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:
1. 2011 Planting: Each of the 275 recombinant inbred lines (RILs) in Nipponbare/HEG4 population plus both parents were directly seeded in the field at USDA ARS facility in Stuttgart, AR, using a Hege 1000 planter resulting in 9 equally spaced plants using a 0.3- x 0.6-m grid. The commercial cultivars Presidio or Rondo were inserted every 20 plots as repeated checks for monitoring environmental variation in the field. About 100 RILs failed to establish 5 or more plants in the field, thus they were restarted in the greenhouse, and transplanted to their respective field plots after fertilizer and permanent flood were applied. 2. Data recording and seed harvesting during 2011: Days to heading (flowering) and plant height in the field were determined, and 10 panicles from a single plant of each line were harvested before bulk-harvesting the remainder of the plot. After cleaning, parents Nipponbare and HEG4 yielded 387 and 340 g of seed, respectively. Except for RIL GN-228 and GN-245, which had low yields of 71 and 175 g, the other 273 RILs had good but variable yields, with 200-299 g among 13 lines, 300-399 among 51 lines, 400-499 g among 85 lines, 500-599 g among 78 lines, 600-699 g among 37 lines, and 700-799 g among 8 lines. GN-253 yielded the most, 873 g. The seeds of each RIL plus both parents have been deposited in the Genetic Stock Oryza (GSOR) collection managed by ARS and will be available for distribution to the public at a later point in time. The remainder of seeds are being used for phenotypic characterization as part of this project. 3. Cold tolerance evaluation: A laboratory protocol for evaluating seed from the field-grown RILs for tolerance to cold temperature at germination was established. Using the protocol, the 275 RILs have been evaluated at 26 C with two replications (30 seeds per replication) as an indicator of baseline germination. The study was divided into seven subsets that were initiated on different days. A cold-resistant germplasm 'Quilla' and the parentals HEG4 and Nipponbare were included in each run and each replication as checks. The checks were found to have 83 to 90% germination under the warm temperatures, and the RILs ranged from 62 to 100%. Then, the same checks and three of the RIL families were evaluated at 12 C with 15 replications (10 seeds per replication) to determine the experimental variability under the cold stress. The Quilla, HEG4, and the three RILs have significantly higher germination rates than the Nipponbare parent. The 275 RILs and repeated checks will next be evaluated under the cold temperature regime. Data will be used to determine genetic variability in the population for response to cold stress at the germination stage. 4. Agronomic evaluation and salt tolerance evaluation: The 275 RILs were field-planted at Stuttgart, AR, and Beaumont, TX, for evaluation of plant height and days to heading. The data will be used to assess the genotype by environment interaction for these traits. At the Stuttgart location the progeny will also be evaluated for tillering ability. In addition, a series of experiments have been designed and implemented for developing a protocol to evaluate the RILs for tolerance to salt during the seedling stage under hydroponic conditions. A methodology for growing the plants successfully prior to salinization has been successful. Currently different salt concentration rates are being tested on the check cultivars and three RILs to determine the rate most effective for screening differences among the lines. 5. Reporting: Conference calls were conducted among the PI's on the grant on a quarterly basis. Documentation on the status of the project was presented as poster and oral presentation at an annual National Science Foundation meeting.