Progress 08/01/07 to 07/31/11
Outputs
OUTPUTS: TILLING population development The project required a substantial change in tactics when we discovered that even if we optimized mutagenesis conditions for tomato variety VFNT Cherry the mutation rate was not sufficiently high to warrant establishing a TILLING service. This was a large setback since the construction of a 7,000-9,000 individual population required substantial efforts and resources. We were able to retrace our experimental path and identified that the problem was the variety itself, VFNT Cherry, which was determined to be extraordinarily resistant to mutagens. A similar experimental protocol was applied to Heinz 1706 and we were pleased to find that this variety, the one used for genome sequencing, is more amenable to mutagenesis. Using Heinz 1706, we produced two batches of mutagenized progeny. The first batch resulted from 3200 seeds treated with 100mM EMS resulting in 1,470 M2 families, two sibs/family were transplanted resulting in 1,300 M2 families, from which 1,160 lineages produced M3 seeds. From this population, 600 DNA preps were made and tested in a TILLING-by-sequencing approach (Fig. 5, also see below). The second batch was mutagenized with 110mM EMS, resulting in ~4,500 M2 seed families from ~12,000 treated seed. M2 plants from this second batch are in seed form: they have not been grown and we are currently looking for support to characterize this second population. We determined the mutation rate using TILLING-by-sequencing. We sequenced 19 genes in 24 indexed libraries in a single HiSeq lane, aligned the reads to the target sequences and identified mutants using the CAMBa2 software. The amplicons for the 19 genes added to 26.6kb, which multiplied by 512 individuals resulted in a total searched sequence of 13.6Mb of DNA. We found 31 highly supported mutations, or 2.27/Mb of DNA (1/440kb). Of these, 15 caused missense changes, some predicted severe, the rest were silent or in introns. Twelve of these mutations were subjected to Sanger sequencing and were confirmed. The 2.3/Mb rate compares favorably to the 1 mutation/Mb we detected in the VFNT Cherry population (data not shown). Correspondingly, we detected a distinct difference in the phenotypic mutation rate of these two populations. Two publications describing our experience with tomato mutagenesis are in preparation. PARTICIPANTS: Project Director (Name, institution, email): Luca Comai, Plant Biology and Genome Center, UC Davis, lcomai@ucdavis.edu Co-PDs (Name, institution, email): Roger Chetelat, Allen Van Deynze, Department of Plant Sciences, HYPERLINK "mailto:trchetelat@ucdavis.edu" trchetelat@ucdavis.edu, avandeynze@ucdavis.edu Training: Dr. Junda Jiang - Specialist. Responsible for overall development and testing of the populations Helen Tsai - TILLING high through-put technician. Manages the TILLING laboratory Tyson Howell, Brian Watson - TILLING technicians. High throughput TILLING Kathy Ngo - bioinformatics specialist, High throughput TILLING Dr. Asif Ali - visiting professor, Faisalabad Agricultural University, characterization of TILLED populations Dario Rossi - Visiting Graduate Student, University Cattolica di Pavia, Italy Andrea De la Garza - undergraduate from Heritage University, WA. Characterization of TILLED populations Davis Darren Ma, Steven Heisey, Kelly Kerr, Robert Castillo, Mien Phan, Brian Bush, Momo Yasutake:- undergraduate from UC Davis, development of TILLED populations Collaborations Julin Maloof: shade avoidance in tomato Ann Powell, Alan Bennett: genes related to Uniform Ripening Brian Staskwaicz: screen for mutations affecting disease responses TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Mutations Multiple presumably novel mutants were identified phenotypically and collected by several laboratories for further characterization. One mutation in VFNT Cherry altering shade avoidance and affecting a previously uncharacterized locus is being studied by the laboratory of Julin Maloof at Davis. Other mutations affecting greening responses are being studied in the laboraty of Ann Powell and Alan Bennett. Brian Staskawicz at UC Berkeley has obtained from us a full replicate of the last population of tomato H1706 produced. Other products/ outcomes: The demonstrated recalcitrance of certain genotypes has added important information about the strategy necessary for efficient TILLING population development. For example, because of this, we have developed methods that allow measurement of the mutation density in single individuals. By this approach, feasibility can be determined before substantial investments have been committed. The method for detection of induced deletions has been applied with great success to poplar where currently our population covers 60% of the genome. The research in poplar is funded by DOE grant 201118510 "Creation of High-Precision Characterization of Novel Poplar Biomass Germplasm". Induced mutation via γ-ray irradiation of pollen We have determined optimal conditions for the induction of deletions through ionizing radiation. We have used a γ-ray source (Cs137) to irradiate pollen using 50 to 500 Gy doses. Lethality of the treatment increased to 150Gy. Higher doses resulted in no viable seed. The resulting plants were sequenced at low coverage. We determined that 2/2 plants treated with 150 Gy had deletions, while 0/7 and 0/6 had deletions at 100 and 75-25 Gy, respectively. In conclusion, the work demonstrated the feasibility of producing and scoring deletions in tomato. This work opened the method for applications in clonally propagated crops such as poplar.
Publications
- No publications reported this period
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