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G

eneral enquiries on this form should be made to:

Defra, Science Directorate, Management Support and Finance Team,

Telephone No. 020 7238 1612
E-mail: research.competitions@defra.gsi.gov.uk




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.



  • This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATION

The information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.





Project identification




1. Defra Project code

GC0139

2. Project title


Fingerprinting the National Fruit Collection - a demonstration study on pear 



3. Contractor
organisation(s)

East Malling Research

New Road

East Malling

Kent


ME19 6BJ

     







54. Total Defra project costs

£ 19,041

(agreed fixed price)




5. Project: start date

1 October 2006







end date

31 March 2007

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.

Aims and Objectives
Defra holds the National Fruit Collections (NFC), which are located at Brogdale, Kent, and curated scientifically by Imperial College, London. These collections constitute valuable genetic resources for projects concerned with the genetic improvement of the UK's principal fruit crops with respect to the requirements of, eg sustainable production and climate change. They contain ~ 545 accessions of pear (Pyrus), as well as collections of apple, cherry, currants and gooseberries (Ribes) and plum. In recent years, DNA markers known as microsatellites or SSRs have been developed which are proving very useful for 'fingerprinting' accessions. Such fingerprints are invaluable aids to the management of collections, eg when checking for trueness to type after propagation or for detecting likely duplicates.

Building on a successful pilot project to fingerprint the cherry collection, Defra's NFC Advisory Committee recommended fingerprinting the pear collection, which is in the process of being repropagated. This proposal followed that advice, with the aim of fingerprinting half of the collection.


The main objective is:


  • To use microsatellites to fingerprint 275 pear accessions in the National Fruit Collections and provide curators with a valuable data set which distinguishes clearly all or most of the varieties tested thus enabling checking of identities and detection of synonyms



Methods
The work was broken down into the following technical aims:


  1. to extract DNA samples from 275 accessions of the pear collection

  2. to optimise PCR conditions for ~12 informative microsatellite primers, developing multiplexes if appropriate

  3. to determine the microsatellite fingerprints of 275 accessions of the pear collection

  4. to collate the data into an Excel spreadsheet, e.g. to allow the curator to search for duplicates, to submit the data to the freely-accessible ECPGR Pyrus database and to draft a paper on pear fingerprinting for e.g. the Eucarpia Fruit Breeding Symposium


Main findings
This technique has proved suitable for distinguishing 229 accessions from the 276 tested. The remaining 47 can be divided into a group of 12 accessions of ‘Williams’ type, a group of five accessions of ‘Conference’ type, two groups of three accessions and 12 groups of two accessions where the accessions within the groups are indistinguishable with the SSRs. These are listed in Appendix 3. These data have been sent to the Curator of the National Fruit Collection where morphological data will be used to determine if the accessions within the groups are in fact identical. These fingerprints will prove to be an extremely valuable reference set for testing the trueness-to-type of the recently re-propagated pear collection in the future. The re-propagated pears can be similarly fingerprinted and the two data sets compared – thus avoiding the need for laborious and time-consuming morphological comparison.
Other significant events and options for new work
Within the duration of this project, EMR organised and hosted an ECPGR workshop where the priority core set of SSRs as used within this project were agreed with other international experts. This core set will be used when other pear collections are fingerprinted thus allowing comparisons between the different National Collections e.g. to confirm identities and, perhaps, to reduce duplication. There has already been wide international approval of this standardised approach. A set of eight control accessions were also chosen from the Brogdale collection which will aid comparison of results.

Options for new work should include fingerprinting the rest of the pear collection to enable the full extent of the duplication of accessions to be realised. A full set of data will also provide the appropriate reference data set for verification of the newly re-propagated pear collection. The ECPGR group also agreed the priority core set of SSRs for fingerprinting apple. As with the pear collection, fingerprinting the apple collection would enable the Curator to confirm duplicates and also test any future re-propagated collections for trueness-to-type.

Regarding the wider context, there is scope for characterising the pear collection for features relevant to climate change, by molecular and phenotypic approaches, and, indeed, using the available genetic resources in a targeted programme of genetic investigation and improvement.








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).




Scientific objectives of the contract
To use microsatellites to fingerprint 275 pear accessions in the National Fruit Collections and provide curators with a valuable data set which distinguishes clearly all or most of the varieties tested thus enabling checking of identities and detection of synonyms
Extent to which the objective set out in the contract was met
The objective was met in full and, in fact, exceeded. In all, 276 accessions were fingerprinted not 275. DNA was extracted from the 276 accessions; the extracts were amplified with a set of thirteen microsatellite primers that were agreed at the ECPGR workshop attended by international experts; the amplification products were separated on an automated sequencer; and the genotypes recorded into an Excel spreadsheet that was provided to the scientific curator at Imperial College.

Materials and methods
Technical aim 1- DNA extraction

Leafy shoots were collected from one of each pair of accessions of the pear collection at Brogdale by the Sub-contractor and were labelled. Leaves were removed, frozen in liquid nitrogen and stored in a -80oC freezer at EMR. DNA was extracted from half the samples (276 accessions) using 0.2g tissue following a modified CTAB protocol (James et al. 2003). Leaves were also collected and DNA extracted from the eight Brogdale accessions selected as controls at the ECPGR workshop organised by EMR. The extracts were quantified and partially qualified by electrophoresis through agarose. The quality of the DNA was further assessed by checking the amplification the samples with a fully optimised PCR. A full list of the accessions characterised can be found in Appendix 1.


Technical aim 2 - Choice of primers and optimisation of PCR conditions

The choice of a set of microsatellites for fingerprinting pears was discussed at the recent ECPGR workshop attended by international experts. Markers have been chosen from each linkage group (where possible) that are robust, preferably single locus and that have been proved to be polymorphic in previous studies. From those recommended, we chose 13 that, fluorescently labelled, were combined into four ‘multiplex’ combinations (see Appendix 2). The most informative 12 primers that were chosen as the ECPGR core set (priority groups 1, 2 and 3 in the table) were optimised into three multiplexes for future use. PCR conditions were optimised to obtain robust and reliable amplification. Primer 13 (CH_Vf1) consistently produced a ‘third’ peak which could not be ignored even though we are confident from the rest of the results that the accessions involved are not triploids.


Technical aim 3 - Fingerprinting the pear collection

The DNA samples were amplified with the chosen primers, in multiplex reactions, using a thermocycler machine and ensuring the presence of the selected control samples within each plate. The amplification products were loaded on to an ABI automated sequencer so that the alleles could be sized. Then the data were compiled using GENESCAN and GENOTYPER software. The quality and reliability of the fingerprints was assured by using standardised methodologies with optimised primers and regular control samples. Hard copies of all traces from the software were printed allowing clear comparisons between peak intensity and fragment size of the amplified products. Any variation in peak size between plates was normalised by comparing the control samples. All data were checked twice by two individual staff members and repeats were performed on any sample where the traces were unclear. Data were stored within EMR’s EMQA system.


Technical aim 4 - Collating genotypes

An Excel spreadsheet was prepared giving the genotypes at each microsatellite locus of the pear accessions. This has been provided to the scientific curator at Imperial College. Data have been analysed using GenAlex and PAUP* software to generate dendrograms to help identify duplicates in collaboration with Sub-contractor. The Pyrus database manager has been notified that the data set is now available for inclusion. A draft paper has been prepared for the Eucarpia Fruit Breeding Symposium to be held in September 2007.


Results
Comparisons between the traces have revealed several groups that are indistinguishable by the SSRs used. These are listed in Appendix 3. Some of the names reveal that these are in fact clonal cultivars which we would expect to appear identical using this method.
Discussion of the results and their reliability
Discussion: Information regarding the optimised multiplexes has been distributed to other European groups who have already adopted their use. This should allow comparison of data sets between collections e.g. to confirm identities and, perhaps, reduce duplication. Although the data from primer 13 (CH_Vf1) is included in this report, they have been dropped from the ECPGR priority core set.

Our data set should assist the curator of the pear collection to confirm some duplicated material. Identification of pairs/groups with indistinguishable SSR fingerprints, combined with morphological observations should enable decisions to be made about possible future de-accessions from the collection.


Reliability: We are confident about the reliability of these results. The initial leafy samples were collected by Dr Alison Lean who has worked with the National Fruit Collections for over 10 years. All samples were labelled clearly with a unique identifier code which relates to the physical position of each source tree within the Collection. The quality and reliability of the fingerprints was assured by using standardised methodologies with optimised primers and regular control samples. Hard copies of all traces from the software were printed allowing clear comparisons between peak intensity and fragment size of the amplified products. All data were checked twice by two individual staff members and repeats were performed on any sample where the traces were unclear.
Main implications of the findings
The fingerprinting will be a significant aid to the efficient management of the collections, as has been demonstrated by the cherry pilot project, allowing some duplicates to be detected. The data will be a useful reference should the recently re-propagated pear collection be fingerprinted to compare against the original collection. The traditional method of doing this, using morphological comparison, is time consuming and takes several years. The agreement at European level on standard microsatellites and reference control cultivars is a major advance and makes this work of international, as well as national, relevance.
Possible future work
The first priority for future work is to complete this fingerprinting exercise with the rest of the pear collection. This data set would identify further duplicates and would complete the reference data set. Fingerprinting the recently re-propagated pear collection would allow the newly propagated trees to be verified against the established collection. There is scope for characterising the pear collection for features relevant to climate change e.g. flowering time or winter chill, by molecular and phenotypic approaches, and, indeed, using the available genetic resources to investigate the genetic control of such important traits.
Action resulting from the research
The primary action from this research is to enable the curator of the National Fruit Collections to identify and remove duplicates. As the ECPGR initiative to suggest a core set of microsatellites for fingerprinting fruit collections coincided with this project, the reference data set that we have presented to the ECPGR Pyrus database is the first data set of this size. Initiation of this work has helped to promote this co-ordinated movement internationally, with interest from both New Zealand and the U.S.A.
Presentations and industry interaction
This project was briefly described during discussions at the ECPGR microsatellite workshop at East Malling Research in December 2006 and at the National Fruit Collections Advisory Committee meeting in January 2007. Some of the data will be presented at the Eucarpia Fruit Breeding Symposium in September 2007 and at the planned EMRA ‘DNA day’ at EMR in November 2007.

Appendix 1: List of the accessions characterised


Plot reference

Cultivar name

Plot reference

Cultivar name

01-09

President Drouard

08-31

Illinois

01-11

Laxtons Superb

08-38

Madame Millet

01-13

San Giovanni

08-41

Santa Claus

01-16

Beurre Clairgeau

09-01

Ananas de Courtrai

01-21

Emile d'Heyst

09-06

Coloree de Juillet

02-01

Bambinella

09-08

Double Williams

02-05

Kruidenierspeer

09-09

Harvester

02-07

Moonglow

09-13

Tany Kisil

02-09

Beth

09-15

Alexandre Delfosse

02-12

Mercer

09-17

Conference

03-04

Early Seckel

09-19

Beurre Baltet Pere

03-07

No Blight

09-25

Certeau d'Automne

03-09

Fair Maid

09-27

Constant Lesueur

03-11

Leonie Bouvier

09-29

Merton Star

03-19

Doyenne du Comice

09-33

Glou Morceau

04-05

Magness

09-35

Josephine de Malines

04-09

Fin Juillet

09-37

Marie Benoist

04-11

Mademoiselle Solange

09-39

President Heron

04-16

Beurre Jean Van Geert

09-41

Scipiona

04-21

Ferdinand Gaillard

10-F

Instone 1

04-27

Robert de Neufville

10-01

Arabitka

05-01

Botzi Blanc

10-04

Bon Chretien Walraevens

05-07

Precoce de Trevoux

10-05

Comte de Lambertye

05-17

Colette

10-08

Max Red Bartlett

05-19

Dubbele Kreeftpeer

10-09

10-09 Hessle

05-21

Fertility

10-13

Triomphe de Vienne

05-29

Comice Bodson

10-15

Alexandre Lambre

06-01

Bristol Cross

10-17

Conference Russet Wheldon

06-03

Gorham

10-19

Beurre Bedford

06-05

Maxine

10-21

Beurre de Beugny

06-07

Nye Russet Bartlett

10-25

Cinq Grappes

06-11

Maltese

10-27

Coscia Tadiva

06-13

Spadona d'Estate

10-34

Doyenne d'Alencon

06-17

Conference Bronzee

10-35

Levard

06-35

Furedi

10-37

Marlioz

07-03

Gregoire Bordillon

10-39

Winter Nelis

07-05

Starking Delicious

11-G

Van Mons Leon Leclerc

07-08

Parburton

11-01

Arpaval Ero

07-26

Theodore van Mons

11-03

Roem van Wijngarden

07-27

Beurre Hardy

11-05

Coscia

07-29

Surprise

11-07

Redbald

07-31

Hermansverk

11-09

Illinka

07-33

Beurre d'Anjou

11-11

Petite Marguerite

07-35

Huyshes Victoria

11-15

Alexandrine Douillard

07-38

Madame Ballett

11-17

Bergamotte Fondante d'Ete

08-08

Arnold

11-23

Beurre Francois

08-14

Poirier Fleurissant Tard

11-27

Crassane Panachee

08-16

Abbe Fetel

11-29

Southworth

08-17

Conference Van Wetten

11-33

Antoine Bouvant

08-20

Cascade

11-37

Rogue Red

08-21

08-21 Beurre d'Avalon

12-H

Seckel Robarts

08-23

Beurre Dumont

12-02

Aspasie Aucourt

08-27

Comte de Paris

12-03

Calcina dal Corbel

12-07

Biggar Russet Bartlett

15-13

EM 19

12-11

Phillippe Chauveau

15-15

Avocat Allard

12-17

Bergamotte Heimbourg

15-21

Gansels Bergamot

12-20

Beurre Capiaumont

15-25

Magyar Kobak

12-21

Beurre Dilly

15-27

Napoleon

12-23

Beurre Superfin

15-29

President d'Osmonville

12-25

Comte de Lamy

15-31

Italy

12-27

Doyenne de Poitiers

15-33

Bergamotte Philippot

12-29

Fagg 1

15-35

Bon Chretien d'Hiver

12-31

Vermont Beauty

15-37

Fauvanelle

12-33

Belle des Abres

15-39

Lehoux-Grignon

12-36

Beurre Sterckmans

16-E

Barney

12-39

General Tottleben

16-G

Butirra di Roma

12-41

Sos

16-01

Belle de Jumet

13-E

Krystali

16-03

Citron des Carmes

13-H

Johnny Mount

16-05

Doctor Stark

13-01

Augusztusi Nagy

16-07

Williams Bon-Chretien

13-04

Bonne d'Ezee

16-11

Robin

13-05

Coscia Precoce

16-13

Howlett 1

13-07

Knock-out Russet Bartlett

16-19

Dutch Holland

13-09

Jazzolo

16-23

Laxtons Victor

13-11

Reine des Poires

16-27

Nargilia

13-16

Autumn Bergamot

16-39

Le Lectier

13-17

Danas Hovey

17-H

Butirra Rosata Morettini

13-25

Louise Bonne Sannier

17-03

Citron des Carmes Panache

13-27

Miskolci

17-06

Doyenne d'Ete

13-29

Pero Nobile

17-07

Williams Bon-Chretien

13-31

White Doyenne

17-11

Sainte-Anne

13-35

Beurre d'Avril

17-15

Belle Guerandaise

13-37

Dorsel

17-19

English Bergamot

13-39

Girogile

17-21

Grosse Calebasse

14-H

Condo

17-23

17-23 Le Brun

14-01

Ayrshire Lass

17-25

Marie-Louise d'Uccle

14-07

Moyer Russet Bartlett

17-29

Princess

14-09

Jean Cottineau

17-35

Bonneserre de Saint-Denis

14-12

Revesz Balint dr

17-37

Duchesse Panachee

14-15

Autumn Nelis

18-03

Claude Blanchet

14 17

De Duvergnies

18-05

English Caillot Rosat

14-19

Duchesse Bererd

18-07

Russet Bartlett

14-22

Foucouba

18-17

Doctor Desportes

14-25

Magnate

18-19

Ewart

14-27

Mora

18-21

Hacons Incomparable

14-29

Phelps

18-23

Legipont

14-31

Barnham

18-27

Nurun Burun

14-33

Bergamotte Esperen

18-30

Mere Perrier

14-35

Blickling

18-35

Broompark

14-39

Highland

18-37

Easter Beurre

14-41

Swans Egg

18-39

Martin Sec

15-F

Harrow Delight

19-01

Beurre de Conitz

15-G

Fulva

19-03

Clapps Favourite

15-01

Beacon

19-05

Espiki

15-03

Cheltenham Cross

19-09

Laxtons Foremost

15-05

Crawford

19-11

Saint Luke
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