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.
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