Bonafide BDI™ – Pure PRG:

A Novel Alternative to Ryegrass

Seedling Root Fluorescence Test

Pegadaraju Venkatramana

Quentin Schultz &

Benjamin (Beni) Kaufman*

Annual ryegrass, Lolium multiflorum

• Forage crop

• Rapid growing ability.

• Flowering independent of

photoperiod and vernalization.

Introduction

Perennial ryegrass, Lolium perenne

• Preferred for permanent lawns,

• Over-winters -does not require

seeding each year.

The ProblemContamination of annual ryegrass in

perennial seed lots:

annual ryegrass is not desirable for permanent turf areas like

golf courses or home lawns.

Annual ryegrass has limited blending ability with other

desirable grass types, and

by definition, annual ryegrass has a limited life span unlike

perennial ryegrass.

The inadvertent mixing, or hybridization of annual in

perennial ryegrass lots results in huge economic losses.

The Challenge

To establish a quality control test

to estimate the presence of annual

ryegrass in perennial seed lots.

Current methods to estimate

annual ryegrass contamination

Biochemical markers

Isozyme Markers:• Phosphoglucose isomerase

• Superoxide dismutase

• Esterase

Annuloline based-Seedling

Root Fluorescence (SRF)

Phenotypic markers

Grow-out test (GOT).

Seedling Root Fluorescence

test

The test based on detecting by UV light the presence of a fluorescent pigment

annulonine, assumed/known to accumulate in the roots of annual ryegrass

seedlings.

A B

Key concerns with SRF test

Under some environmental conditions perennial

ryegrass varieties can express SRF trait

interspecies hybridization between annual and perennial ryegrass

resulted in sporadic introgression of the fluorescent pigment into

perennial ryegrass.

Consequently,

the SRF test exhibits high false positive error rate and

overestimates annual ryegrass contamination thereby is

not an adequate test.

Molecular markers as alternative tool

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

Annual and perennial ryegrass display different responses to

vernalization and photoperiod,

hence,

we focused on identifying functional markers in genes

involved in the vernalization pathway to distinguish annual from

perennial ryegrass

The

Rationale

Behind

The

Test

Characterization of

LpCO & LpVRN2_2 gene

sequences in annual and perennial ryegrass

LpCO and LpVRN2 genes were PCR amplified and sequenced

from 10 perennial & 5 annual ryegrass varieties.

Sequence comparison between annual and perennial LpCO gene

did not produced any potential diagnostic marker

11 single nucleotide polymorphisms (SNPs) and 2 insertion/deletion

(In-Del) sites at LpVRN2_2 were identified. One of the In-Dels

resulted in successful differentiation of all the annual from

perennial varieties!

Technology for Diagnostics

Bonafide BDI™ Pure PRG assay is TaqMan probe-based

detection performed on Real-Time PCR systems

32 perennials,

26 annuals, &

2 intermediates

ryegrass varieties were

tested (in triplicates).

Validation of Bonafide BDI™–

Pure PRG

Annual and Intermediate varieties

Perennial varieties

Internal control

Testing individual seedlings was 100% Accurate

The marker distinguished annuals/intermediates from perennials.

End point assay for single sample

applications

Perennial

Annual

NTC

Linear range of Bonafide BDI

DNA based test.

Regression Analysis (>5 % contamination)

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

0.00 20.00 40.00 60.00 80.00 100.00

Actual % Contamination

Esti

mate

d %

Co

nta

min

ati

on

Regression Analysis (<5% contamination)

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0.00 1.00 2.00 3.00 4.00

Actual % Contamination

Es

tim

ate

d %

Co

nta

min

ati

on

Validation key

Bonafide BDILOD= 1/5000

Key steps in performing

Bonafide BDI™ – Pure PRG

Seed Sample

Count 3000 seeds

(15 mins)

Seed Grind (5mins)

Extract DNA from

Samples (3h)

Conduct TaqMan

Quantitative real-time PCR

assay( 2h)

Report the results

Data analysis

Validation of Pure PRG By

Oregon State University Blind

Test Experiment:

21 blind test samples were created by mixing

various proportions of annual (Gulf) in perennial

(Silver dollar) ryegrass seeds at OSU

Each sample consisted of a pool of 3000 seed

The samples were sent to BDI for testing by way of

BDI Pure-PRG; the results were provided back to

OSU for analysis

OSU contamination Levels

BD

I est

imat

es

OSU pooled seed validation: BDI Pure PRG test results plotted

against the OSU contamination levels.

(R2=0.988)

The lowest contamination level of one

seed in 3000 (0.03%) was consistently

detected by Pure Prg pooled DNA test.

The quantification was most accurate

when the contamination ranged

between 0-15%.

100% DNA Spike Calibrator

15% DNA Spike Calibrator

10% DNA Spike Calibrator

1% DNA Spike Calibrator BDI Predicted Value

0.7 0.63 0.67 0.544 0.51.3 1.155 1.23 1 15.9 5.115 5.43 4.417 5

10.8 9.42 10 8.135 1017.2 15 15.92 12.947 15

23.2 20.265 21.5 17.491 2042.3 36.87 39.13 31.831 4060 52.35 55.55 45.19 50

59.7 52.125 55.31 44.992 6080.1 69.915 74.19 60.35 80100 87.24 92.58 75.309 100

Improving the range of accuracy: Using

multiple calibrators

Comparisons between DNA and SRF testsFeatures DNA Test SRF Test SRF+GOT

Accuracy High Low Low

Environmental

Influence

No Yes Yes

Sample Size 3000 seeds 400 seedlings 400 seedlings

Time Required 2 days 14-21 days 90-120 days

Cost effective Yes Yes No

Distinguish

intermediates from

annual

Yes No No

Seed germination Not required Required Required

Seed grinding Required Not Required Not Required

Summary

Bonafide BDI™ – Pure PRG is sensitive, rapid, accurate and

cost effective procedure for detecting annual ryegrass

contamination in perennial ryegrass.

Bonafide BDI™ – Pure PRG DNA test meets the grass seed

industry need for Low level detection of annual ryegrass

contamination

The dual application of this test in both pooled and individual

seedlings makes it a potential tool for both ryegrass growers

and ryegrass breeders.

BioDiagnostics

A.C. Chandra Shekara

Dr. Michael Thompson

Robert Bialozynski

Mark Blackstad

Dr. Denise Thiede

Victor Piazza

Jennifer Pernsteiner

Diandra Viner

Pennington Seeds

Skip Coville

Trevor Abbott

Oregon State University

Dr. Sabry Elias

Daniel curry

University of Minnesota

Dr. Nancy Elke

Dr. Don Velleckson

Northern Excellence

Brent Benicke

Acknowledgements

The End