1a. Objectives (from AD-416)
Aim I: To resequence the high copy mPing strain HEG4 at sufficient coverage (60-75X) to permit alignment with the sequenced Nipponbare (NB) genome. All sequencing in this project will use the Illumina platform. Resequencing will (i) document for the first time the global impact of a TE burst in any organism, (ii) allow the development of markers to genotype the HEG4 X NB RI population, and (iii) provide a scaffold for characterizing additional and independent mPing amplification events. Aim II: To resequence four very closely related rice strains at sufficient coverage (5-10X) to align with HEG4 and characterize the polymorphisms associated with independent amplifications of mPing. These lines will also contribute to the establishment of a reverse genetic collection of mPing insertions. Aim III: To exploit a series of RIL populations that have been developed by our collaborator (Okumoto) to assess the consequences of mPing expansion on quantitative traits. Importantly, the parents for this population (HEG4 and NB) are closely related and likely vary largely in their copy number of mPing insertions. RIs will be genotyped by a vectorette strategy supplemented with additional markers designed based on the outcome of Aim I. These lines, and additional RIL populations that will be generated during this project, will be ideal for directly monitoring the effects of mPing on generating novel allelic diversity. Aim IV: To perform mPing mutagenesis in U.S. cultivars, a functional Ping element will be introduced into US cultivars to mobilize endogenous mPing elements and create up to 1500 lines carrying novel mPing insertions. In this way, this project will exploit the full range of mPing amplification, from very low to very high mPing containing strains. Aim V: To use expression profiling (RNA-seq) of abiotic stress response to examine the role of mPing in reshaping the leaf transcriptome in response to abiotic stress. Aim VI: To channel all outreach activities through The Dynamic Genome Courses of Wessler at UCR where a unique interdisciplinary team will serve as a conduit that integrates the research output with the development of classroom modules for colleges and high schools focused on next generation sequencing methodologies. Further, Brutnell and Wessler’s involvement in iPlant's Education Outreach Teams will facilitate the broader dissemination of successful modules.
1b. Approach (from AD-416)
1. Germplasm quarantine: A total of 600 recombined inbred lines (RILs), 300 each for Nipponbare (NB)/HEG4 and HEG4/HG4, will be introduced in four sets (150 per set) and processed through quarantine regulated by the USDA APHIS, Beltsville, MD, for insect and disease prevention. The quarantined seed will be used for evaluation of maturity, height, and cold tolerance, and DNA extraction for study on mPing. 2. Seed stock preparation: Over the course of the project, seed of 600 RILs released from the quarantine program and 1,000 mPing mutagenesis lines using the U.S. cultivars will be increased in field for sufficient seed using single hill for a plant strategy. One plant will be selected from each line for DNA extraction and stock seed source, and the rest of the plants similar to the selected one will be bulk-harvested for the evaluation. 3. Evaluation of days to flower and plant height: The harvested bulk seed of 600 RILs and 1,000 mPing lines will be in single row plot and two locations, AR and TX, three replications arranged with a Randomized Complete Block Design (RCBD) in each location. Each replication will include cultivar 'Rondo' and 'Presidio' as standard checks for monitoring environmental influence. The date when 50% of seedlings emerge above the ground surface 1 cm in the test field will be recorded for conversion of the days to flower. Right before harvesting, height from the ground surface to the tip of panicles will be measured from the center of plot as an estimate of plant height. Data will be statistically analyzed. 4. Evaluation of cold tolerance: The harvested bulk seed of 600 RILs and 1,000 mPing lines will be used for cold tolerance evaluation using method described by Andaya and Mackill (2003) with some modifications. Six seeds of each line will be sown in 98-cell plug flats. Each flat will include 'Zhe733' and 'PI560281', cold susceptible and tolerant checks, respectively. Lines in each flat will be arranged using the RCBD with three replications. The flats will be placed in a greenhouse at 27 C until the seedlings reach the 3-leaf stage before a cold treatment at 10 C constantly with 12 h light period and 70% relative humidity. Plant reaction to cold stress as exhibited by necrosis and/or wilting will be scored using the scale of 1 (tolerant) to 9 (susceptible) when Lemont reaches 9. Data of the score will be statistically analyzed. 5. Establishment of a germplasm collection: After measuring plant height in the evaluation field, the first replication of 600 RILs and 1,000 mPing lines in AR will be harvested as seed source to form the rice germplasm collection. Each step will be cautiously operated to avoid seed contamination. Each line will be manually cut using a sickle, bundled using a rope, and threshed using Almaco Thresher. The thresher will be thoroughly blown using compressed air after each line and the seed in paper envelop will be air-dried to 12% of humidity content. The dried seed will be well cleaned using Clipper seed cleaner blown by compressed air after each line and then deposited in the USDA Genetic Stocks – Oryza (GSOR) Collection.
3. Progress Report
A mapping population including 275 recombinant inbred lines (RILs) of Nipponbare/HEG4 was introduced from Japan and went through quarantine procedures through the USDA APHIS, Beltsville, MD and through the one-year quarantine greenhouse grow-out at the University of Arkansas, Fayetteville, AR. During the greenhouse quarantine, leaf tissue of each RIL was collected and shipped to the University of California, Riverside (UCR) for genetic analysis on copy number of transposable elements. After release from the quarantine greenhouse, 10 seeds of each RIL were provided to UCR for testing of genetic uniformity and presence of the transposable element mPing. The 275 RILs were then planted at Stuttgart, AR, during the 2011 field season. Days to heading and plant height will be determined for each RIL. Five panicles will be sampled from a single plant as a pure seed source for each RIL, and the remainder of the row will be bulk harvested. Seed will be used in subsequent experiments to determine the impact of transposable elements on seedling cold tolerance and salt stress. Ultimately, seed of each RIL will be deposited in the Genetic Stock Oryza (GSOR) collection managed by ARS for distribution to the public. Teleconferences were periodically conducted among three cooperating institutions (ARS, UCR and Cornell University) to discuss project plans. In addition, the cooperator from Japan who developed the mapping population visited the Arkansas field site to evaluate the population and discuss research plans.