Molecular techniques for certification of genetic purity and varietal

identification

Elisa Serra Negra Vieira – VARCOM Member

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Presentation summary:

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Ø Molecular marker Ø Definition and advantages

Ø SSR Ø SNPs

Ø Applications of molecular markers Ø Genetic purity Ø Varietal identification

Ø Genotyping technologies Ø Information survey in Brazil Ø Final considerations

Molecular marker:

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Molecular phenotype obtained from a gene or an specific segment of DNA.

Ferreira & Grattapaglia (1998)

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Ø Advantages:

Ø Distributed through role genome. Ø No enviromental effect. Ø Highly stable.

Molecular markers

Molecular markers

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Ø AFLP, RAPD, adaptations

Ø SSR (Simple Sequence Repeat) Ø SNP (Single Nucleotide Polymorphism)

90s

Today

SSRs:

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Ø Highly polymorphic. Ø Reproducible. Ø Multiallelic. Ø Co-dominant. Ø High throughput and multiplexing. Ø Public. Ø Easy but time-consuming interpretation. Ø Insufficient to characterize the number of

varieties released.

Microsatellite region

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Sequencing + Next Generation Sequencing (NGS)

Sequencing Information: billions of sequences

Single nucleotide polymorphisms (SNPs) identification

SNPs:

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Ø Genome richness: • Maize: 1SNP / 48 bp no codificant region

1 SNP / 131 bp codificant region

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Ø Biallelic Ø Efficient Ø Precise Ø Cost effective Ø High throughput and automatization Ø Non public

SNPs:

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Ø Seed production and breeding programs:

Ø Genetic Purity Ø Varietal Identification

Molecular marker applications:

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Ø Genetic Purity: Ø Degree of contamination Ø Self and outcrossing in hybrid seed lots Ø Segregation in genotypes (DUS) Ø Trait confirmation (GMOs)

Molecular marker applications:

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Ø Varietal identification

Ø Protection purposes Ø Unique genetic fingerprint (DUS test) Ø Characterization of Essentially Derived Varieties

(EDV)

Molecular marker applications:

Use of molecular markers

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Ø UPOV: wide consensus for use. Ø Brazil – National Service of Varietal Protection

(SNPC) : Ø Soybean: no permitted use SSRs:

Satt002,Satt005, Satt045, Satt009, Satt030, Satt038, Satt070, Satt114, Satt586, Satt431,

Satt216

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Ø Eucalyptus spp. DUS test: molecular marker as an additional information (05/15/2013)

http://www.agricultura.gov.br/arq_editor/EUCALIPTO_FORMULARIO_30set2013P.doc

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Genotyping technologies – SSR Ø Capillary Electrophoresis:

Ø Qiaxcel Ø LICOR Ø ABI Genetic Analyser

DNA Working Group: similar results!

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Genotyping technologies – SSR Ø Capillary electrophoresis:

Ø Qiaxcel

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Genotyping technologies – SSR Ø Capillary electrophoresis:

Ø Sequence plataforms fragment analysis

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Genotyping technologies – SSR

Ø Capillary electrophoresis: Ø Multiplex

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Ø 14 SSR / 4 dye Ø 18 SSR / 5 dye Ø High resolution for

clone fingerprinting

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Genotyping technologies – SNPs Ø High throughput – markers and no. samples Ø Cost effective Ø Accuracy Ø Easy Ø Databases

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Genotyping technologies – SNPs Ø One marker (particular trait) – large sample size

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Genotyping technologies – SNPs

TaqMan KASP

(Kompetitive Allele Specific PCR)

- Real-Time PCR machines - Normal PCR machines +

fluorophore reader

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Genotyping technologies – SNPs

ggg

Results: Allelic Discrimination plot

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Genotyping technologies – SNPs Ø Thousand SNPs – large sample size ® ARRAYS

Ø OpenArray Ø Array Tape

Ø Affymetrix

Ø Illumina Infinium

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Genotyping technologies – SNPs Ø Open Array

Ø No. TaqMan Essays and Samples Ø Several formats (customized) Ø Automated loading

Array

Subarray

Example:

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32 SNPs

2 samples

Color = sample ® 96 samples

Results:

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Genotyping technologies – SNPs

Ø Array Tape Ø Reaction in tape Ø TaqMan Essays or Allele Specific PCR Assays Ø Amplification in waterbath thermal cycler Ø Allele discrimination plots Ø Hundreds of thousands of data points per day

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Genotyping technologies – SNPs Ø Affimetrix

Ø Probes hybridization

Hybridized probe array

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Genotyping technologies – SNPs Ø Illumina infinium

Ø Single base primer extension

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Infinium array for maize varietal ID

Distinguish 276 hybrids.

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America Seed Trade Association (ASTA) + French Maize Breeders Association (UFS): - Illumina chip with 56 000 SNPs - Small set of 16 SNPs distinguished > 400

inbreds - Cost effective

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Information survey in Brazil

Ø Which technology and for what?

Company / Institution Crop Marker Technology Genetic Purity Variety ID

PioneerSoybean and

maize SNP´s Tape array X X

CoodetecSoybean, maize

and wheat SSR and SNP´sCE in ABI genetic

analyser X X

Embrapa Livestock Forrage grasses SSR CE in ABI genetic

analyser XMastuda Forrage

Grass Seeds Forrage grasses SSRCE in ABI genetic

analyser X

Canadian Grain Commission

Barley, Triticum durum , Triticum

spp SSR and SNP´s

CE in ABI genetic analyser, Open

Array X XNetherlands

Inspection Service for Horticulture Tomato SNP´s X X

National Taiwan University Rice SSR

CE in ABI genetic analyser, Open

Array X XMonsanto - Brazil Maize SNP´s TaqMan Assay XSyngenta - Brazil Maize SNP´s TaqMan Assay X

Embrapa Soybean Soybean SSR and SNP´s

TaqMan Assay and CE in Qiaxcel genetic analyser X X

SNPC - MAPA - BrasilSoybean and

eucalyptus SSRCE in ABI genetic

analyser X

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Final considerations: VARCOM future: Ø Handbook of molecular techniques for genetic

purity and varietal ID:

Ø SSR Ø SNPs

Ø Arrays of SNPs for some crops?????????? Ø Database development

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Acknowledgements: Ø VARCOM members Ø Embrapa Livestock Ø Matsuda Forage Grasses Seeds Ø Pioneer Ø Coodetec – DowAgroscience Ø SNPC – Agriculture Ministery – Brazil

Thank you for your attention

www.seedtest.org

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