Use of PCR Technology for Purity Testing

SCSTDean Hemphill

Quality Testing ManagerDuPont Pioneer

Johnston, Iowa, USA

13/13/2016INSERT SENSITIVITY CAPTION HERE

Introduction & Advice

• Understand the industry you are part of• Embrace new technologies• Keep your career path fluid• Maintain business relationships as companies change• Leadership Advice

• Assemble team of knowledgeable experts• Supply Mindset• Expertise• Aptitude

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Purity Testing: Three Basic Needs

• Genetic Purity• Is the product what we think it is - the correct genetics• Does the product contain “offtype”, “self” or “other”• Genetically fixed

• Trait Purity• Is the trait present • Is the trait homozygous (for inbred)• What level of trait purity

• LLP – Low Level Presence – Non GMO• Adventitious presence of a GMO in a non-GMO product• The presence of an unintended GMO in a GMO

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What Are “Traits”?

• Traits are desirable attributes of a plant• e.g. insect or herbicide resistance; “good” oil composition

of seed; drought tolerance; etc.• Can be natural genes or transgenes

• Transgenes• Desirable genes are artificially introduced into plant

genomes (i.e. not by breeding)• Often derived from other species that can not be bred

with the plant of interest (e.g. MON810 / Bt)• Sometimes human-designed genes (e.g. GAT)

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Commercial Product Testing Descriptions

Quality Analysis Tool

Genetic Purity Isozyme Electrophoresis, SNP, Endpoint PCR, Growout

Trait Purity* ELISA, BioAssay, Endpoint PCR

Trait Confirmation* Endpoint PCR, Chemical Rogue

Zygosity Screen* Endpoint PCR, RT PCR

Low level presence of unintended events*

Lateral flow Strip (ELISA), Endpoint PCR, RT PCR

Trait Detection/Grain* Lateral Flow Strip (ELISA), Endpoint PCR

*For GMO containing products 5

TaqMan Chemistry testing for SNPs

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TaqMan more Detail

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What is ddPCR?

• Droplet Digital PCR is a modification of conventional PCR to allow for the direct quantification of nucleic acids.

• It uses limiting dilution, end-point PCR, and Poisson statistics to yield a direct measure of nucleic acid concentration.

• The number of replicates determines the dynamic range of the assay, precision is not limited by PCR chemistry.

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Limiting Dilution by Droplet

Well contains a mixtureof Template (GMO) and no Template (WT) DNA

PCR reaction is emulsified into individual reactions, limiting the template DNA available in each reaction.

WT

WT

WT

WTWT

WTWT

GMO

GMO

GMO

WTWT

WT

WT

WT

WT

GMO

WT

WT

WT

WT

WT

WTWT

WT

WT

WT

GMO

Using end-point PCR, positive reactions are detected.

WTGMOWT

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Comparison of rtPCR and ddPCRReal-Time PCR Droplet-Digital PCR

Emulsification into ~20,000 rxns

Amplification with Real-Time Machine

Amplification

Analyze with droplet readerYields ~20,000 data points

Analyze sample amplification curve against Standard amplification curves

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Molecular Marker Based Trait and Genetic Purity Testing – SNP

Single Nucleotide Polymorphism (SNP)• A difference in DNA sequence of a single nucleotide

• Replace Isozyme testing for genetic purity

• All parent and commercial corn is transferred to SNP testing worldwide

within the DuPont Pioneer organization

• In humans, 99% of the DNA sequence is identical in all persons; 1% varies• The variation can be a large piece of sequence, or a single nucleotide (SNP)• In human SNPs occur every 100 to 300 bases along the 3-billion-base human

genome

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Single Nucleotide Polymorphisms

Insertion/Deletion G->A SNP

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More Definitions…

• Genome• The sum of all genes of an organism. Each cell contains the entire

genome-worth of DNA.

• Genotype• The sum of all gene versions (“alleles”) of an individual organism

(remember, individuals have some differences in their DNA sequences, such as different SNPs).

• Genotyping• Determining the genotype of an organism by testing the DNA of some of

their cells.

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Chromosomes

DNA and histone proteins are packaged into structures called chromosomes.•Humans: 2 sets of 23 chromosomes (one from the mother, one from the father)•Goldfish: 2 x 50 chromosomes•Soybean: 2 x 20 chromosomes•Maize: 2 x 10 chromosomes

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Genes

•Genes are made up of DNA. •Each chromosome contains many genes.•Each gene has a unique sequence of “letters” (i.e. bases).

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SNP Genotyping

In order to detect a particular SNP in the huge genome,we need to “ amplify” (i.e. make many copies of) that genomic regionbefore probing for the SNP.

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SNP Genotype Scores Interpretation

Individuals with heterozygous A/B alleles

Individuals with homozygous A/A alleles

Individuals with homozygous B/B alleles

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Scoring for Inbred Seed Purity Test

Hybrid control

Inbred control

Inbred control

Contamination

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Example of Pioneer Pedigree Profiles Using 8 SNP Markers

PedigreeInbred A G A T C G G G GInbred B G C A C G G G GInbred C G C A C G G G GInbred D A A T T A T C AInbred E A C T T A T G GInbred F G A A T G T C AInbred G A A T T G T C GInbred H G A A C G G G AInbred I A A A T A T C AInbred J A A T T G T C GInbred K G C T C G G G G

Hybrids12345 A,G A,C A,T C,T A,G G,T C,G A,GInbred K G C T C G G G GInbred I A A A T A T C A

98765 A,G A,C T C,T G G,T C,G GInbred K G C T C G G G GInbred J A A T T G T C G 19

Definition of Inbred Offtypes Based on SNP Scores

• Outcross: • different than expected and at least one marker is heterozygous• if GMO product add traits for confirmation

• Other: • other inbred mix - all marker profiles homozygotes but different

than the expected; • if transgenic traits do not line up

• Segregation: • any individual marker that is not fixed • contain heterozygous individuals

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Inbred Data Analysis -- Outcross

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Inbred Data Analysis -- Other

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Definition of Hybrid Offtypes Based on SNP Scores

• Outcross: • wrong male contribution

• Self: • missing the male or female contribution at all heterozygous

markers

• Other: • unexpected genotype

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Hybrid Data Analysis -- Self and OutcrossJOB G

01

G02

G03

G04

G05

G06

G07

G08

G09

G10

G11

G12

G13

G14

G15 T16

T17

T18

T19

T20

T21

T22

T23

T24

T25

FEMALE G T A A C T C T A C T C C C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

MALE A T T A G C T A G C G C G A T NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG

HYBRID G,A T A,T A C,G T,C C,T T,A A,G C T,G C C,G C,A T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G T A A C T C T A C T C C C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G T A A C T C T A C T C C C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A C,T A A C T C,T T A C T C C,G C T NEG NEG POS NEG POS POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NF NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG

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Hybrid Data Analysis –Product Mix, 2 different hybrids

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Why convert?

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Advantages SNP Markers for Seed Testing

• Increased resolution & reliability• Automated & high throughput• Objective scoring and interpretation• Decreased cost per informative datapoint• Trait purity as well as genetic purity can be determined at the

same time on the same seed• Applicable for all crops at all regions• Single inbred profile database used worldwide

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Diagnostic Markers EL vs. SNP -DuPont Pioneer Hybrids Profiled in China

PRODUCTEL Diag Markers

SNP Diag Markers

PRODUCTEL Diag Markers

SNP Diag Markers

X001 0 8 X018 3 7X002 0 12 X019 0 11X003 3 8 X020 0 10X004 0 8 X021 0 9X005 4 11 X022 2 9X006 0 6 X023 2 12X007 0 6 X024 2 10X008 3 9 X025 2 11X009 3 11 X026 1 11X010 0 6 X027 0 6X011 0 7 X028 0 9X012 0 7 X029 2 7X013 0 10 X030 2 7X014 0 9 X031 0 12X015 0 10 X032 4 8X016 3 8 X033 0 9X017 3 6 X034 3 13

•EL diagnostic markers of 0 includes those products with incomplete EL profiles•SNP diagnostic markers out of a standard set of markers across 10 maize chromosomes

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Additional Advantages of SNPs v Isozymes• Workforce

• Training• Availability

• Platform• Scalable (from totally manual to fully automated)• Scalable (test multiple crops on same platform)

• Vendors• Reagent availability

• Startup• MaizeGBD.org

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International Rules for Seed Testing

ISTA rules governing commercial seed lot testing frequencies and requirements

http://www.ingentaconnect.com/content/ista/rules

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Open for Discussion

Thank You

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