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 HHMI 2007

Background: The Market Demand Perennial -Greener -Softer -Finer -Used for Turf We Want the Pretty Perennial in Our Lawn!! 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). Perennial Annual Intercrossing

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 Grrrr ! 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 = Solution Use genetic markers (SNP) to discriminate between annual and perennial ryegrass The gene LpID 1 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 altered 1 A G G C T A G A A T T C C A T T C G A G T C A G G C T A G A A T T G C A T T C G A G T C In our assay, the SNP is used as a diagnostic tool. RE Site 1

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: LpID 1 Gene Exon 1IntronExon Kb SNP 0.85 Kb 0.45 Kb CGTGAGCGA……………………………………………GA[A]TTC…………………………CCTTCTGTGTG CGTGAGCGA……………………………………………GA[T]YTC…………………………CCTTCTGTGTG Annual Consensus Sequence: Perennial Consensus Sequence: EcoR1 Site Notes: -The consensus sequences shown both run 5’ to 3’ (left to right)…they are not complements! -Indeterminate Gene, LpID 1, 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. Ambiguous Pyrimidine (C or T)

Methods: The CAPS Assay DNA Extraction Amplification of Genes of interest by Polymerase Chain Reaction (PCR) 1. Add EcoR1 DNA Marker 1.5 kb- 1.0 kb- 0.8 kb- PerennialAllele Annual or Hybrid Allele 2. Gel Electrophoresis Annual Consensus Sequence: Perennial Consensus Sequence: CGTGAGCGA……………………………………………GA[A]TTC…………………………CCTTCTGTGTG CGTGAGCGA……………………………………………GA[T]YTC…………………………CCTTCTGTGTG EcoR1 Site PCR Product

Methods: AcycloPrime-FP SNP Assay [A/T] Template (PCR Product) Primer A T Acyclonucleotides* * Acyclonucleotides require special polymerase Ideal Results AA TT AT (-) controls A – FP T - FP FP = Fluorescence Polarization

Results: AcycloPrime-FP Assay Difficulties Control ReactionsPlate of 96 Reactions 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 Homozygous Perennial Heterozygotes Homozygous Annual Non-template Controls

Methods: Taqman ® AD-Assay Reference:

Results: Taqman ® AD-Assay Annual Allele Perennial Allele (96 well plate rxn)

Reliability: CAPS vs. Taqman ®

Speed of Detection Methods CAPS Setup 0.5 PCR 1.5 Gel 1.0 Digest 1.5 Gel 1.0 Scoring 0.5 Total 7.0 hrs Taqman Setup 0.75 Run 2.0 Total 2.75 hrs Acyclo-FP Setup 0.75 PCR 1.5 Clean-Up Rxn 0.75 Acyclo Rxn 1.0 Total 4.0 hrs (based on 96 well plate)

Cost of Detection Methods CAPS $0.50/rxn Taq Pol. EcoR1 Agrose Plastics Taqman $0.63/rxn Probe Optical plate Optical film Plastics Acyclo-FP $2.00/rxn Kit (all inclusive) Plastics

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