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SEED TESTING INTERNATIONAL   www.seedtest.org
RULES DEVELOPMENT •
Validation of a New Method for a ‘Microsatellite 
Marker Analysis for Barley Variety Verification’
Verena Peterseil1, Doris Kaiser1, Marie-Claude Gagnon2, Ana Laura Vicario3 and Sean Walkowiak4
1Austrian Agency for Health and Food Safety, Vienna, Austria
2Canadian Food Inspection Agency, Ottawa, Canada; ISTA Variety Committee Chair
3Instituto Nacional de Semillas INASE, Buenos Aires, Argentina; ISTA Variety Committee Vice-Chair
4Canadian Grain Commission, Winnipeg, Canada; ISTA Variety Committee Vice-Chair
ISTA’S STANDARDISED PROCEDURES 
FOR THE DETERMINATION OF VARIETY 
VERIFICATION have traditionally been based 
on the examination of seeds, seedlings or plants 
in a laboratory, glasshouse, growth chamber or 
field plot, to assess morphology (grow-out tests), 
specific substances (biochemical methods) or 
protein characteristics (protein-based methods). 
DNA-based approaches are very useful tools 
for variety verification and for assessment of 
purity. In comparison to traditional variety 
verification methods, DNA-based techniques 
may reveal more polymorphism, thus allowing 
greater resolution among varieties. DNA-
based techniques are also independent of 
environmental conditions or developmental 
stages.
To initiate the process for the inclusion of a new 
DNA-based method for barley variety verification 
by means of microsatellite markers into the 
International Rules for Seed Testing (ISTA Rules), 
comparative test (CT) leader Verena Peterseil 
organised two CTs with the participation of 
laboratories from several countries around the 
world, over a period of three years. The objectives 
of the CTs carried out are summarised below:
	
• The aim of CT1 was to compare results 
among participant laboratories and evaluate 
the possibility of obtaining the same simple 
sequence repeat (SSR) profiles and same allele 
sizes using different reagents, equipment and 
working protocols. Varieties and SSR markers 
were the same for all participant laboratories. The 
expected result of CT1 was to obtain comparable 
results among laboratories.
	
• The aim of CT2 was also to compare results 
among participant laboratories and evaluate the 
possibility of obtaining the same SSR profile and 
same allele sizes when using different reagents, 
equipment and working protocols. Varieties and 
SSR markers were the same for all participant 
laboratories.
	
• During CT2, the applicability of the method 
was evaluated by extending the range of 
varieties tested compared to CT1. In addition, 
reproducibility of the markers was tested through 
comparing the results of different laboratories 
with those of CT1. Repeatability was tested by 
using variety Semper which was also used in 
the first CT. Two out of a total of 12 laboratories 
participated in both CTs.
Materials and Methods
Samples 
For CT1, eight varieties (authorised in Austria) 
were analysed using ten SSR markers obtained 
from Perry et al. (2013) and four SSR markers 
developed by the Austrian Agency for Health and 
Food Safety (AGES; personal communication, 
Doris Kaiser and Verena Peterseil). Each 
participant received six individual crushed seeds 
per variety and two subsamples of a pool of 30–40 
seeds. Samples were provided in sealed tubes 
labelled with the name or code of the variety.
For CT2, nine varieties were analysed (four from 
Iran, two from Estonia, two from Lithuania 
and one from Austria) using the same 14 SSR 

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