Applied molecular genetics testing for quality control in seed testing. Dr. Patrik Stolt ScanBi Diagnostics

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1 Applied molecular genetics testing for quality control in seed testing Dr. Patrik Stolt ScanBi Diagnostics

2 It is hard to find things you do not know that you are looking for! Short reminder about DNA/PCR methodology What can be done with DNA from one extraction? Examples how to interpret DNA marker results Challenges using DNA markers

3 Define questions and identify risks in the seed production chain Gateway 1 Gateway 2 Gateway 4 Gateway 5 Gateway 6 Gateway X Agrobiotech Breeding Production Market A1 Trait discovery, gene isolation, gene cloning, transformation A2 Zygosity, gene expression, gene stability, trait confirmation B1 B2 Trait Back crossing, transmission, stable line no parent material, segregation hybrids Gateway 3 P1 Plants in tents or bags on field. Seed lots of e.g kg seeds P2 Production on field, seed lot of e.g kg seeds. P3 Production for market seed lot of e.g ton. M1 Farmer materials, cleaning, bagging, sales Gateway 1 Gateway 2 Gateway 4 Gateway 5 Gateway 6 Gateway X What methodologies are available to answer the questions?

4 Determine the same trait at different resolution Phenotype Genotype RealTime PCR Gene / nucleotide e.g. NMR Photos: unknown scientific contributers. Not for publication.

5 Short reminder about DNA/Proteins Cell Phenotype Chromosom Protein Promoter when, where and intensity Terminator the endpoint of the gene DNA Storage device for genetic information Start Valine Leucin Isoleucin Proline The code is read Gene The template for the protein With premission from Dr. K. Vamling

6 Short reminder about DNA/Proteins Cell Phenotype 12 Chromosom Protein Probe/Marker Valine Glutamine Leucin Isoleucin Proline GAC CAG..xGTCx....xCTGx.. With premission from Dr. K. Vamling

7 Reminder about PCR Amplification of DNA DNA amplification due to each cycle Source: Suzanne Kennedy and Nick Oswald

8 How to interpret RealTime PCR results Sample canola Positive control Canola: DNA copies Positive control Mustard: 50 DNA copies Sample mustard Fluorescens Ctcanola = = 2 Ctmustard 1 Ct = x2 = = -2 4 x = 50/4 = DNA DNA copies copies Ct 22 Ct 24 Ct 34 Ct 36 PCR-cycles (Ct-values) Mustard in canola = 12.5/ = 0.006% The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e. exceeds background level).

9 Real Time-PCR based marker detection Often the expensive step Grinding of seed material Extraction of DNA Fluorescens Results Amplification of DNA PCR-cycles

10 What can be tested from one DNA extaction? Flour Seeds Leaves Roots Soil Bulk samples Single seed/leaflet samples Extraction of DNA

11 What can be tested with DNA from one extraction? Genotype/purity: Species Variety Hybrid Trait.... The extracted DNA from a sample contains all DNA from all organism. Virus Bacteria Fungi

12 Example; What can be tested with DNA from one extraction? Purity = Absence or Presence Variety Cauliflower mosaic virus, CaMV Surface/systemic infections e.g.: Sclerotinia sclerotiorum Fusarium graminearum Species e.g. mustard in canola GM test DNA markers e.g. low erucic acid Hybridity CMS ogura Rf - ogura

13 What can be tested from one DNA extaction? Example: improve seed purity in CMS breeding programs GMO leaf test CMS test Genotype test No unapproved GMO No pollen prod. plants Pure genotype

14 DNA markers in conventional cross-breeding and back-crossing Plant with desired trait/gene Back-crossing and selection cross cultivar with desired trait/gene(s) crop plant crop plant (elite line) With premission from Dr. K. Vamling

15 DNA markers and genetic engineering and Gene transfer and Back-cross Organism with gene of interest Back-crossing and selection, regulatory compliance Isolation of desired gene Transfer of Gene construct Isolation of suitable promotor Cultivar (GM) with desired traits crop plant Selection of elite event crop plant (elite line(s)) With premission from Dr. K. Vamling

16 The challenge

17 Source: Adenosine Mike Jones; Triangle of U Simple.PNG

18 You can only find what can be measured!

19 Thank you Quality and Service through Knowledge and Understanding Our corporate culture is built upon these four corner stones!