INTERNATIONAL SEED TESTING ASSOCIATION www.seedtest.org
Seed quality is defined and measured along a number of axes:
• Is the seed of the right variety? ...Varietal ID tests • Is it genetically pure? ... Genetic purity tests • Does it contain the right traits …Trait purity tests • with no adventitious presence of unintended GM seed …AP/GMO tests • Is it physically pure (free from debris or inert matter)? …Purity tests • Is the seed free from disease and pathogens …Seed health tests • is the seed vigorous? …Cold test, Accelerated aging test • how well will it germinate? ...Warm test; Tetrazolium test
The way we define seed quality is not likely to change soon;
however, the methods we test it by are evolving constantly
Advances are driven by technology: New technological innovations enables new & Improved modes of testing.
http://www.englishblog.com/2009/03/cartoon-the-evolution-of-communication.html
“Making the leap”: From traditional (e.g. organismal) approaches to new (molecular) methodologies –
http://tomfishburne.com/2010/10/making-the-leap.html
Already employing advanced/new methods… Keeping up with advances: For example the evolution from SSRs to SNPs for genetic purity testing.
“Look Ma no hands… !!!”
ARCGA - Arnold Roth Deposit Collection, The Ohio State University Billy Ireland Cartoon Library & Museum
In this Session some of the new and emerging
technologies will be presented:
Next Generation Seed Quality Testing, Benjamin (Beni) Kaufman SpectraSeed: Seed phenotype database through spectral imaging, Jens Michael Carstensen, Karsten Hartelius and Kaare Jensen Seed identification of medicinal plant species using machine vision, Sepideh Anvarkhah, Mohammad Khajeh-Hosseini, Ali Davari Edalat Panah, Mohammad Hassan Rashed Mohassel. Development of a diagnostic tool and resource for seed identification with Lucid and imaging technologies, Ruojing Wang and Jennifer Neudorf An image analysis for determination of genetic purity of different wheat varieties Esmaeil N. Azar, Ghasem Tohidloo, Samad Mobasser, Majid D. Shoar and Fardin Khazaei Speed of seed imbibition related to seedling evaluation: precocious criteria from image analysis to predict the seedling type in a germination test. Marie-Hélène Wagner, Sylvie Ducournau, Valérie Blouin, Didier Demilly and Joël Lechappe Multiplex detection of plant pathogens, Jan J.H.W. Bergervoet, Marjanne M. de Weerdt, René R.A.A. van der Vlugt, Sharon S. Van Brunschot and José J.R.C.M. van Beckhoven
8 6/26/2013
*
Technical advances as they apply to: Genome analysis.
@Present: DNA Markers, a method for genome sampling
Slide Provided by Dr. Pegadaraju Venkatramana BioDiagnostics Inc.
ØNew Chemistries… § Isothermal amplification
§ Universal Probe chemistry
ØNew Platforms… §Miniaturization, High Through Put
ØNew Approach… § Next Generation Sequencing/ Sequence capture
*NūPCR is a probe-based qPCR technology that utilizes Illumina’s unique NūZyme chemistry
*NūZyme is a multipart nucleic acid enzyme (DNAzyme).
*All oligos must assemble on the target sequence in order for fluorescence to be generated (high specificity).
*Universal substrate results in cost savings
*4 dyes to choose from-allows multiplexing
New Chemistry
3-step real-time PCR protocol
Compatible with instruments from all major manufacturers
*Higher specificity
*Higher PCR efficiency
*Earlier Cq
*Higher multiplexing success rates
*Lower cost
vs.
Mostly by bringing competition to the qPCR Chemistry market. Its impact will depends on: Level of: “Signal to noise ” Variation Cost
EnviroLogix - Confidential Internal Use Only
ü The nicking enzyme nicks the target DNA
ü Polymerase attaches to
open 3’-end of nicked strand ü Polymerase extends and
displaces DNA strand with high processivity
New Chemistry
ü Reverse template anneals and polymerase extends
ü Forward template anneals and polymerase extends beyond Nicking Enzyme Recognition site
ü Nicking enzyme nicks ü Polymerase attaches and
extends
DNAbleTM Schematic
EnviroLogix - Confidential Internal Use Only
ü Formation of DNAbleTM Duplex ü Exponential Amplification
n Nick n Displace n Extend
ü Formation of amplification products
ü Detection
DNAbleTM Schematic
EnviroLogix - Confidential Internal Use Only
ØAssay can detect any reasonable DNA or RNA sequence
Ø“Crude” samples are compatible
ØTrue isothermal reaction
ØUtilizes 2 templates (primers), polymerase and nicking enzyme
* Enzymes allow direct amplification from genomic DNA
ØReaction volumes range from 1 μl to 50 μl.
ØReagents may be stabilized via lyophilization
ØDual detection allows for detection of internal control
ØMultiple readout options
* Real-Time via Molecular Beacons
* Endpoint via Molecular Beacons
* Lateral-flow detection with visual results
DNAbleTM Features:
DNAble “Impact Factor”:
Ø Isothermal reaction x Crude extract : May enable the development of “hand held” /”field” testing devices. Applicable at: Elevators for GMO testing Breeder’s field nursery for trait confirmation.
ØIsothermal x Possible small Reaction volumes Reagents may be stabilized via lyophilization
Dual detection (allows for detection of internal control) Amendable to high through-put in-line platforms
• In line multiple step processing (Assembly line)
• Continues feed ( high through put)
• Tape array (savings in consumables)
• Small reaction volume ( saving in reagents)
• Hands free ( savings in labor)
New Platforms:
15.30 DNA markers in purity testing (Beni Kaufman, Pioneer a DuPont Business, United States of America)
Nested Molecular Markers Battery for the testing of:
• Genetic Purity • Trait Purity • Varietal Identification • Essentially Derived Varieties (EDV) • Plant Varietal Protection
Trait Purity Genetic Purity
Varietal ID Essentially Derived Variety (EDV)
Plant Varietal Protection (PVP)
The number and genomic distribution of
the markers determines the depth and
resolution of the DNA fingerprint
obtained.
The closer the relatedness or genetic
similarity of individuals/ varieties/lines.
The higher the number of markers are
needed to tell them apart.
Slide Provided by Dr. Pegadaraju Venkatramana BioDiagnostics Inc.
Nested Markers to satisfy all purity applications:
Varietal ID
Genetic Purity
EDV
PVP BDI-III-XX
Why sample the genome - why not analyze whole genomic DNA sequences
*
Mark B. Gerstein, Yale University
DNA Segment
Whole Genome Sequencing
Fractional Sequencing Methods
RNA-Seq
Amplicon
RAD-Seq
Whole genome not always necessary…
Courtesy of Rick Nipper
Advantages
•Focus on Region of Interest with high Coverage
oQTL, Exon Only, etc…
•Multiplex many samples
Disadvantages •Reference Sequence must be available •Size restrictions 100kb-10Mb •Library Cost and/or Labor Sequence Capture
Methods/Vendors •Amplicon Pooling and Sequencing
•“Golden Gate” Type Probe Extension
•RainDance MicroDroplets
•NimbleGen On Array and In Solution
•Agilent On Array and In Solution •Upcoming:
wIllumina Custom PCR Plates/cRNA Probes wLife Tech Seq Cap for Ion
Restriction-site Associated DNA Sequencing (RAD-Seq)
*RAD-Seq uses restriction nucleases as a strategy to focus DNA sequencing on a small fraction of the target genome
*Despite the relatively small amount of the genome queried, thousands to hundreds of thousands of genetic variants can be readily identified.
RAD sequences
0.1% to
10%
of a selected genome
Courtesy of Rick Nipper
Chromosome
GTGGTCATGCATCGTAGTGCACATGGCATGTGCAGTCAT CACCAGTACGTAGCATCACGTGTACCGTACACGTCAGTA
enzyme recognition site
genomic sequence
Restriction Enzyme Digestion Sites
Sequence region around digestion site utilizing next-gen sequencing platform
Courtesy of Rick Nipper
CACCAGTACGTAGCACCTCGT CACCAGTACGTAGCACCTCGT CACCAGTACGTAGCACCTCGT CACCAGTACGTAGCACCTCGT
Sample I
CACCAGTACGTAGCACCTCGT CACCAGTACGTAGCACCTCGT CACCAGTACGTAGCAACTCGT CACCAGTACGTAGCAACTCGT
Sample II
Sample 1 Genotype:
C/C
Sample 2 Genotype:
C/A
Examine sequence data from individual locus: Identify variants and determine genotypes
TCAGTACACGTGCACCAGTACGTAGCATCTCGT
Chromosome
Courtesy of Rick Nipper
Nested Molecular Markers Battery for the Testing of:
• Genetic Purity • Trait Purity • Varietal Identification • Essentially Derived Varieties (EDV) • Plant Varietal Protection
By way of TARGATED SEQUENCING
• One run through - will have all the sequence information for all tests. • Data analysis will target sequences associated with test of interest • Stored data can always be accessed and additional mining/analysis done
Why stop here…
Seed testing:
• Is the seed of the right variety? ...Varietal ID tests • Is it genetically pure? ... Genetic purity tests • Does it contain the right traits …Trait purity tests • with no adventitious presence of unintended GM seed …AP/GMO tests • Is it physically pure (free from debris or inert matter)? …Purity tests • Is the seed free from disease and pathogens …Seed health tests • Is the seed vigorous? …Cold test, Accelerated aging test • How well will it germinate? ...Warm test; Tetrazolium test
Ö Ö
Ö
Ö Ö
Whole Genome Association (& QTL) studies identify genomic sequences associated with the traits of interest see for example:
* Bogamuwa, S., Jang, J.-C. The Arabidopsis tandem CCCH zinc finger proteins AtTZF4, 5 and 6 are involved in light-, abscisic acid- and gibberellic acid-mediated regulation of seed germination (2013) Plant, Cell and Environment. Article in Press.
* Abe, A., Takagi, H., Fujibe, T., Aya, K., Kojima, M., Sakakibara, H., Uemura, A., Matsuoka, M., Terauchi, R. OsGA20ox1, a candidate gene for a major QTL controlling seedling vigor in rice (2012) Theoretical and Applied Genetics, 125 (4), pp. 647-657.
* Davar, R., Majd, A., Darvishzadeh, R., Sarrafi, A. Mapping quantitative trait loci for seedling vigour and development in sunflower (Helianthus annuus L.) using recombinant inbred line population (2011) Plant OMICS, 4 (7), pp. 418-427.
* Quantitative trait loci analysis for rice seed vigor during the germination stage. Wang ZhouFei; Wang JianFei; Bao YongMei; Wang FuHua; Zhang HongSheng; Zhejiang University Press, Hangzhou, China, Journal of Zhejiang University (Science B), 2010, 11, 12, 958-964
Is This Feasible ?
The information is there, so is the technology – someone needs to put it together
Is This Feasible ?
Economically – The more testing needs are satisfied per sequencing effort the more economical feasible/favorable it is: • Varietal ID tests $100
• Genetic purity tests $100 • Trait purity tests $60 • AP/GMO tests $150 • Cold test $35 • Germ test $40 • Total: $485
Targeted sequencing: RAD: $175 Other: $200
37
Data2Bio’s tGBS technology stringently controls the fraction of genome that is sequenced and genotyped. Reads are clustered at few sites: • Minimizes missing data across lines; enhances repeatability • 100s-60,000 SNPs (“tunable”); adequate for most applications,
including QC • High confidence SNP calls • Can confidently genotype heterozygous loci, even in species
without a reference genome
Line 1
Line 2
*Potentially it is possible to test simultaneously all the seed attributes.
*Highest possible resolution *It has the promise to be economical BUT:
Not everything is genetics! Environmental factors can not be predicted
therefore
Some “Standard Seed Testing” will always be required!
“Impact Factor” ?
Gina Zastrow-Hayes DuPont Pioneer
Rick Nipper Floragenex
Satish Rai Douglas Scientific
Daniel Shaffer Envirologix
Todd Deppe Margaret Esser Illumina
Pat Schnable Data2Bio Iowa State University Pegadaraju Venkatramana
BioDiagnostics Inc.
43
Line 1
Line 2
• ~1M sites, i.e., more than is required for most applications, including seed quality applications
• SNP calls based on few reads (error rate). Can not call heterozygotes
• Limited overlap of sites among lines leads to missing data across samples
• Requires imputation (heavy-duty bioinformatics) - difficult in species without a reference genome