STRESS TOLERANCE RESPONSES IN RICE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1008603
Grant No.
2016-67013-24587
Project No.
WISW-2015-06587
Proposal No.
2015-06587
Multistate No.
(N/A)
Program Code
A1101
Project Start Date
Dec 1, 2015
Project End Date
Nov 30, 2020
Grant Year
2016
Project Director
Schlappi, M.
Recipient Organization
MARQUETTE UNIVERSITY
915 W WISCONSIN AVE RM 175
MILWAUKEE,WI 53233
Performing Department
Biological Sciences
Non Technical Summary
The proposal "Mechanisms of Cold Stress Tolerance Responses in Rice" is submitted by Program Director (PD) Michael Schläppi from Marquette University and co-PD Georgia Eizenga from the USDA-ARS, and fits the A1101 program area "Plant Growth and Development, Composition and Stress Tolerance". Rice (Oryza sativa) yield would be enhanced if growers could plant two weeks earlier in the season to better utilize the spring rain and avoid the high nighttime temperatures of mid-summer, which decrease grain quality and yield. Our objective is to use two diverse and well-characterized rice collections, the USDA Rice Mini-Core (RMC) consisting of 203 accessions, and Rice Diversity Panel 1 (RDP1) composed of 420 accessions, to provide a better understanding of the basic genetic and cellular mechanisms responsible for tolerance to cold temperatures at germination and the seedling stage in rice. To accomplish this, we will (i) validate cold tolerance phenotypes of the RMC in field experiments; (ii) test hypotheses for mechanisms of rice cold tolerance by examining membrane damage, levels of antioxidants and enzymes, and photoperiod response to cold in the RMC, and by conducting genome-wide association studies (GWAS); (iii) fine-map some of the previously identified 37 quantitative trait loci (QTL) for cold tolerance using two bi-parental mapping populations genotyped with genotyping-by-sequencing (GBS); (iv) identify putative candidate genes associated with cold tolerance using SNP based linkage disequilibrium (LD) data, gene expression profiles, and public databases; and (v) validate selected candidate genes in transgenic plants with the CRISPR/Cas9 system and by overexpression approaches. This information will be the basis for gene discovery, and ultimately, will identify cold-associated DNA markers and rice germplasm with improved seedling vigor early in the growing season for use in variety improvement programs. In addition, by planting earlier, growers will use less irrigation water from aquifers, thus decreasing the environmental impact.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20115301080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1530 - Rice;

Field Of Science
1080 - Genetics;
Goals / Objectives
The overarching goal of the project, "Mechanisms of Cold Stress Tolerance Responses in Rice", is to understand the molecular and cellular mechanisms governing chilling tolerance in rice (Oryza sativa) and to identify strong-effect rice cold tolerance genes as candidates to enhance chilling tolerance of commercial rice grown in the United States. This will be a major achievement for sustainable rice cultivation in the United States, because rice yield would be enhanced if growers could plant two weeks earlier in the season to better utilize the spring rain and avoid the high nighttime temperatures of mid-summer, which decrease grain quality and yield. By planting earlier, growers will also use less irrigation water from aquifers, thus decreasing the environmental impact. Moreover, this will allow for rice to be grown in colder climates should there be such a need in current production areas due to climate change such as drought or inclement weather patterns.The project has three specific aims or goals:To validate growth chamber determined chilling tolerance traits at the germination and seedling stages in field experiments.To test various hypotheses regarding chilling tolerance phenologies that might lead to gene discovery.To fine-map preliminarily mapped chilling tolerance quantitative trait loci (QTL) for gene discovery.Within these specific aims or goals, we have five objectives:To quantify chilling tolerance phenotypes of the USDA rice mini-core collection (RMC) consisting of 203 varieties in field experiments and compare the results to growth chamber based data to assess the heritability of chilling tolerance traits.To test hypotheses for mechanisms of rice cold tolerance by examining membrane damage, levels of antioxidants and enzymes, and photoperiod response to cold in the RMC, and to generate quantitative trait data for genome-wide association studies (GWAS).To fine-map some of the previously identified 37 QTL for chilling tolerance using two bi-parental mapping populations that will be genotyped using the genotyping-by-sequencing (GBS) approach.To identify putative candidate genes associated with chilling tolerance using single nucleotide polymorphism (SNP) based linkage disequilibrium (LD) data, considerations of the tolerance trait associated with the QTL, genomics data, and gene expression profile data.To validate selected candidate genes by performing functional studies in transgenic plants, by either overexpressing genes in chilling sensitive rice lines or by knocking them out in chilling tolerant lines using the CRISPR/Cas9 system, and by complementing respective orthologous gene mutants in Arabidopsis.This information will be the basis for cold-associated DNA marker and gene discovery, and ultimately, the production of rice germplasm with improved seedling vigor early in the growing season for use in variety improvement programs.
Project Methods
Validation of Cold Tolerance Phenotypes in Field Experiments: Several hundred rice varieties will be reevaluated in growth chamber based assays to select the most chilling tolerant varieties. Those varieties will then be tested over several years in paddies on farmland near Milwaukee, Wisconsin. The varieties will be subjected to early plantings in April and germination rates and seedling survival rates recorded. Statistical analyses will be done to determine the heritability of the cold tolerance traits by comparing controlled growth chamber results with field assays. Theses results will increase in the selection of chilling tolerant rice varieties available to rice farmers that can be directly used for earlier spring planting or serve as breeding lines for elite cultivars that are more chilling sensitive.Test Hypotheses for Mechanisms of Rice Cold Tolerance: Biochemical assays to measure the levels of antioxidant enzymes and physiological assay to measure the degree of membrane damage will be done to determine whether chilling tolerant rice varieties use different mechanisms to achieve the same end goal. A major effort will be to address our recently discovered phenomenon that some, but not all, chilling tolerant rice varieties become more chilling sensitive under longer photoperiods. This will allow us to incorporate photoperiodic effects on chilling tolerance into new models for the mechanisms of chilling tolerance. This will generate critically important information for farmers so they can use the earliest possible spring planting times to avoid negative effects of longer photoperiods.Fine-Map QTL Using Two Bi-parental Mapping Populations: Currently there are two mapping populations available at the F5 and BC1F2 stages, respectively: a recombinant inbred line (RIL) and a backcross inbred line (BIL) population. Those lines will be propagated to the F7 and BC1F5 stages. For fine-mapping of QTL, we will select chilling tolerant RILs and BILs containing few chromosomal segments from the tolerant parent and backcross them with the cold sensitive parent to generate BC4F3 chromosome segment substitution (CSSL) lines. For each backcross generation, we will select chilling tolerant progeny and genotype them with targeted single nucleotide polymorphism (SNP) markers for QTL region using the Fluidigem system. F5 RILs, BC1F5 BILs, and BC4F3 CSSLs will be genotyped using the genotyping-by-sequencing (GBS) system. Thus, sophisticated marker assisted mapping protocols will be used to reveal the chromosomal regions of the chilling tolerant parent that are associated with various cold tolerance QTL. These mapping lines will provide the groundwork for germplasm enhancement and marker assisted breeding programs to improve not only the chilling tolerance of elite rice lines, but also for mapping of other abiotic stress tolerance QTL (high temperature, drought, salinity, oxidative stress).Identification of Putative Candidate Genes Associated with Cold Tolerance QTL: We will use various resources to select candidate genes in genomic regions that logically associate with various cold tolerance QTL. We will use linkage disequilibirium (LD) data from our various mapping and fine-mapping results to identify small chromosomal segments that tend to cosegregate with the various cold tolerance phenotypes. We will use genome annotation data, in silico gene expression data (to estimate developmental stage, tissue, and subspecies specific expression differences), and various databases and our own GBS data to identify SNPs in putative candidate genes. We will not only use data from our two mapping populations, but also from our genome wide association studies (GWAS) using several hundred rice cultivars from the USDA mini-core collection and the Rice Diversity 1 panel. Any newly identified putative candidate gene will significantly increase in the number of genes associated with chilling tolerance in rice, because only approximately 12.5% of previously identified QTL has thus far led to the positive identification of associated chilling tolerance genes.Functional Assays of Selected Candidate Genes in Transgenic Plants: To positively identify how chilling tolerance genes from candidate genes associated with various QTL, we will overexpress them from a strong promoter in a cold sensitive rice variety and knock them out in chilling tolerant rice variety using the new CRISPR/Cas9 system. We will use Agrobacterium-mediated transformation of either a chilling sensitive or tolerant variety that can be transformed and regenerated using this system. Candidate genes will be ligated into appropriate expression cassettes containing strong promoters for overexpression or producing guide RNAs for knockout using binary vectors that yield high frequency rice transformation when introduced into high efficiency Agrobacterium strains. Only genes that, reciprocally, improve chilling tolerance when overexpressed in a sensitive background and reduce chilling tolerance when knocked out in a tolerant background will be considered new chilling tolerance genes in rice. Various molecular and statistical methods will be used to assess the effect of those genes. We expect only moderate effects of single genes and will assess the additive or synergistic effects of introducing two or more candidate genes from different QTL regions into rice. In parallel, we will use Arabidopsis mutant lines with loss-of-function alleles of the closest rice homologs to determine whether complementation improves the cold temperature germinability of transformed Arabidopsis lines. These concerted efforts will positively identify new chilling tolerance genes in rice that can be used directly to improve the chilling tolerance of elite rice lines used by US farmers.

Progress 12/01/16 to 11/30/17

Outputs
Target Audience:As in 2016, data of this NIFA funded project were presented during the 2017 reporting period to a broad audience of fellow researchers, students, industry representatives, the general public, and academics engaged in community research activities. Specifically, data were presented in a talk at the annual USDA-NIFA Physiology of Agricultural Plants PD meeting in San Diego, CA (January 13); two talks at the Wisconsin Lutheran College Milwaukee-area Undergraduate Research conference (April 22); a talk at the Milwaukee Public Museum in Milwaukee, WI (May 9); a poster at the 28th International Conference on Arabidopsis Research in St. Louis, MO (June 19-23); a talk to Lake Park Friends, Milwaukee WI (November 18); and in interactions with farmers and members of local business incubators interested in establishing rice cultivation in the cold climate of Wisconsin. A wide range of audiences were reached through news and media features on establishing rice cultivation in Wisconsin, a project funded by the Strategic Innovation Fund of Marquette University based on data generated by this USDA-NIFA project. Newspaper articles in the Milwaukee Journal Sentinel were featured on June 29 and November 2; radio shows on Wisconsin Public Radio and WUWM Milwaukee Public Radio 89.7 FM were aired on July 10 and July 14, respectively; and TV shows on FOX 6 NOW and CBS 58 News on October 30 and November 12, respectively, and on Nyob Zoo Milwaukee Hmong TV on September 29 and November 3. Lastly, the rice research and cultivation projects were featured in a student article in the Marquette Tribune. Additional efforts were undertaken to deliver science-based knowledge by mentoring two undergraduate students and one rotation graduate student during independent research projects that were connected to the funded research project, and by supervising undergraduate volunteers and interns in rice related lab and field activities. Moreover, gardeners and farmers of diverse racial and ethnic backgrounds (African-American, Caucasian, Hmong, and Middle Eastern, among others) were again exposed during this funding period to parts of this "mechanisms of cold stress tolerance responses in rice" research proposal. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities: Two graduate students are involved in this project and were being mentored by the PD. They provided most of the progress reported for 2017. Moreover, both graduate students were mentoring two undergraduate (UG) students working on project related independent studies and five UG student interns that directly assisted them with experiments related to their PhD thesis projects. How have the results been disseminated to communities of interest?Outreach activities: In 2017, the PD expanded his work with a non-profit organization called Fondy Food Center having the mission of brining fresh food to Milwaukee inner city residents by leasing land to small scale farmers who will sell their produce at inner city farmers markets. The PD is working with the Fondy farm manager and Hmong minority farmers to establish a rice cultivation business in the Milwaukee area. With the help of the farm manager and Hmong farmers, the PD established, to the best of our knowledge, the first-ever one-acre rice farm in the state of Wisconsin using imported Japanese rice farming equipment, resulting in a 1,200 pound rice harvest in later October of 2017. This had enormous cultural significant for the Hmong farmers who are not interested to partner with the PD in establishing sustainable rice farming in the cold climate of Wisconsin (see also media reports under "Products"). What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Perform another field validation experiments using selected RMC and RDP1 accessions to better model field performances of rice accessions based on growth chamber assays. Goal 2: Continue testing additional hypotheses for mechanisms of rice cold tolerance by analyzing the role of antioxidants in cold tolerance; by investigating the photoperiodic and light quality effects on seedling survivability and use the data for GWAS mapping; and by mining genes within LTSS and EL QTL to determine whether they can be assigned to genetic pathways involved in abiotic stress tolerance. Goal 3: To have most RILs at the F9 generation and most BILs at the F6 generation; to phenotype thoseRILs and BILs using growth chamber based assays; and map those lines using a 6K SNP array for allele calling and for QTL analysis (instead of using a genotyping-by-sequencing approach, as previously intended). Goal 4: To publish a major paper on a putative anthocyanin UDP-glycosyltransferase gene as a novel cold tolerance gene in rice; and to continue to identify additional cold tolerance candidate genes. Goal 5: First, to receive sabbatical training in establishing a high-efficiency transformation system of standard and recalcitrant rice cultivars; to use the acquired technique to generate transgenic calli containing several probable rice cold tolerance genes; and to initiate regeneration of transgenic T0 plants. Second, to use recombinant glycosyltransferase protein to determine its enzyme activity; to identify its enzyme substrate; and to determine whether different alleles code for proteins with different enzyme activities.

Impacts
What was accomplished under these goals? IMPACT A major impact of the proposal "Mechanisms of Cold Stress Tolerance Responses in Rice" for US farmers is that the data it generates can be translated into breeding or engineering programs to improve the cold tolerance of elite rice cultivars. For instance, rice yield would be enhanced if growers could plant two weeks earlier in the season to better utilize the spring rain and avoid the high nighttime temperatures of mid-summer, which decrease grain quality and yield. Our objective is to use over 600 rice varieties from the USDA collection to enhance our understanding of the basic genetic and cellular mechanisms responsible for tolerance to cold temperatures at the germination and seedling stages in rice. By planting earlier, growers will also use less irrigation water from aquifers, thus decreasing the environmental impact. Moreover, it will allow to grow rice in colder climates should there be such a need due to climate change such as drought or inclement weather patterns in current production areas. To accomplish this, we will, first, select the most-cold tolerant rice varieties in growth chamber-based experiments and validate them in field experiments, so farmers could use them directly if they suit their quality and yield requirements. Second, we will test hypotheses for mechanisms of rice cold tolerance by examining cell membrane damage in leaves of cold stressed plants; by analyzing the role of antioxidants in cold tolerance; by measuring the photoperiod response of rice plants to cold; and by performing genetic studies. Third, we will generate gene mapping populations made from one cold tolerant and one cold sensitive parent. Fourth, we will identify putative cold tolerance candidate genes using those mapping populations and bioinformatics approaches. And fifth, we will validate selected candidate genes using molecular biology and genetic approaches. This information willnot only be the basis for gene discovery, but will also identify chilling-tolerance associated DNA markers that could be used for rice variety improvement programs. PROGRESS REPORT ON PROJECT OBJECTIVES Goal 1: In 2017, last year's goal was mostly met. Two-week-old seedlings of 202 Rice Mini-Core (RMC) accessions were transplanted between April19-26 into raised bed paddies on the campus of Marquette University in Milwaukee, WI, and seedling survival was determined on May 25. During the first three weeks after transplanting, the average water temperature at 9 am was 8.5°C ± 3.1°C, and the average temperature at 5 pm was 18.2°C ± 4.7°C. Each accession was planted in groups of 2-8 plants at three paddy locations in a randomized complete block design. The survival rate of the five subgroups Admixes, Aromatic, Aus, Indica, Temperate Japonica, and Tropical Japonica was 30% (7/23), 67% (4/6), 2.6% (1/38), 4.4% (3/68), 44% (15/34), and 39% (13/33), respectively. Taken together, the Japonica (Aro, TeJ & TrJ) and Indica (Aus, Ind) subspecies had a 44% (32/73) and 3.8% (4/106) survival rate, respectively, which was consistent with the field results using a smaller number of RMC accessions in 2016 [43% (27/63) and 7.2% (6/83), respectively]. These results suggest that in general terms, growth chamber based assessments of low temperature survivability of different rice accessions predict the field performance of their respective subpopulations during early spring planting in a cold climate such as Wisconsin's. However, survivability of individual accession varies from year to year, indicating that heritability of the quantitative trait of chilling tolerance is strongly influenced by the environment. Goal 2: Two of the goals set last year were mostly met. In 2017, we performed electrolyte leakage (EL) analyses at 10°C on RMC accession and identified by genome-wide association study (GWAS) mapping 29 EL Quantitative Trait Loci (QTL) on all chromosomes except chromosome 10. We then analyzed seedling survivability and membrane damage in leaves of approximately 400 rice accessions from the Rice Diversity Panel 1 (RDP1) at 16°C, 12°C, 8°C, and 4°C. Cold-induced membrane damage was measured after one week of cold treatment using the EL assay. At 12°C and 8°C, there was a relatively good inverse correlation between survivability and membrane damage, but not at 4°C, indicating that additional mechanisms strongly contribute to overall low seedling survivability at 4°C. At 16°C, overall survivability of all subgroups was >90%, which correlated only weakly with overall low EL values. Initial GWAS mapping analyses were done for the survivability and EL scores of the 400 RDP1 accessions using the 700K HDRA single nucleotide polymorphism SNP open access pipeline developed by Cornell University for the RDP1 panel. By defining QTL as having a least 3 single SNPs within a 1 Mb region, 11 Low Temperature Seedling Survivability (LTSS) and 19 EL QTL were identified at 8°C. LTSS and EL QTL mapping for other temperatures is in progress. Genes near peak SNPs are currently investigated to associate candidate genes with the QTL. Goal 3: Last year's goals were partially met. In 2017, most F8 recombinant inbred lines (RILs) and F5 backcross inbred lines (BILs) generations were generated, but some lines were discontinued or removed from the pool due to fertility and heading date issues. As a backup, new crosses between different parents were initiated at the end of the reporting period. We moreover decided to advance the RIL and BIL lines one more generation and finish the remaining goals in 2018. Goal 4: Last year's goals were all met. In 2017, we finished the in-depth analysis of a putative glycosyltransferase-encoding gene as a novel cold tolerance gene in rice (for more details see goal 5 reporting). Based on our SNP based GWAS mapping of LTSS and EL QTL (see goal 2 reporting), a list of approximately 30 putative cold tolerance candidate genes containing SNPs in promoter and coding regions was generated, 10 of which will be pursued in more detail during 2018. Goal 5: Two of three goals set last year were met and the third goal is in progress. In 2017, preliminary results indicated that overexpression in Arabidopsis of a putative anthocyanin UDP-glycosyltransferase encoding gene we tentatively identified as a novel cold tolerance gene in rice improved abiotic stress tolerance measured by cold germination and whole plant freezing assays. More importantly, functional assays done with transgenic rice plants overexpressing this gene in a cold sensitive Indica-type background as well as in a more cold tolerant Japonica background showed that certain lines reproducibly had less membrane damage (as measured by EL assays) after cold stress. Conversely, two lines having CRISPR/Cas9 generated knockout mutations in the anthocyanin UDP-glycosyltransferase gene had reproducible more membrane damage after cold stress. Taken together, these results are in agreement with our hypothesis that this gene is a novel cold tolerance gene in rice, possibly by functioning in anthocyanin mediated antioxidant pathways. We also succeeded in expressing the protein in E. coli, but have not yet identified its substrate and definitive enzyme activity.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Schl�ppi, M., Jackson, A.K., Eizenga, G.C., Wang, A., Chu, C., Shi, Y., Shimoyama, N., and Boykin, D. 2017. Assessment of five chilling tolerance traits and GWAS mapping in rice using the USDA mini-core collection. Front. Plant Sci. 8: 957.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Schl�ppi, M. 2017. Undergraduate Student Research Opportunities and Economic Revitalization through Urban Agriculture Initiatives. Metropolitan Universities Journal 28: 37-45.
  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Liu, C., Ou, S., Mao, B., Tang, J., Wang, W., Wang, H., Cao, S., Schl�ppi, M.R., Zhao, B., Xiao, G., Wang, X., and Chu, C. Early domestication of bZIP73 improved japonica rice adaptation to cold climate.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Investigating cold tolerance in rice by assessing anthocyanin and ROS content. Wisconsin Lutheran College Milwaukee-area Undergraduate Research Conference (MUBRC), Milwaukee, WI, April 22, 2017. (Plenary Lecture presented by Lisa Vlach).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Effect of plant density and climate change on yield of Asian rice (Oryza sativa L.). Wisconsin Lutheran College Milwaukee-area Undergraduate Research Conference (MUBRC), Milwaukee, WI, April 22, 2017. (Poster presented by Brooke Jeffery).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Promotion of urban and suburban agriculture through basic research, education, and community outreach programs. 28th International Conference on Arabidopsis Research, St. Louis, MO, June 19-23, 2017. (Poster presented by M. Schl�ppi).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mechanisms of Cold Stress Tolerance Responses in Rice. USDA-NIFA Physiology of Agricultural Plants Project Directors Meeting, San Diego, CA, January 13, 2017. (Talk presented by M. Schl�ppi).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Undergraduate Student Research Opportunities and Economic Revitalization through Urban Agriculture Initiatives. Milwaukee Public Museum, Milwaukee, WI, May 9, 2017. (Talk presented by M. Schl�ppi).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Establishing Rice Cultivation in Wisconsin: a Long-term Vision. Lake Park Friends, Milwaukee, WI, November 18, 2017. (Talk presented by M. Schl�ppi).


Progress 12/01/15 to 11/30/16

Outputs
Target Audience:Data of this NIFA funded project were presented in 2016 in a talk and poster at the international Gordon Research Conference of Plant Molecular Biology in Holderness, NH (June 16), in an invited seminar at the Dale Bumpers National Rice Research Center in Stuttgart, AR (August 8), and in two posters at the 18th Annual Fall Symposium on Genetics of Crop Improvement in St. Louis, MO (September 29). General parts of the project were moreover introduced to retired faculty and staff at Marquette University in Milwaukee, WI (May 25), and at the annual meeting of the Coalition of Urban and Metropolitan Universities in Washington DC (October 24). In this 2016 reporting period, the project was thus presented to a broad audience of fellow researchers, students, industry representatives, the general public, and academics engaged in community research activities. Additional efforts were undertaken to deliver science-based knowledge by mentoring three undergraduate students during independent research projects that were connected to the funded research project, and by supervising undergraduate volunteers and interns in rice related lab and field activities. Moreover, through a community engaged research project funded by Marquette University to introduce rice farming to urban gardeners and small scale farmers near Milwaukee, gardeners and farmers of diverse racial and ethnic backgrounds (African-American, Caucasian, Hmong, and Middle Eastern, among others) were exposed to parts of this "mechanisms of cold stress tolerance responses in rice" research proposal. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities: Two graduate students are involved in this project and were being mentored by the PD. They provided most of the progress reported for 2016. Moreover, both graduate students were mentoring three undergraduates working on project related independent studies and five student interns that directly assisted them with their PhD thesis work related to this project. How have the results been disseminated to communities of interest?Outreach activities: The PD is working with a non-profit organization called Fondy Food Center having the mission of providing fresh food to Milwaukee inner city residents by leasing land to small scale farmers who will sell their produce at inner city farmers markets. Fondy is leasing farm land to the PD who is working with the Fondy farm manager and Hmong minority farmers to establish a rice cultivation business in the Milwaukee area. Results from the lab and field trials to identify cold tolerant rice varieties and using mapping populations as potential sources for improved rice varieties wasdisseminated during the reporting period to Fondy associates andto members of an urban garden in Milwaukeeinterested in growing rice in urban agriculture settings. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: To repeat the validation experiments using all 203 RMC accessions and to better quantify the data for heritability analyses. Goal 2: (i.) To finish the membrane damage analysis of allRMC accessions and to perform GWAS analyses using currently released RMC SNP data. (ii.) To perform membrane damage experiments with approximately 400 RDP1 accessions and to map genes using GWAS and 70,000 publically available SNP data. (iii.) To start investigating the photoperiodic and light quality effects on seedling survivability and use the data for GWAS mapping. Goal 3:To have the RILs at the F8 generation and the BILs at the F5 generation and phenotype thoseRILs and BILs using growth chamber based assays. At the same time, the lab of the co-PD will map the RILs and BILs using genotyping-by-sequencing (GBS) approaches. Goal 4:To finish the genomics and gene expression analysis for the glycosyltransferase cold tolerance candidate gene and to identify additional genes to pursue for detailed analysistoward the end of the next reporting period. Goal 5: To analyze transgenic Arabidopsis plants overexpressing different alleles of the putative glycosyltransferase cold tolerance gene,to determine whether thisrice gene complements orthologous gene mutants and improves the chilling and freezing tolerance of transgenic Arabidopsis plants, and to regenerate transgenic rice plants overexpressing different glycosyltransferease alleles or having knockout mutations in the gene. Potentially, chilling tolerance phenotypes of transgenic rice plants could be analyzed at the end of the next reporting period. Lastly, to express the putative glycosyltransferase protein in E. coli and to perform enzyme assays to (i.), identify the enzyme substrate, and (ii.), to determine whether different alleles code for proteins with different enzyme activities.

Impacts
What was accomplished under these goals? IMPACT A major impact of the proposal "Mechanisms of Cold Stress Tolerance Responses in Rice" for US farmers is that the data it generates can be translated into breeding or engineering programs to improve the cold tolerance of elite rice cultivars. For instance, rice yield would be enhanced if growers could plant two weeks earlier in the season to better utilize the spring rain and avoid the high nighttime temperatures of mid-summer, which decrease grain quality and yield. Our objective is to use over 600 rice varieties from the USDA collection to enhance our understanding of the basic genetic and cellular mechanisms responsible for tolerance to cold temperatures at the germination and seedling stages in rice. By planting earlier, growers will also use less irrigation water from aquifers, thus decreasing the environmental impact. Moreover, it will allow to grow rice in colder climates should there be such a need due to climate change such as drought or inclement weather patterns in current production areas. To accomplish this, we will, first, select the most-cold tolerant rice varieties in growth chamber-based experiments and validate them in field experiments, so farmers could use them directly if they suit their quality and yield requirements. Second, we will test hypotheses for mechanisms of rice cold tolerance by examining cell membrane damage in leaves of cold stressed plants; by analyzing the role of antioxidants in cold tolerance; by measuring the photoperiod response of rice plants to cold; and by performing genetic studies. Third, we will generate gene mapping populations made from one cold tolerant and one cold sensitive parent. Fourth, we will identify putative cold tolerance candidate genes using those mapping populations and bioinformatics approaches. And fifth, we will validate selected candidate genes using molecular biology and genetic approaches. This information willnot only be the basis for gene discovery, but will also identify chilling-tolerance associated DNA markers that could be used for rice variety improvement programs. PROGRESS REPORT ON PROJECT OBJECTIVES Goal 1: "To validate growth chamber determined chilling tolerance traits at the germination and seedling stages in field experiments". A first set of 165 rice mini-core (RMC) accessions were planted between April 15 and May 3into outside paddies in Milwaukee, WI. The average water temperature at 9 am was 9.4 ± 2.4°C, and the average temperature at 5 pm was 17.6 ± 4.4°C. The survival rates of Temperate Japonica and Indica accessions was 70% and 10%, respectively, which was very similar to growth chamber-based assays usinga constant 10°C for one week (76% and 9%, respectively). Interestingly, the survival rates for Tropical Japonica (23% vs 67%), Aus (3% vs 15%), Aromatics (17% vs 54%), and Admixes (27% vs 43%) was lower in the field than in growth chambers. This preliminary experiment suggests that results from growth chamber experiments for Temperate Japonica and Indica, but not for the other subpopulations, might predict field performance during early spring planting. Goal 2: "To test various hypotheses regarding chilling tolerance phenologies that might lead to gene discovery". Due to growth chamber space limitations, in 2016 the main mechanismtested repeatedly was examination of membrane damage in leaves of all 203 RMC accessions exposed for one week to 10°C or two days to 4°C. Preliminary results were used for genome-wide association study (GWAS) mapping, yielding thus far 16 quantitative trait loci (QTL) , some of which overlapped with previously identified seedling survival QTL. Interestingly, although there was a relatively good correlation between cold induced membrane damage and seedling survivability, some accessions died with little membrane damage and vice versa, indicating that other mechanisms than membrane damage can lead to death and that some accession can efficiently repair such damage, which might be under genetic control. Goal 3: "To fine-map some of the previously identified 37 QTL for chilling tolerance using two bi-parental mapping populations that will be genotyped using the genotyping-by-sequencing (GBS) approach". In 2016, the main effort for this goal was to continue generating recombinant inbred lines (RILs) and backcross inbred line (BILs) for the genetic mapping. An F7 RIL and F4 BIL generation was generated during the growing seasoninMilwaukee and progeny was sent to the co-PD in Arkansas for greenhouse propagation to the F8 and F5 generations, respectively. Goal 4: "To identify putative candidate genes associated with chilling tolerance using single nucleotide polymorphism (SNP) based linkage disequilibrium (LD) data, considerations of the tolerance trait associated with the QTL, genomics data, and gene expression profile data". Since this is the first year of the grant, only one potential seedling survivability gene was pursued based on preliminary mapping and gene expression data. To determine whether the putative glycosyltransferase encoding gene is involved on cold tolerance, quantitative reverse transcription polymerase chain reaction (qPCR) analyses were done with leaf and root materials from cold tolerant and cold sensitive plants. The results showed that this gene is leaf specific,has a higher baseline level in cold tolerant thancold sensitive plants, and is cold induced. Moreover, cloning and sequencing of alleles from cold tolerant and cold sensitive plants not only identified promoter polymorphisms that might explain the gene expression differences, but also three non-synonymous single nucleotide polymorphism (SNPs) in the genecoding region, which might affect enzyme function of the protein. Goal 5: "To validate selected candidate genes by performing functional studies in transgenic plants, by either overexpressing genes in chilling sensitive rice lines or by knocking them out in chilling tolerant lines using the CRISPR/Cas9 system, and by complementing respective orthologous gene mutants in Arabidopsis". Since this is the first year of the grant, only the above mentioned putative glycosyltransferase encoding gene was tested for this goal. Different alleles of the gene from cold tolerant and sensitive rice varieties were cloned, fused to a strong promoter, and introduced not only into Arabidopsis lines with orthologous glucosyltransferase gene mutations, but also into wildtype accessions differing in freezing tolerance. Homozygous transgenic plants with single transgene loci were being identified at the end of the reporting period. The overexpression constructs were also tentatively introduced into rice calli made from cold tolerant and sensitive rice varieties, and a CRISPR/Cas9 construct to knock out the gene was introduced into callus made from cold tolerant rice varieties. The rice transformation experiment was at an early stage at the end of this reporting period.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Michael R. Schl�ppi, Aaron K. Jackson, Georgia C. Eizenga, Aiju Wang, Chengcai Chu, Yao Shi, Naoki Shimoyama, Debbie L. Boykin. Assessment of Five Chilling Tolerance Traits and GWA Mapping in Rice Using the USDA Mini-Core Collection. Frontiers in Plant Sciences (under review).