b'ASSOCIATION NEWS Variety CommitteeMarie-Claude Gagnon (Chair) and Ana Laura Vicario (Vice-Chair)www.seedtest.org/en/technical-committees/variety-committee.htmlTHE PURPOSE OF THE ISTA VARIETYand barley. These activities are in accordanceselection of annual, perennial and hybrid seedCOMMITTEE IS TO DEVELOP TESTINGwith ISTAs vision of uniformity in seed qualitysamples, the selected SNPs (single nucleotideMETHODS FOR SPECIES AND VARIETYevaluation worldwide and ISTAs mission topolymorphisms) were tested using digital PCRVERIFICATION, to determine the extent aproduce internationally agreed rules for seed(dPCR). Indeed, previous tests demonstratedsubmitted sample conforms to the species ortesting. that real-time PCR was often, but not always,variety as requested by the applicant, usingable to distinguish ARG contamination in PRG.methods not permissible in a purity testHighlights of the YearThe objective of testing dPCR was to see if betteraccording to Chapter 3 of the International Rulesdetection could be obtained with the use of thisfor Seed Testing (ISTA Rules). Within Chapter 8 of(January 2024 to present) technology when compared to real-time PCR.the ISTA Rules it is possible to find conventionalNew Markers for Perennial Rye GrassInstead of creating a threshold where samplesmethods like morphological traits that can beVariety Detection become detectable (as for real-time PCR), dPCRseen under daylight or ultraviolet (UV) light,The objective of this work is to develop markersconducts thousands of mini-PCR reactions inby naked eye or using magnification, with orfor the detection of annual rye grass (ARG)each well and counts + or , well for well. Thewithout previous treatment; protein-based testswithin perennial rye grass (PRG) varieties. Theanalysis of pure seeds using this technologylike those for obtaining storage protein bandadmixture detection is important to preventgives fluorescence patterns as displayed in Fig. 1. patterns that allow variety verification; and DNA- recurrent sowing of lawns. This collaborativeA pure ARG variety shows a pattern of many based tests like the semi-performance-basedwork began in 2020 and is led by Giovanny LpezFAM amplifications and very few SUN (HEX) methods for obtaining DNA patterns that providefrom the Advanced Technologies Committee,amplifications (Fig. 1a), while a pure PRG variety a tool for assessing laboratory performancewith the collaboration of Shaun Bushman fromshows a pattern of few FAM amplifications and aimed at laboratory accreditation, includingthe United States Department of Agriculturemany SUN (HEX) amplifications (Fig. 1b). When the organisation of Proficiency Tests which are(USDA), who developed the markers and isdifferent percentages of ARG in PRG are mixed mandatory for ISTA accredited laboratories. Therunning the tests; Daniel Curry from Oregonand analysed, the intensity of the fluorescence Committee is currently developing DNA-basedState University, who provided seed sampleschanges from SUN (HEX) to FAM. Different tests for specific traits for variety identification,for the tests and technical support; and Ingocontamination levels of ARG in PRG were tested like the Lolium test to discriminate annualLenk from DLF (Denmark) providing technical(2%, 4%, 6%, 10%, 20% and 50%) and the results rye grass from perennial rye grass varieties,support. After the initial development of are displayed in Fig. 2. The digital PCR results and morphological-based tests using newmarkers, validation using a KASP (kompetitive obtained so far show consistency across the technologies equivalent to the human analystallele specific polymerase chain reaction, three-probe sets tested. The dPCR instrument is for seed analysis, such as the use of neuronalPCR) and quantitative PCR approaches, as more quantitative and sensitive than real-time networks for variety identification in wheatwell as real-time PCR tests using a larger PCR. This instrument will continue to be used SEED TESTING INTERNATIONAL OCTOBER 2025 47'