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TRANSCRIPT
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…..