DNA Markers in

Purity Testing

Dr. Benjamin (Beni) Kaufman

Nucleic Acid Analysis Laboratory

DuPont Pioneer

Table of Content:

What are DNA markers

The concept

Types

applications

What is purity

Purity by context

Purity & identity

Markers in purity testing

Various harmonization efforts:

ISTA

UFS

An optimal utilization &

harmonization scenario

The future ?

In 25 min.

Historically and conceptually an extension of morphometrics used in evolution, taxonomy, population genetics studies …breeding

Answering the same questions:

How similar are my subjects (individuals, populations, operational taxonomic units)

How are the differences among my subjects associated with…

Brought to the molecular level – Measuring similarities at the DNA level

Can be “local”, a specific sequence/gene

or “global”, comparing whole genome

What are DNA markers?

Chromosomal map positions of BioDiagnostics corn SNPs panel

Ideally: Comparing the whole DNA sequence/genome

The compromise: DNA Markers, a method for genome

sampling

Slide Provided by

Dr. Pegadaraju Venkatramana

BioDiagnostics Inc.

DNA Molecular Marker Systems in Plant

Genome Analysis (partial list)

Single or low copy probes

Restriction fragment length polymorphism (RFLP)

Restriction landmark genomic scanning (RLGS)

Sequence-tagged sites (STS)

Allele-specific associated primers (ASAPs)

Expressed sequence tag markers (EST)

Single strand conformation polymorphism (SSCP)

Single Nucleotide Polymorphism (SNP)

Arbitrary sequence markers

•Randomly-amplified polymorphic DNA markers

(RAPD).

•DNA amplification fingerprinting (DAF).

•Arbitrary primed polymerase chain reaction (AP-PCR).

•Sequence characterized amplified regions for amplification

of specific band (SCAR)

•Cleaved amplified polymorphic sequences (CAPs)

•Randomly amplified microsatellite polymorphisms

(RAMPO)

•Amplified fragment length polymorphism (AFLP)

Multi locus probes & repetitive DNA markerskers

•Microsatellites (SSR, STR, VNTR, HVR)

•Minisatellites

•Sequence-tagged microsatellite site markers (STMS).

•Direct amplification of minisatellite DNA markers

(DAMD-PCR).

•Inter simple sequence repeat markers (ISSR).

•Transposable elements.

•Alu-repeats..

•Repeat complementary primers.

Slide Provided by

Dr. Pegadaraju Venkatramana

BioDiagnostics Inc.

Microsatellites [Short Tandem Repeats (STRs), Simple Sequence Repeats (SSRs)]

•The repeat region is variable between samples while the flanking

regions conserved

•Alleles differ by the number of repeats

•Repeats made of motifs of various nucleotide lengths

•Highly polymorphic (2-15 alleles/locus). Mendelian. Co-dominant.

7 repeats

8 repeats

AATG

Single Nucleotide Polymorphisms ( SNP)

Detects single nucleotides

changes

— Co-dominant in nature

—Mendelian inheritance

— Dispersed throughout

the genome

— Highly abundant in

number

Multiplexed effectively,

hence - cost effective

Application of

DNA Markers:

Genotyping

QTL Analysis

Association Mapping

Phylogenetic Analysis

Marker Assisted Selection

Marker Assisted Backcrossing

Trait Stacking

Patenting

Genome Mapping

Genetic Purity Analysis

Genome-wide Selection

Bulk Segregant Selection

Essentially Derived Varieties

Plant Variety Protection

Trait Purity Analysis

Genetic

Studies

Breeding

Tools

Legal

Applications

Production QA/QC

Genetic Purity Testing Are all individuals of a line/lot/bag the desired/declared

genetic make-up?

(In actuality = above a predetermined relative proportion)

Used by seed producers as a quality assurance tool to identify:

Out crosses

Selves (where applies)

Seed mixes

Seed swaps

By seed producers, regulators, growers as:

Regulatory compliance testing

Using DNA Markers for Genetic Purity Testing

Based on DNA fingerprint, that is, genotyping

N loci/markers

Requirements:

• Polymorphic, have more then one allele

represented in the population.

• ~Even Allele frequency

• No null alleles (where applies)

• Diagnostic

• Minimal number of markers (cost, efficiency)

• Robust performance

Sample of SSR results for wheat:

•8 markers shown

• Multiplexed PCR protocol

•Visualization system: LiCor *indicates lane for size marker

Duilio Iride Mieti Simeto

* *

* *

Line A Line B Line C Line D Line E Line F Line N SSR 1 A G A G A B G SSR 2 A B A B C A B SSR 3 B A B A D B A SSR 4 C A C A F A A SSR 5 D C D C H B C SSR 6 A D A D B A D SSR 7 F D F F C A D SSR 8 H F H H D C F SSR 9 B D B B F D A

SSR 10 B G A B H F F SSR 11 B D B B D H D SSR 12 F B A F F B B

Similarity(A_C) = 9/12 = 75%

Genetic Distance

When characterizing the genetic make-up

of a crop variety or line in terms of

molecular markers genotypes:

• The number and genomic distribution of

the markers determines the depth and

resolution of the DNA fingerprint

obtained.

•Translated into relatedness or genetic

similarity of individuals, varieties,

populations, lines.

Slide Provided by

Dr. Pegadaraju Venkatramana

BioDiagnostics Inc.

Biotechnology Trait Purity Testing

In Research: In production:

Trait confirmation Trait confirmation

Copy number testing Adventitious Presence testing

Event characterization Low Level Presence

Adventitious Presence testing

Trait Purity: Based on genotyping particular genes of interest

To confirm the presence of some

To verify the presence of others

Purity & Identity

Genetic purity is a population concept and it is

assessed by analyzing the identity of its members

The interplay between Purity and Identity is sometimes

a cause of confusion.

Varietal identification

Identification:

Using diagnostic traits (markers) to:

• Recognize an individual as being part of a particular sub-species division

• Separate one such sub-species cluster from others.

Plant Variety

(http://www.upov.int/about/en/upov_system.html#iv_c ):

•A plant grouping within a single botanical taxon of the lowest known rank.

•Can be defined by traits resulting from (a ) given genotype/s

•Distinguished from other plant grouping by at least one of the said traits

•Considered as a unit with regard to its suitability for being propagated

unchanged

TITLE ISTA’s Variety ID Technical Committee initiated work

towards development of standardized DNA based methods for ISTA that can be used for testing variety

identity.

- Strategy:

SSRs

Comparative tests between laboratories

- the same band patterns (allele sizes) when using different reagents, equipment and working protocols?

- select the most suitable SSRs for variety identification.

- Validate method by having additional laboratories perform testing

Working group Number of

selected SSR

Amount of

tested Varieties

Tested Areas

Soybean 11 74 Argentina

Brazil

Wheat 8 84 Canada

Brazil

France

Italy

Maize 12 72 France

Canada

Rice 15 192 Asia

Italy

USA

Summary of Results:

Summary of Results:

For all crops:

• different numbers of SSRs could be selected

• more SSR should be selected from some crops to identify larger sets of varieties

• Different visualization systems do give the same banding patterns (even if allele sizes are slightly different)

• Allele calling a challenge!

Need to define a set of “reference varieties”/crop that should be used by laboratories wishing to establish these protocols for variety verification.

Essentially Derived Varieties (EDV)

Definition :

A plant variety is taken to be an essentially derived variety of another plant

variety if:

(a) it is predominantly derived from that other plant variety; and

(b) it retains the essential characteristics that result from the genotype

or combination of genotypes of that other variety; and

(c) it does not exhibit any important (as distinct from cosmetic)

features that differentiate it from that other variety.

PLANT BREEDER'S RIGHTS ACT 1994 - SECT 4

Essentially Derived Variety - a variety predominantly derived from an

initial variety and therefore under the ownership of the breeder of the

initial variety.

Identification of SNP Markers to Aid Assessment

of Essential Derivation in Maize

Essential derivation threshold established by SSRs:

• 82% SSRs = “orange” threshold, possible EDV

• 90% SSRs = “red” threshold, an EDV or reversal of burden of proof

• Scope of current project: Use existing SSR thresholds

to determine equivalent thresholds using Illumina 56K

SNP chip

• Joint ASTA / USF initiative under ISF umbrella • Monsanto, Syngenta, DOW, Pioneer, Caussade Semences, RAGT,

Limagrain, Euralis, KWS, Maisador

• Biogenetics and Trait Genetics providing SSR and SNP data

Correlation between 163 UFS SSRs and 26,874 SNPs

26,8

47 S

NP

s

R2 = 0.92,

Slope 0.50

163 UFS SSRs

Accuracy of Genetic Distances for Different Sub-

Sets of SNPs and SSRs

384, 768, 1536 and 3072 SNPs were sampled from 26,847 SNPs 100 times and CVs assessed at a GD of 0.1 for SNPs, and 0.2 for SSRs (ie close to red zone):

Marker

Set

CV SD Mean correlation with

full set SNPs

3072 0.05 0.004 0.99 (0.99-1)

1536 0.07 0.006 0.98 (0.98-0.99)

768 0.10 0.008 0.98 (0.96-0.98)

384 0.15 0.012 0.95

163 UFS

SSRs

0.16 0.031

CVs are lower for SNP

sets than SSR set

3072 and 1536 SNP

sets give almost as

much information as

26,768 SNPs

Next steps • Select SNP set by June 2012

• Formal proposal to ISF October 2012

• Request formal ISF approval at assembly of

field crops section May 2013

The SNP set selected for EDV will be useful for

developing other sub-sets eg for managing

reference collections and for variety

identification

Relation between number of markers and

resolution in purity and identity applications:

Trait purity

Genetic purity

Varietal ID

Essentially Derived Variety

Plant Varietal Protection

Number of markers

Genetic resolution

Nested Markers Idea

to satisfy all DNA marker applications:

Varietal ID

Genetic Purity

EDV

PVP BDI-III-XX

What’s in the future?

Ideally: Comparing the whole DNA sequence/genome

The compromise: DNA Markers, a method for genome sampling

Is next generation sequencing on the

purity testing horizon?

Contributions to Sequencing Project Overall Costs

Across Time

Mark B. Gerstein,

Yale University

The $1,000 Genome

Thank You

Acknowledgments:

Dr. Liz Jones

DuPont Pioneer

Her update on UFS project/s

Cheryl Dollard and Variety TCOM.

Update and work on ISTA’s DNA purity efforts

Dr. Pegadaraju Venkatramana (Venki)

BioDiagnostics INC.

Dr. Gregory May

DuPont Pioneer