Ryegrass Fluorescence Testing Why differentiate? Annual ryegrass, - - PowerPoint PPT Presentation

Ryegrass Fluorescence Testing

Traditional and Genetic Methods

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

Traditional Method Seedling Root Fluorescence

 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)

How to:

Tilt boxes in chamber –orientation of roots

Dark box with UV light

Fluorescence of roots under UV light-in dark box

Fluorescence of roots under UV light.

Evalution

Customer ID: Warm Planted: 5/28/10 Office: Initial Read: 6/9/10 Final Read : 6/16/10 Date Received: Analyst: Analyst: Sample No. Variety Lot Seed Size Componen t Rep 1 Rep 2 Rep 3 Rep 4 Rep 1 Rep 2 Rep 3 Rep 4 Total (for Purity) HALO Normal fl 1 2 1 1 3 4 12 Comments : Normal non-fl 86 87 85 89 Abnormal 2 3 5 6 Dead 10 5 6 4 Firm ungerm Rep total - all Normals 88 92 89 90 359

• Ave. Normal

89.75 (Test fluorescence) TFL=3.34% total normal fl/total of all normals *100 (Varietal fluorescence) VFL=2.87%

Data Sheet Example:

AOSCA value

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 100%



Assume VFlP to be 0%

 SRF =Seedling Root fluorescence, the method

%VFL (annual) - % TFL % Perennial Ryegrass= %VFL (annual) - % VFL (perennial) x % pure ryegrass

The Equation

So, In our example: %PRG= 100-3.34 x 99.5 100-2.87

% PRG= 96.66 x 99.5 = 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%

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 5% annual ryegrass, in AOSA=Mixture



• L. perenne and L. multiflorum are both reported as pure seed kinds

Perennial Ryegrass Referee 2009

Seedling Root Fluorescence Test

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

Referee Setup

 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)

Current provisions-Cultivar Purity Handbook (Version 2008)

 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.

Survey Results

 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

Survey Results Continued

• 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

Survey Results Continued

 Light intensity

• 67% use 700-1250 lux.
• others = 30-40wattsLux not measured

 Length of Test

• 73% do 1st 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

Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 1 8 82.25 1.82 86.5 0.29 2 perennial 0 annual Estimated ARG = 0.95% 1 10 92.75 0.81 89.5 0.28 1 6 81.5 2.15 88.5 1.41 1 9 90.5 0.27 1 2 96 1.56 89.75 1 5 92.75 1.08 91.25 1.37 1 12 92.75 0.27 93.75 1.33 1 3 87 0.57 94.5 0.53 1 4 84.5 0.89 88.75 2.25 Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 2 8 94 74.47 96 82.55 210 perennial 16 annual Estimated ARG=19.41% 2 10 96.75 97.41 98.75 81.01 2 6 94.75 59.1 94.5 56.88 2 9 94.75 59.1 2 2 93.75 38.4 95.25 35.43 2 5 91.25 60 89.5 61.73 2 12 96.25 84.42 95.75 66.58 2 3 90 56.94 95 60 2 4 94.5 60.05 94.75 59.1

Results: Samples 1 and 2

Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 3 8 97 98.71 97.5 96.15 (Growout of NFL) 3 perennial 1 annual Estimated ARG=61.68% 3 10 97 99.22 98.25 100 3 6 96 99.74 98 98.47 3 9 98.5 100 3 2 98.25 96.69 94.25 92.57 3 5 98 96.92 97 97.43 3 12 97.25 99.23 98.75 99.24 3 3 96 99.74 95.5 98.95 3 4 98.25 100 97.75 99.49 Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 4 8 84.75 0.59 85.75 2 perennial 0 annual Estimated ARG=0.14% 4 10 87.5 0.29 93.5 0.27 4 6 88 0.57 90.5 0.55 4 9 85.5 4 2 96 0.26 93.75 0.27 4 5 91 0.27 87.5 0.29 4 12 93.75 0.8 91.25 0.27 4 3 91.5 93 0.54 4 4 89.5 0.56 88.75 0.85

Results: Samples 3 and 4

Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 5 8 94 2.93 96.75 3.1 13 perennial annual Estimated ARG=1.78% 5 10 95.75 4.18 96 2.08 5 6 94.75 2.9 93.5 5.08 5 9 94 4.78 5 2 96.75 1.55 96 2.34 5 5 93 4.03 94.25 2.65 5 12 96.5 3.89 94.25 2.39 5 3 94 1.86 95.25 3.41 5 4 94.75 2.9 96.5 4.15 Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 6 8 90.5 0.28 90 0.56 2 perennial 0 annual Estimated ARG=0.18% 6 10 91.75 0.27 94.75 6 6 92.25 0.54 89.75 0.56 6 9 90.25 0.27 6 2 93.5 0.53 91.25 6 5 93 0.27 93 0.27 6 12 94.5 0.26 91.5 0.55 6 3 88.5 90.75 0.55 6 4 92.75 0.81 91.75 0.27 Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PC Growout from SRF (No PC) DNA on 3,000 seeds 7 8 94.25 0.53 90 0.28 No growout Estimated ARG=0.13% 7 10 92.75 97 7 6 90.25 91.5 0.27 7 9 91 0.27 7 2 93 91.75 0.27 7 5 89.25 92.25 0.81 7 12 93.25 90.75 0.28 7 3 89.25 89.75 7 4 90.25 0.28 92.75

Results: Samples 5, 6 and 7

TFL: Prechill vs No Prechill

Perennials Annuals

1 2 3 4 5 6

TFL-PC TFL - No PC TFL %

20 40 60 80 100 120

TFL-PC TFL - No PC TFL %

Sample Sample Sample Sample Sample Sample Sample

TFL vs VFL (for samples with VFL)

1 2 3 4 5 6

TFL-PC TFL-No PC VFL

Fluorescence %

Variety=Silver Dollar Variety=Prelude

1.72% 0.04%

Germination Across Laboratories

No Prechill Prechill

10 20 30 40 50 60

75 80 85 90 95 100

Sample

10 20 30 40 50 60 70

80 82 84 86 88 90 92 94 96 98 100

Sample

Germination Results-Ryegrass Referee 2009

No Prechill Prechill Sample Average Range Tolerance Within? Average Range Tolerance Within? 1 88.75 14.5 6 No 91 8.5 5 No 2 93.5 7 4 No 94.25 9.5 4 No 3 97.5 3 3 Yes 96.5 4.5 3 No 4 90.5 11 5 No 90 8 6 No 5 95 4 4 Yes 95 4 4 Yes 6 91.5 6 5 No 92.25 5.5 5 No 7 91.75 5.5 5 No 93.25 7.5 5 No

Germination Results Between Laboratories- How do we compare?

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.

Ryegrass Testing-

Genetic Testing Methods

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

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

2 New Available Genetic Methods

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

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

• ffering the test until there is more demand in the

marketplace.

Single sample (pooled) applications Bonafide BDI

 Perenni

al

 Annu

al

 N

T C

TaqMan based Technology

Validation of Individual plants with Bonafide method

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

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

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 (≈$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

Thank you! Questions?

Click icon to add picture