DNA Markers in Purity Testing - ISTA Online Markers in Purity Testing Dr. Benjamin (Beni) ......
Transcript of DNA Markers in Purity Testing - ISTA Online Markers in Purity Testing Dr. Benjamin (Beni) ......
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