DEVELOPMENT OF AN

INTERNATIONAL SEED TESTING

ASSOCIATION (ISTA) DNA-

BASED APPROACH FOR TESTING

VARIETY IDENTITYVicario, A., Casarini, E., Perry D., Zhang D., Dollard, C., Hwu K.

Cheryl DollardCanadian Food Inspection Agency

Ottawa - Canada

The strategy for incorporating DNA-based tests into

the ISTA Rules.

• Establishing the DNA WG in 2007

• Selection of microsatellites (SSRs) for markers, as they have proven

to be one of the most appropriate for variety identity and genetic

relationship studies

• Selection of crops based on those representing the most important

ones in terms of cultivated area and world production

• Conducting comparative tests (CTs) for the selection of the most

reliable markers per crop

• These CTs were performed initially by laboratories having experience

with each crop and/or technique

The current organization of the Variety TCOM DNA working group:

WG Leader: Emanuela Casarini (Italy)

Soybean WG and vice chair of the Var Com: Ana Laura Vicario (Argentina)

Rice WG: Cheryl Dollard (Canada) & Kae-Kang Hwu (Taiwan)

Wheat WG: Daniel Perry (Canada)

Maize WG: Benjamin Kaufman (United States of America)

The DNA-WG – the past 3 years…

Iguassu Falls – Brazil on May 2007

Bologna – Italy on June 2008

Zurich – Switzerland on June 2009

The strategy for incorporating DNA-based tests

into the ISTA Rules.

• AIMS:

• to compare results between participant laboratories, and

evaluate whether it is possible to obtain the same band

patterns and allele sizes even when using different reagents,

equipment and working protocols

• to select the most suitable SSRs for variety verification

• to verify if the marker set was polymorphic enough to provide

unique DNA-based patterns for a larger variety set

• to prove robustness of the method by including additional

laboratories that would run the selected SSRs and the same

varieties tested by the original participating laboratories

Summary of results

Crop group

Participating laboratories per CT Visualization methods used

CT1 CT2 CT3

Wheat Canada x2

France

Italy

Canada x2

France

Italy

Austria, Argentina

Canada x3

France, Italy

Licor 4200 and 4300, ABI 3130xl and Silver stain

Rice Canada, ItalyTaiwan

Canada, ItalyTaiwan

Canada, India

Italy, USAx2, Taiwan

ABI3100 and 3130, Licor 4300 and Agarose

Soy Argentina

Brazil, Canada

Argentina

Brazil, Canada, USA

Argentina x3

Brazil, Canada, USA

Silver stain and ABI 3100

Maize Argentina

Brazil, Canada

France

Argentina

Brazil, Canada

France

Brazil, Canada

China, France x2, Italy, USA x2

ABI3130xl, Silver stain, ABI 3100 and Silver Stain

Summary of results

• SSR were qualified as “good” and “very good”, meaning that

reproducable results were obtained for that marker during 2-3

rounds of comparative tests

• For all crops different numbers of SSR could be selected

• For most crops, more SSR should be selected so as to identify large

sets of varieties in many countries

• Different visualization systems prove to give the same banding

patterns (even when the allele sizes may not have been the same)

Examples of analytical data generated from different equipment

CA17

CA18

CA09

CA10

CWSW

S1

CWSW

S2

CA17

CA18

CA09

CA10

CWSW

S1

CWSW

S2

Xgwm003

Xgwm052

Xbarc074

Xbarc184

Xbarc374

Xgwm372

Silver Stain

Licor

28 varieties from France

30 varieties from Canada

20 varieties from Italy

6 from Brazil

3 pairs of varieties with the same SSR pattern

Summary of results: wheat

30 varieties from Argentina

22 varieties from Brazil

4 varieties from Canada

2 pairs of varieties with the same SSR pattern

Summary of results: soybean

Summary of results: rice

192 varieties from Asia, USA and Italy evaluated

with 50 SSR

From this, it is planned to select a set of 15 SSR

and 24 core varieties (plus seven optional Indian

varieties) for further work.

24 varieties from France

24 varieties from Canada

96 varieties from China

96 varieties from other countries

No pairs of varieties with the same SSR pattern

Summary of results: maize

Summary of results

• The maize group has established an SSR set that is ready to be

applied for the organization of a Performance Test

• For the Performance Test it is necessary to have a core set of

varieties to be used as reference material (RM)

• ISTA is working on a strategy for the development of RM suitable

for these tests

• The PT will allow the development of an appropriate approach for

the accreditation of laboratories wishing to carry out variety

verification tests

The Performance Tests

The Reference Material (RM) must have the following properties:

• Pure seed, possibly pre-base seed

• Public or commercial varieties, distributed as flour or crushed

seed

• RM preparation and distribution must be done in collaboration

and with the supervision of ISTA

• PTs will be organized preparing two variety „sets‟ of reference

material.

• First set: a small set of varieties representing allele variability

• This fixed set of varieties will be the control of many performance

factors: DNA extraction, PCR, electrophoresis, comparison of allele

sizes, etc.

• Participant laboratories will receive an accompanying

documentation with the description of the expected allele profiles

• This will be the calibration reference material

The Performance Tests

• Second set: a larger set of varieties (twenty or more) that will be

used as unknown samples during the PTs

• Sets of 10 varieties (or more) would be chosen to be used in a

given PT, each sample variety would have a blind code.

• Allele names (A, B, C, etc) and sizes (in bp) reported for the test

sample and allele profiles would be the same as were defined for

the first set of reference samples.

• A large number of possible combinations could then be prepared

from this set before the same 10 varieties would have to be

repeated.

The Performance Tests

•Define a set of “reference varieties” for each crop that can used

by laboratories to establish protocols for variety verification

•Define a strategy together with ISTA‟s Statistics Committee for the

accreditation of laboratories performing DNA-based variety

verification tests and for the data analysis.

• Establish 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

Next Steps…..

On behalf of Emanuela, I wish to thank all the WG leaders and all of our

collaborating laboratories.

Special acknowledgments for their special collaboration with the DNA

WG activity go to:

Ana-Laura Vicario (Argentina)

Cheryl Dollard (Canada)

Daniel Perry (Canada)

Berta Killerman (Germany)

We would like to also acknowledge the many private and public

institutions kindly collaborated with these tests and also to ISTA who

gave technical and administrative support.

Acknowledgements…..

Thank you for your attention

www.seedtest.org