1. Traditional and Genetic Methods

2. Annual ryegrass, Lolium multiflorum Forage crop Rapid growing ability. Flowering independent of photoperiod and vernalization. Why differentiate? Perennial ryegrass, Lolium perenne Preferred for permanent lawns, Over-winters -does not require seeding each year. About twice the value of annual ryegrass Different Uses and Values

3.  Distinguish Lolium multiflorum and Lolium perenne  Idea: Annual ryegrass roots fluoresce under UV light. (1930’s)  Needs=filter paper+UV light  Seedlings with fluorescent roots recorded and removed at first count (7d) and final count (14d)

10. Customer ID: Warm Planted: 5/28/10 Office:Initial Read:6/9/10 Final Read : 6/16/10 Date Received:Analyst: Sample No. VarietyLot Seed Size Componen t Rep 1Rep 2Rep 3Rep 4Rep 1Rep 2Rep 3Rep 4 Total (for Purity) HALO Normal fl1201134012 Comments : Normal non-fl 86878589 Abnormal 2356 Dead 10564 Firm ungerm 0000 Rep total - all Normals88928990359 Ave. Normal89.75 (Test fluorescence)TFL=3.34% total normal fl/total of all normals *100 (Varietal fluorescence)VFL=2.87% Data Sheet Example: AOSCA value

11.  Report % PRG and % ARG on Purity Report  Pure seed and crop percentages may adjust based on the germination fluorescence test.  ARG = Annual ryegrass (Lolium multiflorum)  PRG = Perennial ryegrass (Lolium perenne)  TFl = Test fluorescence (lab determined)  VFl = Variety fluorescence (AOSCA value)  VFl A = Variety fluorescence for annual ryegrass  VFl P = Variety fluorescence for perennial ryegrass  If you are not given the variety name, the variety is not listed by AOSCA, or you are not testing that species, then:  Assume VFl A to be 100%  Assume VFl P to be 0%  SRF =Seedling Root fluorescence, the method

12. %VFL (annual) - % TFL % Perennial Ryegrass=%VFL (annual) - % VFL (perennial)x % pure ryegrass The Equation So, In our example: %PRG=100-3.34x99.5 100-2.87 % PRG= 96.66x99.5= 99.02% 97.13 (From purity) let’s say it’s 99.5% with 0.50% inert matter Complete the equation: Report of Analysis Pure Seed Lolium perenne 99.02% Inert matter 0.50% Other crop Lolium multiflorum 0.48% Weed Seed 0.00% Report of Analysis Pure Seed Lolium perenne 99.02% Inert matter 0.50% Other crop Lolium multiflorum 0.48% Weed Seed 0.00%

13.  If No VFL has been described and accepted by AOSCA, all fluorescent normals are considered annual contamination and go against pure perennial ryegrass %.  If TFL is less than VFL, report no annual in a perennial lot.  If over 5% annual ryegrass, in AOSA=Mixture  L. perenne and L. multiflorum are both reported as pure seed kinds

14. Seedling Root Fluorescence Test

15.  Determine the uniformity of test results from lab to lab  Review method as described in Cultivar Purity Handbook  Goal:  To help clarify the method and foster uniformity ▪ Lifting vs non-lifting of roots ▪ Intensity of Fluorescence

16.  Capture Environmental Differences  Production Environment  Lab Environment-Variation of up to 6% in TFL over a period of less than one year ( Sharon Davidson )  Referee Study:  Seven samples-varying in annuality and production area.  Prechill vs. No Prechill  Completed within one month  Cultivar Purity Handbook (version 2008)

17.  All fluorescent root traces should be counted regardless of the intensity of fluorescence.  Non-fluorescent seedlings should not be lifted to observe fluorescence.  Fluorescence for abnormal seedlings should not be recorded.

18.  80% of the participants=experienced/very experienced.  42% test 1,000 samples/year or more  58% using most current version 2008-09 of CPH; 42% had older versions

19.  PreChill ▪ 80% labs do prechill (majority being when it’s fresh) ▪ 77% use 10C and 23% use 5C  Media ▪ 47% use filter paper ▪ 29% use blotters ▪ 24%=combination ▪ 80% tilt boxes ▪ 71% use KNO3; 27% water; 2% distilled H2O  Light ▪ 80% = 8 hours light ▪ 13% = 16 hours light ▪ 6% = 12 hours light

20.  Light intensity  67% use 700-1250 lux.  others = 30-40watts  Lux not measured  Length of Test  73% do 1 st read at 7 days: 20% at 10 days; 7% don’t do first count  100% do final read at 14 days  Fluorescence  31% remove all seedlings at final count;69% do not  40% look underneath root for path of fluorescence*  94% do not discriminate based on intensity

21. SampleLabGerm (PC)TFL-PCGerm (no PC)TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 1882.251.8286.50.29 2 perennial 0 annual Estimated ARG = 0.95% 11092.750.8189.50.28 1681.52.1588.51.41 1990.50.27 12961.5689.750 1592.751.0891.251.37 11292.750.2793.751.33 13870.5794.50.53 1484.50.8988.752.25 SampleLabGerm (PC)TFL-PCGerm (no PC)TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 289474.479682.55 210 perennial 16 annual Estimated ARG=19.41% 21096.7597.4198.7581.01 2694.7559.194.556.88 2994.7559.1 2293.7538.495.2535.43 2591.256089.561.73 21296.2584.4295.7566.58 239056.949560 2494.560.0594.7559.1

22. SampleLabGerm (PC)TFL-PC Germ (no PC)TFL - No PC Growout from SRF (No PC)DNA on 3,000 seeds 389798.7197.596.15 (Growout of NFL) 3 perennial 1 annual Estimated ARG=61.68% 3109799.2298.25100 369699.749898.47 3998.5100 3298.2596.6994.2592.57 359896.929797.43 31297.2599.2398.7599.24 339699.7495.598.95 3498.2510097.7599.49 SampleLabGerm (PC)TFL-PC Germ (no PC)TFL - No PC Growout from SRF (No PC)DNA on 3,000 seeds 4884.750.5985.750 2 perennial 0 annualEstimated ARG=0.14% 41087.50.2993.50.27 46880.5790.50.55 4985.50 42960.2693.750.27 45910.2787.50.29 41293.750.891.250.27 4391.50930.54 4489.50.5688.750.85

23. SampleLab Germ (PC)TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 58942.9396.753.1 13 perennial 0 annual Estimated ARG=1.78% 51095.754.18962.08 5694.752.993.55.08 59944.78 5296.751.55962.34 55934.0394.252.65 51296.53.8994.252.39 53941.8695.253.41 5494.752.996.54.15 SampleLab Germ (PC)TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 6890.50.28900.56 2 perennial 0 annual Estimated ARG=0.18% 61091.750.2794.750 6692.250.5489.750.56 6990.250.27 6293.50.5391.250 65930.27930.27 61294.50.2691.50.55 6388.5090.750.55 6492.750.8191.750.27 SampleLab Germ (PC)TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 7894.250.53900.28 No growout Estimated ARG=0.13% 71092.750970 7690.25091.50.27 79910.27 7293091.750.27 7589.25092.250.81 71293.25090.750.28 7389.25089.750 7490.250.2892.750 Results: Samples 5, 6 and 7

24. PERENNIALSANNUALS Sample

26. NO PRECHILLPRECHILL

27. Germination Results-Ryegrass Referee 2009 No Prechill Prechill SampleAverageRangeToleranceWithin?AverageRangeToleranceWithin? 188.7514.56No918.55No 293.574No94.259.54No 397.533Yes96.54.53No 490.5115No9086No 59544Yes9544Yes 691.565No92.255.55No 791.755.55No93.257.55No Germination Results Between Laboratories- How do we compare?

28. Referee Conclusions  All treatments and interactions among them affected the results of both germination and fluorescence (except prechill vs no prechill)  Goal was to bring about uniformity in the existing test, but  1. Still room for improvement ▪ Education? ▪ Inherent variability each time you test a lot  2. Move on-DNA? One year post institution of SRF  learned that fluorescence not tightly linked to annuality.

29. Genetic Testing Methods

30.  Inadvertent mixing of annual in perennial ryegrass lots, resulting in huge economic losses.  Until now: Lack of accurate quality assurance tools to estimate annual contamination in perennial seed lots.  Good lots of perennial ryegrass rejected each year  Contaminated seed wrongly diagnosed as pure seed

31. LIMITATIONS OF CURRENT METHODS  Both SRF and GOT are:  Time consuming  Labor intensive  Environmentally influenced  SRF is inaccurate :  High false positive error rate  Overestimates annual ryegrass contamination MOLECULAR ADVANTAGES Tightly linked to the traits of interest. Independent of stage of development Reliable and not influenced by external environment Cost effective and less time consuming

32. BY PURITY  1. Allelic Discrimination  Reed Barker  Test seedlings from SRF test by DNA method to confirm whether or not they are on- types.  Individualized method  May have value when there is a need for characterizing individual seedlings to type BY IMPURITY  Bonafide BDI Pure PRG  BioDiagnostics, Inc.  Test 3,000 seeds to find exact level of contamination of annual in perennial ryegrass  Pooled seed method  No Bias from Fluorescence test Both Developed using different genes for flowering

33.  Currently, this test is offered by BioDiagnostics, Inc.  Goal- to license method to other labs.  To ensure accuracy and uniformity in testing, Oregon State Seed Lab will partner BDI to validate all labs providing this service in the future.  Start up costs will limit the number of labs offering the test until there is more demand in the marketplace.

34. Perennial Annual NTC TaqMan based Technology

35. Annual and Intermediate varieties Internal control Perennial varieties Testing individual seedlings was 100% Accurate The marker distinguished annuals/intermediates from perennials.

36.  What is the benefit to producers?  Rapid, accurate assessment of quality  (as opposed to an inaccurate, variable SRF test)  Use this test to produce a premium product  Start with a clean seed source using DNA test  Take advantage cold winters that eliminate annual contamination  Replace a commodity with a premium product  Demand a premium price  Enhance profitability by creating a niche in the marketplace

37.  The SRF test originally designed to detect annual ryegrass contamination is no longer a reliable indicator of annual types.  Bonafide BDI™ – Pure PRG™ is highly sensitive, rapid, accurate and cost effective (≈$70/sample) procedure for detecting annual ryegrass contamination in perennial ryegrass.  The ability of this test to detect contamination in both pooled and individual seedlings makes it an attractive tool for both ryegrass growers and ryegrass breeders.  Meeting legal requirements keeps fluorescence test in use.  In the works  Getting DNA methods into Cultivar Purity Handbook for use on tags and reports-gathering and using collection of valuable information

38. Thank you! Questions?