Tomato TILLING

Overview

The UCD Genome Center TILLING laboratory has been funded by USDA-NRI Genome to produce a population of tomato suitable for TILLING and to implement a TILLING service in tomato. The project participants have been Junda Jiang (Tomato breeder and molecular biologist), Allen Van Deynze (co-PI), Roger Chetelat (co-PI), and Luca Comai (PI). We have developed methods to achieve satisfactory mutation rate in Heinz-1706 and demonstrated it in a pilot TILLING using ~500 M2 individuals. We have produced ~4000 more M2 seed families and are awaiting funding to complete the population. This will involve growing one M2 per family, preparing DNA and harvesting M3 seeds for inventory. We will then need to array these DNAs for TILLING by sequencing.

Strategy and Results

History of tomato populations

• Population VFNT-140: 140 mM EMS, 9,000 families at the M2 stage. A pilot test to determine the mutation rate in ~800 M2 plants is in progress.
• Population VFNT-140×2: 1 M2 seed from each of the 9,000 families of population “VFNT – 140” was retreated with 140 mM EMS. From this seed, 5,000 of the M2M1 individuals produced fruits and M3M2 seeds were collected. We planted approximately 12 seeds per family in germination trays, and transplanted up to 6 survivors per family into the field. Sib redundancy is used to increase the probability of obtaining fertile M2s. We had about 20,000 plants growing in the field during the summer of 2009, and hoped to take at least 4,000 families into production.
• Population A1. H1706-100: 100 mM EMS. M2 plants grown in field in Summer 2011. ~1000 individuals. Pilot tested 500.
• Population H1706-m5: 5 mM MNU. M1 plants in greenhouse in Summer 2011.
• Population A2. H1706-110: 110 mM EMS. M2 seed was harvested in the Fall of 2012. We have about 4000 families.
• Population B. H1706-110: 110 mM EMS. M2 seed was grown and harvested in Chile in the Spring of 2016. We have about 10,000 families. This population was funded by a grant from industry. UC Davis has the right to TILL it and distribute the seed. However, the funding partners maintain reasonable IP and commercial rights to genes and products developed. Contact the office of research at UCD (College of Biological Sciences) for further information.
• See page 2 (below) for population production

Phenotypes in Population A (Heinz 1706, EMS)

Quick history and current status

• September 2010. The mutation rate for the VFNT – 140×2 population was unexpectedly low (1 mutation/Mb or less). A new population had to be started.
• Summer 2011. M2 Heinz 1706 treated with 100 mM EMS is grown in the field. M1 Heinz 1706 treated with 5 mM MNU was also grown in the field. MNU turned out to be too harsh a mutagen giving severe phenotypes on most plants.
• Winter 2012. M2 Heinz 1706 plate of 512 individuals have been screened. The mutation rate is good: 2.2 mutations/Mb. In total. this population (the “A” population) consists of 1000 individuals.
• Winter 2013. We have scaled the population by extending the mutagenesis to additional individuals. We have obtained over 4000 additional M2 families (the “B” population). These are currently at the seed stage as M2. The mutation rate has not been measured, but we expect it to be good as the B population was treated at a higher concentration of EMS than the A.
• Winter 2014. We are currently in the process of securing funding to create the screening population. We hope to provide our services to the community soon.
• Summer 2015. With funding from an industry consortium we are expanding the H1706 population.
• Summer 2016. A total of 14,000 M2 individuals are available for TILLING. This downloadble POSTER was presented at the 2016 Solgenomics meeting (Davis, Sep 2016). It presents the details.

Choice of mutagen and variety

We have tested several mutagens (MNU, EMS, UV) by producing populations of different size (30 to 10,000) and in different background. We finally chose Heinz 1706, which is the variety sequenced by the Tomato Genome Consortium.

Funding

Funding for tomato TILLING and the forthcoming service is by grant NRI 2007-02747 “TILLING resources for the tomato functional genomics community” from USDA Cooperative State Research, Education and Extension Service, NRI Plant Genome Program.

See page 2 for phases of population production