Tomato Tilling
From 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 are Junda Jiang (Tomato breeder and molecular biologist), Allen Van Deynze (co-PI), Roger Chetelat (co-PI), and Luca Comai (PI).
Status
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 have focused on VFNT Cherry because of the following advantages:
- Compact growth habit allows fruiting in a 1-inch pot
- Grows well in greenhouse and field, highly productive
- Resistant to TMV
- Indeterminate growth habit allows easy propagation
- Semiglabrous stem allows easy identification of contaminants
- Strong cleistogamy prevents accidental outcrossing
- Responds well to mutagen treatment
Population production
- 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 VNT -140x2: 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 about 12 seeds per family in germination trays, and have transplanted up to 6 survivors per family into the field. Sib redundancy is used to increase the probability of obtaining fertile M2. As of July 15 2009 we have about 20,000 plants growing in the field. We hope to take at least 4,000 families to maturity and seed production.
- More information on population production
Mutation Detection Approach
We have converted from CELI-LiCor assays for detection of mismatches to massively parallel sequencing of pooled genes through the Illumina GAII platform. This entails the following steps:
- Two-dimensional pooling of individuals DNAs
- PCR amplification of 20 to 100 genes from each pool
- Construction and sequencing of "Solexa" libraries
- Identification of mutations. The present assay resolves them to 8x pools
- Interesting mutations are deconvolved by a simple, gel-based assays or by direct sequencing
- The seed coordinate of the mutant individuals are given to the user
- The user orders seed tomato genetics cooperative at UC Davis
How to get TILLING
We yet do not have a developed TILLING population for tomato. We may have the population in the Fall of 2009. In the mean time, we are happy to use genes suggested by the tomato research community for testing our pilot populations. Note that the probability of finding good mutations is low until a developed population is available. If you are interested in having your genes included in pilot tests, here are the steps:
Design and test primers for your gene
Design primers that amplify 1 kb to 1.5 kb target and have 68°C thermal stability using Primer3. We suggest the use of CODDLE to target the optimally suited target region in your gene. Make sure by BLASTing your primers against available genomic sequence of tomato to ensure, as much as possible, that they target a single sequence. Test your primers on VFNT Cherry tomato (LA1221) DNA, if available, using the the following PCR conditions (CONDITIONS MAY CHANGE AS WE OPTIMIZE OUR PROTOCOLS):
Final concentration of reagents
- Ex Taq Buffer 0.5X
- MgCl2 2.5mM
- ExTaq buffer comes in two versions: with or without Mg++. We use the Ex Taq Buffer with 2mM of MgCl2 at 1X. We add additional MgCl2 to reach 2.5mM.
- dNTP 0.2mM
- primers 0.15µM
- Ex-Taq polymerase 0.15µl/30 ul rxn
- template DNA 1.5 ng/rx
Rx volume: 30 ul
Cycling program
- 95° 2’
- Loop 1: 8 cycles
- 94° 20”
- 73° 30”
- Increment -1°C/cycle
- 72° 1’
- Loop 2: 35 cycles
- 94° 20”
- 65° 30”
- 72° 1’
- Hold 8°
Polymerase: Takara Ex Taq Hotstart
CHANGES from the above protocol:
- Our high-throughput operation does not allow us to incorporate protocol changes. The only exception is in the case of high GC content templates. If the above conditions do not work for you, you can try adding a co-solvent such as 3% TMSO. In that case, please indicate it to us.
For details on co-solvents, see [1]
- ↑ Chakrabarti R & Schutt CE (2001) The enhancement of PCR amplification by low molecular weight amides. Nucleic Acids Res 29: 2377-81 PubMed
Placing order
Please refer to our placing order information page for step by step instructions to place orders.
What happens next
We will amplify your target and add it to our sequencing queue. Once mutations are found you will be notified. We expect to charge a reasonable fee for deconvolution of your mutations.
Frequently Asked Questions
1.How many mutations can I expect?
We are testing populations that have been treated with mutagens. The mutation density may vary and so would the number of discovered mutations. At this stage, we do not promise anything.
2.What type of phenotypic changes will these mutations cause?
If the action of the targeted gene is required for a process that can be scored phenotypically or biochemically, you should detect a change in this process if any of your mutations causes loss of function.
3.How many of my mutations will result in a loss of function of the protein encoded by the targeted gene?
In average, we expect about 45% of the mutations in a coding region to be missense, i.e. to cause a codon and corresponding amino acid change. About a third of these changes should impact protein function. We also expect 4-5% nonsense and splice site mutations. These mutations results in premature termination of the encoded polypeptide. The above are approximations; the structure and nucleotide composition of your gene will determine the impact of the expected mutations.
4.All summed, will TILLING be useful for my analysis?
Utility will emerge as a suitable population is developed. This is a population in which mutations are found every 500 kb of diploid genomic DNA or more frequently.
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.
