Traditional and Genetic Methods. Ryegrass Fluorescence Testing

Transcription

1 Traditional and Genetic Methods Ryegrass Fluorescence Testing

2 Why differentiate? Annual ryegrass, Lolium multiflorum Forage crop Rapid growing ability. Flowering independent of photoperiod and vernalization. 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 Traditional Method Seedling Root Fluorescence Distinguish Lolium multiflorum and Lolium perenne Idea: Annual ryegrass roots fluoresce under UV light. (19 s) Needs=filter paper+uv light Seedlings with fluorescent roots recorded and removed at first count (7d) and final count (1d)

4 How to:

5 Tilt boxes in chamber orientation of roots

6 Dark box with UV light

7 Fluorescence of roots under UV light-in dark box

8 Fluorescence of roots under UV light.

9 Evalution

10 Data Sheet Example: Customer ID: Warm Planted: Office: Initial Read: /9/1 Final Read : Date Received: Analyst: Analyst: Sample No. Variety Lot HALO Comments : /8/1 Seed Componen Rep 1 Size t Normal fl 1 /1/1 Rep Rep Rep Rep 1 Rep 1 1 Normal non-fl Abnormal Dead Firm ungerm Rep total - all Normals Ave. Normal Rep Rep (Test fluorescence) AOSCA value Total (for Purity) 1 TFL=.% total normal fl/total of all normals *1 (Varietal fluorescence) VFL=.87%

11 Key: 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) VFlA = Variety fluorescence for annual ryegrass VFlP = 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 VFlA to be 1% Assume VFlP to be % SRF =Seedling Root fluorescence, the method

12 The Equation %VFL (annual) - % TFL % Perennial Ryegrass= %VFL (annual) - % VFL (perennial) x % pure ryegrass So, In our example: %PRG= 1-. x 99. (From purity) let s say it s 99.% with.% inert matter Complete the equation: % PRG= 9. x 99. = 99.% 97.1 Report of Analysis Pure Seed Lolium perenne 99.% Inert matter.% Other crop Lolium multiflorum.8% Weed Seed.%

13 What this means: 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 % annual ryegrass, in AOSA=Mixture L. perenne and L. multiflorum are both reported as pure seed kinds

14 Seedling Root Fluorescence Test Perennial Ryegrass Referee 9

15 Motivation 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 Referee Setup Capture Environmental Differences Production Environment Lab Environment-Variation of up to % 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 8)

17 Current provisions-cultivar Purity Handbook (Version 8) 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 Survey Results 8% of the participants=experienced/very experienced. % test 1, samples/year or more 8% using most current version 8-9 of CPH; % had older versions

19 Survey Results Continued PreChill 8% labs do prechill (majority being when it s fresh) 77% use 1C and % use C Media 7% use filter paper 9% use blotters %=combination 8% tilt boxes 71% use KNO; 7% water; % distilled HO Light 8% = 8 hours light 1% = 1 hours light % = 1 hours light

20 Survey Results Continued Light intensity 7% use 7-1 lux. others = -watts Lux not measured Length of Test 7% do 1st read at 7 days: % at 1 days; 7% don t do first count 1% do final read at 1 days Fluorescence 1% remove all seedlings at final count;9% do not % look underneath root for path of fluorescence* 9% do not discriminate based on intensity

21 Results: Samples 1 and Sample Sample Lab Lab Germ (PC) Germ (PC) TFL-PC TFL-PC Germ (no PC) TFL - No PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on, seeds perennial annual Growout from SRF (No PC) 1 perennial 1 annual Estimated ARG =.9% DNA on, seeds Estimated ARG=19.1%

22 Results: Samples and Sample Sample Lab Lab Germ (PC) Germ (PC) TFL-PC TFL-PC Germ (no PC) TFL - No PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on, seeds (Growout of NFL) perennial 1 annual Estimated ARG=1.8% Growout from SRF (No PC) DNA on, seeds perennial annual Estimated ARG=.1%

23 Sample Sample Sample Lab Germ (PC) Lab Germ (PC) Lab Germ (PC) Growout Germ (no TFL - No from SRF DNA on, TFL-PC PC) PC (No PC) seeds perennial Estimated annual ARG=1.78% Growout Germ (no TFL - No from SRF DNA on, TFL-PC PC) PC (No PC) seeds perennial 9.7. Estimated annual ARG=.18% Growout Germ (no TFL - No from SRF DNA on, TFL-PC PC) PC (No PC) seeds No Estimated Results: Samples, and 7

24 TFL: Prechill vs No Prechill TFL-PC TFL - No PC Perennials Annuals TFL - No PC TFL % TFL % 1 Sample Sample Sample Sample Sample TFL-PC Sample Sample

25 TFL vs VFL (for samples with VFL) TFL-PC TFL-No PC VFL Fluorescence % 1.7% 1.% Variety=Silver Dollar Variety=Prelude

26 Germination Across Laboratories No Prechill Prechill Sample 8 1 Sample 7

27 Germination Results Between LaboratoriesHow do we compare? Germination Results-Ryegrass Referee 9 No Prechill Sample Average Range Prechill Tolerance Within? Average Range Tolerance Within? No No No No Yes 9.. No No 9 8 No 9 Yes 9 Yes 91. No 9.. No No No

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. Move on-dna? One year post institution of SRF learned that fluorescence not tightly linked to annuality.

29 Genetic Testing Methods Ryegrass Testing-

30 The Challenge 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 Why DNA? 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 New Available Genetic Methods By Purity By Impurity 1. Allelic Discrimination Reed Barker Test seedlings from SRF test by DNA method to confirm whether or not they are ontypes. Individualized method May have value when there is a need for characterizing individual seedlings to type Bonafide BDI Pure PRG BioDiagnostics, Inc. Test, 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 Bonafide BDI-Pure PRG 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 Single sample (pooled) applications Bonafide BDI Perenni al TaqMan based Technology N T C Annu al

35 Validation of Individual plants with Bonafide method Annual and Intermediate varieties Internal control Perennial varieties Testing individual seedlings was 1% Accurate The marker distinguished annuals/intermediates from perennials.

36 How? DNA for Producing Pure PRG 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 Conclusions 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 ( $7/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

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