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 MethodSeedling 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: 6/9/10Final Read :

6/16/10

Date Received: Analyst: Analyst:

SampleNo.

Variety LotSeedSize

Component

Rep 1 Rep 2 Rep 3 Rep 4 Rep 1 Rep 2 Rep 3 Rep 4Total (for Purity)

  HALO     Normal fl 1 2 0 1 1 3 4 0 12

Comments:

     Normal non-fl         86 87 85 89  

    Abnormal         2 3 5 6  

  Dead         10 5 6 4  

       Firm

ungerm         0 0 0 0  

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 AnalysisPure 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 = 30-40wattsLux 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 PCGrowout from

SRF (No PC)DNA on 3,000

seeds1 8 82.25 1.82 86.5 0.29

2 perennial

0 annualEstimated ARG =

0.95%

1 10 92.75 0.81 89.5 0.281 6 81.5 2.15 88.5 1.411 9 90.5 0.27 1 2 96 1.56 89.75 01 5 92.75 1.08 91.25 1.371 12 92.75 0.27 93.75 1.331 3 87 0.57 94.5 0.531 4 84.5 0.89 88.75 2.25

Sample Lab Germ (PC) TFL-PC Germ (no PC) TFL - No PCGrowout from

SRF (No PC)DNA on 3,000

seeds2 8 94 74.47 96 82.55

210 perennial

16 annualEstimated

ARG=19.41%

2 10 96.75 97.41 98.75 81.012 6 94.75 59.1 94.5 56.882 9 94.75 59.1 2 2 93.75 38.4 95.25 35.432 5 91.25 60 89.5 61.732 12 96.25 84.42 95.75 66.582 3 90 56.94 95 602 4 94.5 60.05 94.75 59.1

Results: Samples 1 and 2

Sample Lab Germ (PC) TFL-PCGerm (no

PC) TFL - No PCGrowout from

SRF (No PC) DNA on 3,000 seeds3 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 1003 6 96 99.74 98 98.473 9 98.5 100 3 2 98.25 96.69 94.25 92.573 5 98 96.92 97 97.433 12 97.25 99.23 98.75 99.243 3 96 99.74 95.5 98.953 4 98.25 100 97.75 99.49

Sample Lab Germ (PC) TFL-PCGerm (no

PC) TFL - No PCGrowout from

SRF (No PC) DNA on 3,000 seeds4 8 84.75 0.59 85.75 0

2 perennial 0 annual Estimated ARG=0.14%

4 10 87.5 0.29 93.5 0.274 6 88 0.57 90.5 0.554 9 85.5 0 4 2 96 0.26 93.75 0.274 5 91 0.27 87.5 0.294 12 93.75 0.8 91.25 0.274 3 91.5 0 93 0.544 4 89.5 0.56 88.75 0.85

Results: Samples 3 and 4

Sample LabGerm (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

0 annual

Estimated ARG=1.78%

5 10 95.75 4.18 96 2.085 6 94.75 2.9 93.5 5.085 9 94 4.78 5 2 96.75 1.55 96 2.345 5 93 4.03 94.25 2.655 12 96.5 3.89 94.25 2.395 3 94 1.86 95.25 3.415 4 94.75 2.9 96.5 4.15

Sample LabGerm (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 06 6 92.25 0.54 89.75 0.566 9 90.25 0.27 6 2 93.5 0.53 91.25 06 5 93 0.27 93 0.276 12 94.5 0.26 91.5 0.556 3 88.5 0 90.75 0.556 4 92.75 0.81 91.75 0.27

Sample LabGerm (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 0 97 07 6 90.25 0 91.5 0.277 9 91 0.27 7 2 93 0 91.75 0.277 5 89.25 0 92.25 0.817 12 93.25 0 90.75 0.287 3 89.25 0 89.75 07 4 90.25 0.28 92.75 0

Results: Samples 5, 6 and 7

TFL: Prechill vs No Prechill

PERENNIALS ANNUALS

1 5 9 13 17 21 25 29 33 370

1

2

3

4

5

6

TFL-PCTFL - No PC

TFL %

1 3 5 7 9 11 13 150

20

40

60

80

100

120

TFL-PC

TFL - No PC

TFL %

SampleSample Sample Sample Sample Sample Sample

TFL vs VFL (for samples with VFL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 240

1

2

3

4

5

6

TFL-PCTFL-No PCVFL

Flu

ore

scen

ce %

Variety=Silver Dollar

Variety=Prelude

1.72%

0.04%

Germination Across Laboratories

NO PRECHILL PRECHILL

0 1 2 3 4 5 6 7 875

80

85

90

95

100

Sample0 1 2 3 4 5 6 7 8

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 offering the test until there is more demand in the marketplace.

Single sample (pooled) applications Bonafide BDI

Perennial

Annual N

TC

TaqMan based Technology

Validation of Individual plants with Bonafide method

Annual and Intermediate varieties

Internal control

Perennial varieties

Testing individual seedlings was 100% AccurateThe 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 ryegrasscontamination 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 annualryegrass contamination in perennial ryegrass.

The ability of this test to detect contamination in both pooled

and individual seedlings makes it an attractive tool for bothryegrass growers and ryegrass breeders.

Meeting legal requirements keeps fluorescence test in use.

In the worksGetting DNA methods into Cultivar Purity Handbook for use on tags and reports-gathering and using collection of valuable information

Thank you!

Questions?