PROGRESS TOWARDS DEVELOPMENT OF AN INTERNATIONAL SEED TESTING ASSOCIATION (ISTA) DNA-BASED APPROACH FOR TESTING VARIETY IDENTITY

DNA Working Group Update:

No DNA-Methodology is standardized in the ISTA International Rules for Seed Testing.

Methods being included within the ISTA International Rules for Seed Testing must be validated internationally to ensure an acceptable level of accuracy, repeatability and reproducibility, regardles of the purpose of testing, or the place where the testing is done.

This shows the value of the method at the international level which in turn,helps facilitate trade.

Current status: ISTA and DNA based methods

There is more and more interest in using DNA based methods for variety identification purposes.

Many markers are now available and DNA based methods are increasingly being used and developed for this purpose.

Recognizing this, ISTA’s Variety Technical Committee initiated work towards development of standardized DNA based methods for ISTA that can be used for testing variety identity.

Background: Why this work is being done:

DNA Working Group was established: 2007 Marker system selected: microsatellites

- proven one of the most appropriate for variety identity and genetic relationship studies

Crops selected: Wheat, Rice, Corn, Soy- most important in terms of cultivated area/world production

Process: Comparative tests (CTs) to select the most reliable markers/crop and to validate and standardize the protocols.

The DNA Working Group:

Challenge: DNA Methodologies can have

complexities!• All DNA based methods require a ‘set’ of different protocols/procedures:

• Extraction of DNA• Purification of DNA• Selection of Primers and PCR conditions• Visualisation systems• There are many variables: chemistries, protocols and procedures,

equipment (e.g.PCR machines) and visualization systems. • DNA methods can be sensitive to contamination. • Repeatability within the same laboratory (through the same analyst or

through a different analyst) can be difficult to establish.

DNA WG strategy for incorporating DNA-based tests

into the ISTA Rules: AIMS

- compare results between laboratories through comparative tests, and evaluate if it is possible to obtain the same band patterns/allele sizes even when using different reagents, equipment and working protocols.

- select the most suitable SSRs for variety identification.

- verify the marker set is polymorphic enough to provide unique DNA-based patterns for a larger variety set.

- prove robustness of the method by having additional laboratories to run the selected SSRs and the same varieties tested by the original collaborators.

Acceptance criteria for the chosen SSRs :

- Repeatable between laboratories and between CTs

- Sharp band profile

- Without faint bands or null alleles

- Without stuttering

- Polymorphic with the most common technical equipments

SSR that did not present these characteristics were rejected and substituted in the following CTs

DNA WG strategy for incorporating DNA-based tests

into the ISTA Rules:

Working group Number of selected SSR

Amount of tested Varieties

Tested Areas

Soybean 11 74 Argentina Brazil

Wheat 8 84 Canada Brazil

FranceItaly

Maize 12 72 FranceCanada

Rice 15 192 AsiaItalyUSA

Summary of Results:

Summary of Results:

SSRs: classified as “good” and “very good” - meaning that repeatable and reproducible results were obtained for that marker during 2-3 rounds of comparative tests

For all crops: - different numbers of SSRs could be selected- more SSR should be selected from some crops to identify larger sets of

varieties

Different visualization systems do give the same banding patterns (even if allele sizes are slightly different)

•ISTA has now the chance to offer Proficiency Test with DNA based markers

•The PT will allow the development of an appropriate approach for the accreditation of laboratories willing to carry out variety identification tests

•The DNA WG has established a proficiency testing (PT) program to assist laboratories develop testing capacity for varietal verification for these crops using molecular markers and to promote use of these internationally standardized methods

•For the Performance Test it is necessary to have a core set of varieties to be used as reference material (RM)

Next Steps

The Proficiency Tests

•The RM will be composed by more or less 30 varieties representing as much as possible the allele variability of the specie

•Seed lot must be pure seed, possibly pre-base seed or “breeder seed”

•Varieties can be public or commercial ones

•Participant laboratories will receive a sub set of i.e. 12 varieties to be analysed. Two varieties will be known and accompanied by documentation with the description of the expected allele profiles

•The two known varieties will be the control of many performance factors: DNA extraction, PCR performance, electrophoresis conditions, comparison of allele sizes, etc.

•The remaining 10 varieties will be the Performance Test

The DNA WG together with Rita Zecchinelli from the ECOM has established a list of priorities:

1)PTs will be based on a performance based approach

2) Devitalized or grounded seeds will be distributed

3) 5 kilos of seeds for each variety included in the RM list will be stored

4) PTs will be organized storing over the years the 30 varieties and each PT will be organized choosing randomly a subset of varieties that will be sent to the laboratories with a code. This would give a large number of possible combinations and many PTs can be organized before the same 10 variety set have to be repeated.

The Performance Tests

5) one or two labs that performed the last maize CTs will set up the SSR profiles to be sent to the participating laboratories as reference sample.

6) RM preparation and distribution must be done in collaboration and with the supervision of ISTA

7) the Variety Committee will ask the support of the ECOM to keep in contact with Companies or Institutions in order to facilitate the Committee in providing the needed reference materials

8) the VAR Committee will ask the opinion of the ECOM on the inclusion in the ISTA Rules of the performance based approach for this kind of testing

The Performance Tests

The Reference Matherial

• The WG has composed a proposal with a variety list to be submitted to important Institution as USDA and ESA in order to obtain the seed lots to be used as RM

• The DNA WG is open to work with ISTA’s Statistics Committee to develop a strategy for accreditation of laboratories performing DNA-based variety verification tests.

• Up to now the WG is waiting for the answer from USDA and ESA and for an official involvement of ISTA

We would like to acknowledge to all our collaborating colleagues, the private and public institutions that kindly collaborated with these tests, and ISTA, for technical and administrative support.

Acknowledgements

www.seedtest.org