SNPs Assays:
Discriminating Between
Annual and
Perennial Ryegrass Cultivars
Travis Behrend
Dr. Laurel Cooper
Dr. Reed Barker
USDA-ARS Grass Genetics
3450 SW Campus Way
Corvallis, OR 97330
HHMI 2007
Background: The Market Demand
We Want the Pretty Perennial
in Our Lawn!!
Perennial
-Greener
-Softer
-Finer
-Used for Turf
The Problem ?
As a result of both accidental and deliberate
intercrossing, many L. perenne (perennial)
cultivars contain a certain percentage of alleles
from L. multiflorum (annual).
Intercrossing
Perennial
Annual
Background: Farmer Economics
-Plants with the appearance of annual ryegrass are unacceptable in high-quality
turf and seed producers are penalized for annual ryegrass contamination
Farmer Joe
100% Perennial
0% Contamination
$50 per bushel
Farmer Bob
90% Perennial
10% Contamination
$45 per bushel
Farmer Bill
80% Perennial
20% Contamination
$40 per bushel
Seed Growers lose $5-7 million annually!
Background: Seed Testing
Currently, the Fluorescence test is used to determine seed purity
Problem
Fluorescence test is less effective as a result of the two species
intermingling and tends to overestimate the annual types.
i.e It docks the farmer’s pay more than necessary
=
Grrrr
!
Solution
Use genetic markers (SNP) to discriminate between annual and perennial
ryegrass
The gene LpID1 is involved in the transition from flowering to vegetative
growth, a distinguishing characteristic between annuals and perennials
SNP = Single Nucleotide Polymorphism
SNP = DNA sequence variations that occur when a single nucleotide (A, T, G, or C) in
the genome sequence is altered1
RE Site
AGGCTAGAATTCCATTCGAGTC
AGGCTAGAATTGCATTCGAGTC
In our assay, the SNP is used as a diagnostic tool.
1
http://www.everythingbio.com/glos/definition.php?word=single+nucleotide+polymorphism+(SNP)
Project Purpose: SNP Detection
Objective: Compare and contrast the speed, reliability, and costs of three
different methods of SNP detection
SNP Detection
Methods
Cleaved Amplified
Polymorphic Sequence
(CAPS)
Taqman® Allelic
Discrimination
Assay
(Real-Time PCR)
AcycloPrime®-FP
SNP Detection Assay
(VICTOR®)
Predictions
1. The CAPS assay will be the cheapest.
2. The Taqman Allelic Discrimination Assay will be the fastest.
Note: 1. Results may vary depending on the
amount of throughput.
2. Results may vary depending upon the
unknowns of the design process.
Background: LpID1 Gene
0.85 Kb
Exon 1
0.45 Kb
Intron
SNP
Exon 2
1.3 Kb
EcoR1 Site
Annual Consensus Sequence:
CGTGAGCGA……………………………………………GA[A]TTC…………………………CCTTCTGTGTG
Perennial Consensus Sequence:
CGTGAGCGA……………………………………………GA[T]YTC…………………………CCTTCTGTGTG
Ambiguous Pyrimidine (C or T)
Notes:
-The consensus sequences shown both run 5’ to 3’ (left to right)…they are not complements!
-Indeterminate Gene, LpID1, is involved in the transition from flowering to vegetative growth
-There are more differences (SNPs, deletions, etc…) than shown in the figure; this is a simplification.
Methods: The CAPS Assay
PCR Product
EcoR1 Site
Perennial Consensus Sequence:
CGTGAGCGA……………………………………………GA[T]YTC…………………………CCTTCTGTGTG
Amplification of Genes
of interest by
Polymerase Chain
Reaction (PCR)
DNA
Extraction
1. Add EcoR1
2. Gel Electrophoresis
1.5 kb1.0 kb0.8 kb-
Perennial
Allele
Annual or Hybrid Allele
CGTGAGCGA……………………………………………GA[A]TTC…………………………CCTTCTGTGTG
DNA Marker
Annual Consensus Sequence:
Methods: AcycloPrime-FP SNP Assay
A
Primer
Acyclonucleotides*
T
[A/T]
* Acyclonucleotides require special polymerase
Template
(PCR Product)
Ideal Results
AT
A – FP
AA
FP = Fluorescence Polarization
TT
(-) controls
T - FP
Results: AcycloPrime-FP Assay
Difficulties
Control Reactions
Assign Clusters
Plate of 96 Reactions
150
140
Assign Clusters
Homozygous Perennial
150
130
120
110
Heterozygotes
TAMRA (mP)
100
Homozygous Annual
TAMRA (mP)
100
90
80
70
60
50
40
50
30
Non-template
Controls
20
10
0
0
40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80
0
50
100
150
R110 (mP)
R (Intensity Ratio)
Problem: 1. Heterozygotes are between the homozygotes
2. Too much variability
Troubleshooting: 1. Varied number of cycles
2. Varied concentrations and amounts of reagents
3. Florescent plate reader recalibration
Methods: Taqman® AD-Assay
Reference: http://www.appliedbiosystems.com/
Results: Taqman® AD-Assay
Perennial Allele
(96 well plate rxn)
Annual Allele
Reliability: CAPS vs. Taqman®
SNP Detection Reliability
100
90
80
70
60
CAPS Assay Homozygous Annual
Percent
50
CAPS Assay Heterozygous
CAPS Assay Homozygous Perennial
40
Taqman AD Assay Homozygous Annual
Taqman AD Assay Heterozygous
30
Taqman AD Assay Homozygous Perennial
20
10
0
Perennial-like
Intermediate
Annual-like
Morphologies
Gulf
Speed of Detection Methods
(based on 96 well plate)
Taqman
Setup
Run
Total
0.5
1.5
1.0
1.5
1.0
0.5
7.0 hrs
Time Calculations of Detection Methodologies
8
7
6
5
0.75
2.0
2.75 hrs
Acyclo-FP
Setup
PCR
Clean-Up Rxn
Acyclo Rxn
Total
Time (hrs)
CAPS
Setup
PCR
Gel
Digest
Gel
Scoring
Total
4
3
2
0.75
1.5
0.75
1.0
4.0 hrs
1
0
CAPS
Taqman
Detection Methodologies
Acyclo-FP
Cost of Detection Methods
Cost of Detection Methodologies
CAPS $0.50/rxn
Taq Pol.
EcoR1
Agrose
Plastics
(dollars)
2
Cost
Taqman $0.63/rxn
Probe
Optical plate
Optical film
Plastics
2.5
1.5
1
0.5
Acyclo-FP $2.00/rxn
Kit (all inclusive)
Plastics
0
CAPS
Taqman
Detection Methodologies
Acyclo-FP
Conclusions
• CAPS assay is cheapest, but takes a long time
(PCR, restriction digestion, gel electrophoresis),
good reliability
• AcycloPrime®-FP was expensive, needed a lot
of troubleshooting, much more difficult to
perform, questionable reliability.
• LpID1 Taqman® assay is more expensive, but
much faster, is easy to perform, good reliability
Acknowledgments
Special THANKS! to the following:
• Dr. Laurel (Lol) Cooper
• Dr. Reed Barker
• Dr. Kevin Ahern
• Dr. Mary Slabaugh
• Lori Evans-Marks
• Howard Hughes Medical Institute