Download General enquiries on this form should be made to

General enquiries on this form should be made to:
Defra, Science Directorate, Management Support and Finance Team,
Telephone No. 020 7238 1612
E-mail:
[email protected]
SID 5



Research Project Final Report
Note
In line with the Freedom of Information
Act 2000, Defra aims to place the results
of its completed research projects in the
public domain wherever possible. The
SID 5 (Research Project Final Report) is
designed to capture the information on
the results and outputs of Defra-funded
research in a format that is easily
publishable through the Defra website. A
SID 5 must be completed for all projects.
1.
Defra Project code
2.
Project title
This form is in Word format and the
boxes may be expanded or reduced, as
appropriate.
3.
ACCESS TO INFORMATION
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to any part of Defra, or to individual
researchers or organisations outside
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process final research reports on its
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exemptions under the Environmental
Information Regulations or the Freedom
of Information Act 2000.
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the Environmental Information
Regulations or the Freedom of
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its obligations under the Data Protection
Act 1998. Defra or its appointed agents
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research aimed at improving the
processes through which Defra works
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SID 5 (Rev. 3/06)
Project identification
IF0156
Production of a TILLING population and DNA pools as
strategic resources for lettuce crop improvement
Contractor
organisation(s)
Warwick HRI
University of Warwick
Wellesbourne
Warwick
Warwickshire
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 4
£
40,000
start date ................
01 December 2008
end date .................
31 March 2009
6. It is Defra’s intention to publish this form.
Please confirm your agreement to do so. ................................................................................... YES
NO
(a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They
should be written in a clear and concise manner and represent a full account of the research project
which someone not closely associated with the project can follow.
Defra recognises that in a small minority of cases there may be information, such as intellectual property
or commercially confidential data, used in or generated by the research project, which should not be
disclosed. In these cases, such information should be detailed in a separate annex (not to be published)
so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report
without including references to any sensitive or confidential data, the information should be included and
section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No"
answer.
In all cases, reasons for withholding information must be fully in line with exemptions under the
Environmental Information Regulations or the Freedom of Information Act 2000.
(b) If you have answered NO, please explain why the Final report should not be released into public domain
Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
TILLING (Targetted Local Lesions IN Genomes) offers a route for carrying out what is termed a ‘reverse
genetics’ approach to investigate whether a specific ‘candidate gene’ has an effect on a trait of interest. A
TILLING approach requires two strategic resources; a TILLING population of seed derived from individual
mutagenised plants and a set of DNA samples produced from the individual parent plants of the seed
population organised into pools for molecular screening. It is then possible to screen the DNA to identify
plants in which the ‘candidate gene’ has been mutated and then to go to the seed from those plants to see
if the changes in the gene have any effect on the trait of interest.
The development of a lettuce TILLING population was initiated prior to the commencement of this project.
The population was developed from the cv Saladin which is one of the parents of the Warwick HRI
mapping population and also a parent of a mapping population developed by Prof. Richard Michelmore at
UC Davis. Seed was treated with the chemical mutagen ethyl-methyl sulphonate (EMS) followed by two
rounds of producing seed by self pollination to produce what is termed the M2 population. Approximately
four thousand M2 plants were grown in 2008 and duplicate leaf samples were take from each plant and
freeze-dried for DNA extraction. Fertile plants surviving to maturity were bagged for self pollination and
seed was collected and dried.
In this project DNA has been extracted from a total of 3,800 DNA samples. Five of the original tissue
samples failed to give DNA but extraction from backup samples (collected & freeze dried at the same time
as the originals) was successful. The DNA samples were placed in the Warwick HRI freezer archive
facility for secure long-term storage.
In order to make a strategic resource that can be used for the foreseeable future these DNA samples
needed to be amplified. This was successfully done using a technique called GenomiPhi amplification;
yielding high concentrations of DNA. This amplified DNA will be the main resource for future screening of
the TILLING population for mutations in genes of interest. The strategy for doing this is to make ‘pools’ of
DNA from individual plants, in this way the number of DNA samples in any initial screen is reduced by the
factor of the size of the pools. Once a pool is identified as having a mutation of interest the individual DNA
samples making up the pool are then screened to identify which plant (and hence which seed lot) was
carrying the mutation of interest. Initial DNA pools of 4 samples were prepared in 10 indexed 96 well
plates. This pool size was chosen to optimise the identification of mutations as it is likely that lettuce
cannot tolerate a high ‘mutant load’. (NB In our initial experiments on the production of the population we
had to reduce the concentration of EMS significantly as the treated plants either died or were infertile).
M3 seed produced by self pollination of the M2 plants has been hand cleaned as individual seed lots and
will be placed in to secure storage within the Warwick HRI -200C genomics seed store.
SID 5 (Rev. 3/06)
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Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
Objectives
The objectives of the project were to:
 Complete the development of a lettuce TILLING population
 Produce the complementary DNA pools
For use as strategic resources in future work investigating genetic variation in candidate genes influencing traits
of interest.
The objective to produce the DNA pools has been fully achieved. The development of the lettuce TILLING
population still requires approximately have been achieved.
Background
TILLING (Targetted Local Lesions IN Genomes) offers a route for carrying out reverse genetics (Comai and
Henikoff, 2006) without the need for extensive genomic information. A TILLING approach to reverse genetics
requires two strategic resources; a TILLING population of seed derived from individual mutagenised plants and a
set of genomiphied DNA samples produced from the individual parent plants of the seed population organised
into pools for molecular screening. Development of a TILLING population for lettuce is timely as new genomic
resources are rapidly becoming available; combining a TILLING approach with microarray/gene expression and
QTL studies will facilitate research to identify genes underlying QTL for important traits for sustainability. We
derived a TILLING population from the cv Saladin which is one of the parents of the W HRI mapping population
and a mapping population developed by Prof. Richard Michelmore at UC Davis. Seed was treated with the
chemical mutagen ethyl-methyl sulphonate (EMS) followed by two rounds of selfing. Four thousand M2 plants
were grown in 2008 and duplicate leaf samples were take from each plant and freeze-dried for DNA extraction.
Fertile plants surviving to maturity were bagged for self pollination and seed was collected and dried.
Methods
Seed Cleaning
Dried seed heads previously harvested from individual lettuce plants grown in a Haygrove ‘Spanish’ tunnel at
Warwick HRI were threshed by hand with thorough cleaning of the equipment between samples to avoid crosscontamination. Seed from individual plants will be packed in foil pouches, catalogued and placed in the Warwick
HRI -200 genomics seed store for secure storage.
DNA Extraction
Lettuce DNA was extracted using the DNeasy 96 plant kit (Qiagen Ltd., Crawley, W. Sussex). Two 5 mm
diameter leaf disks from each plant had previously been placed in racked 1.2 ml collection tubes, freeze-dried and
stored at -800C. Extraction followed the manufacturer’s protocol except that the tissue was rehydrated for 5 min in
sample lysis solution before tissue disruption in the mixer mill by two pulses of 90 s at 30 Hz. Purified DNA was
eluted in a final volume of 100 μl. DNA yield and quality were measured by NanoDrop spectrophotometer and
agarose gel analysis. DNA samples were stored at -800C in the Warwick HRI genomic centre freezer archive.
SID 5 (Rev. 3/06)
Page 3 of 4
DNA Amplification & Pooling
The DNA was amplified and normalised using the GenomiPhiTM V2 amplification kit (GE Healthcare, Little
Chalfont, Bucks.). This uses proofreading Phi29 DNA polymerase to amplify genomic DNA, producing high
molecular weight products (>10 kb). Genomic DNA (1 μl) was added to 9 μl sample buffer on ice, the mixture was
denatured at 950C for 3 min & cooled on ice. A master mix containing 9 μl reaction buffer and 1 μl enzyme mix
per reaction was prepared on ice, added to each sample & incubated at 30 0C for 90 min. The enzyme was
deactivated by heating at 650C for 10 min. Amplified DNA was stored at -800C.
Amplified DNA samples were pooled for subsequent screening of the TILLING DNA for mutation detection. Pools
of 4 samples were prepared by combining 5 μl of amplified DNA in 96 well plates.
Results
Seed Cleaning
Approximately 75% of the M2 plants yielded M3 seed; ranging form a few seeds up to >50gms per plant.
Currently there are still approximately 200 seed lots that require threshing and cleaning. Seed has been packed
into individual packets and will be placed in the Warwick HRI -200C genomics seed store and indexed in a
database.
DNA Extraction
A total of 3,800 DNA samples were extracted in 40 96-well plates. Five of the original tissue samples failed to give
DNA but extraction from backup samples (collected & freeze dried at the same time as the originals) was
successful. Yields of DNA ranged from approx. 30 – 120 ng/μl in 100 μl total volume with a mean 260/280 ratio of
1.74 (pure DNA has a 260/280 ratio of 1.8). Gel analysis showed that the DNA was high molecular weight with no
evidence of shearing or degradation. The DNA samples were placed in the Warwick HRI freezer archive facility
for secure long-term storage.
DNA Amplification & Pooling
GenomiPhi amplification of the DNA was successful, yielding high concentrations of DNA with high molecular
weight when analysed on agarose gels. This DNA will be the main resource for future screening of the TILLING
population for mutations in genes of interest. Initial DNA pools of 4 samples were prepared in 10 indexed 96 well
plates using 5 μl of each undiluted GenomiPhi DNA sample. This pool size was chosen as a basis for subsequent
optimisation of screening procedures.
Future Work
Screening protocols for the TILLING DNA will be optimised. PCR primers which amplify known regions of lettuce
genes will be used to verify the DNA sequence from the wild type Saladin and individuals from selected pools.
The pooled DNA at various dilutions will be screened for detection of known mutations found in individuals by
formation of heteroduplexes between DNA strands of mixed wildtype and mutant amplicons, followed by detection
using the Roche LightCycler real time PCR system and/or FAME dHPLC analysis.
The objective will be to successfully detect mutations in pools of 8 individuals.
The cleaning of the M3 seed will be completed and repackaged into foil packets prior to depositing in the Warwick
HRI -200Cgenomics seed store and recording in a database. This will be completed in IF0157 - Vegetable
Genetic Improvement Network (VeGIN): Pre-breeding research to support sustainable farming of leafy vegetables
and salads
References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
Hand P, McClement S, Horridge J and Pink D.A.C. (2007). Development of a lettuce TILLING population
for functional genomics. Eucarpia Leafy Vegetables 2007, University of Warwick 18-20 April. (Poster).
Lettuce TILLING research highlighted in press and news item broadcast on BBC ‘Midlands Today’
(http://news.bbc.co.uk/1/hi/england/7490042.stm).
SID 5 (Rev. 3/06)
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