Molecular techniques for certification of genetic purity and varietal identification
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1 Molecular techniques for certification of genetic purity and varietal identification Elisa Serra Negra Vieira VARCOM Member Elisa Vieira 1
2 Presentation summary: Molecular marker SSR SNPs Definition and advantages Applications of molecular markers Genetic purity Varietal identification Genotyping technologies Information survey in Brazil Final considerations Elisa Vieira 2
3 Molecular marker: Molecular phenotype obtained from a gene or an specific segment of DNA. Ferreira & Grattapaglia (1998) Elisa Vieira 3
4 Molecular markers Advantages: Distributed through role genome. No enviromental effect. Highly stable. Elisa Vieira 4
5 Molecular markers 90s AFLP, RAPD, adaptations SSR (Simple Sequence Repeat) SNP (Single Nucleotide Polymorphism) Today Elisa Vieira 5
6 SSRs: Microsatellite region Highly polymorphic. Reproducible. Multiallelic. Co-dominant. High throughput and multiplexing. Public. Easy but time-consuming interpretation. Insufficient to characterize the number of varieties released. Elisa Vieira 6
7 Sequencing + Next Generation Sequencing (NGS) Sequencing Information: billions of sequences Single nucleotide polymorphisms (SNPs) identification Elisa Vieira 7
8 SNPs: Genome richness: Maize: 1SNP / 48 bp no codificant region 1 SNP / 131 bp codificant region Elisa Vieira 8
9 SNPs: Biallelic Efficient Precise Cost effective High throughput and automatization Non public Elisa Vieira 9
10 Molecular marker applications: Seed production and breeding programs: Genetic Purity Varietal Identification Elisa Vieira 10
11 Molecular marker applications: Genetic Purity: Degree of contamination Self and outcrossing in hybrid seed lots Segregation in genotypes (DUS) Trait confirmation (GMOs) Elisa Vieira 11
12 Elisa Vieira 12
13 Molecular marker applications: Varietal identification Protection purposes Unique genetic fingerprint (DUS test) Characterization of Essentially Derived Varieties (EDV) Elisa Vieira 13
14 Use of molecular markers UPOV: wide consensus for use. Brazil National Service of Varietal Protection (SNPC) : Soybean: no permitted use SSRs: Satt002,Satt005, Satt045, Satt009, Satt030, Satt038, Satt070, Satt114, Satt586, Satt431, Satt216 Elisa Vieira 14
15 Eucalyptus spp. DUS test: molecular marker as an additional information (05/15/2013) RIO_30set2013P.doc Elisa Vieira 15
16 Genotyping technologies SSR Capillary Electrophoresis: Qiaxcel LICOR ABI Genetic Analyser DNA Working Group: similar results! Elisa Vieira 16
17 Genotyping technologies SSR Capillary electrophoresis: Qiaxcel Elisa Vieira 17
18 Genotyping technologies SSR Capillary electrophoresis: Sequence plataforms fragment analysis Elisa Vieira 18
19 Genotyping technologies SSR Capillary electrophoresis: Multiplex Elisa Vieira 19
20 14 SSR / 4 dye 18 SSR / 5 dye High resolution for clone fingerprinting Elisa Vieira 20
21 Genotyping technologies SNPs High throughput markers and no. samples Cost effective Accuracy Easy Databases Elisa Vieira 21
22 Genotyping technologies SNPs One marker (particular trait) large sample size Name of presenter 22
23 Genotyping technologies SNPs TaqMan KASP (Kompetitive Allele Specific PCR) - Real-Time PCR machines - Normal PCR machines + fluorophore reader Elisa Vieira 23
24 Genotyping technologies SNPs ggg Elisa Vieira 24
25 Results: Allelic Discrimination plot Elisa Vieira 25
26 Genotyping technologies SNPs Thousand SNPs large sample size fi ARRAYS OpenArray Array Tape Affymetrix Illumina Infinium Elisa Vieira 26
27 Genotyping technologies SNPs Open Array No. TaqMan Essays and Samples Several formats (customized) Automated loading Subarray Array Elisa Vieira 27
28 Example: 32 SNPs 2 samples Color = sample fi 96 samples Elisa Vieira 28
29 Results: Elisa Vieira 29
30 Genotyping technologies SNPs Array Tape Reaction in tape TaqMan Essays or Allele Specific PCR Assays Amplification in waterbath thermal cycler Allele discrimination plots Hundreds of thousands of data points per day Elisa Vieira 30
31 Genotyping technologies SNPs Affimetrix Probes hybridization Hybridized probe array Elisa Vieira 31
32 Genotyping technologies SNPs Illumina infinium Single base primer extension Elisa Vieira 32
33 Infinium array for maize varietal ID Distinguish 276 hybrids. Elisa Vieira 33
34 America Seed Trade Association (ASTA) + French Maize Breeders Association (UFS): - Illumina chip with SNPs - Small set of 16 SNPs distinguished > 400 inbreds - Cost effective Elisa Vieira 34
35 Information survey in Brazil Which technology and for what? Company / Institution Crop Marker Technology Genetic Purity Variety ID Soybean and Pioneer maize SNP s Tape array X X Coodetec Soybean, maize and wheat SSR and SNP s CE in ABI genetic analyser X X Embrapa Livestock Forrage grasses SSR CE in ABI genetic analyser X Mastuda Forrage Grass Seeds Forrage grasses SSR CE 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 X Netherlands Inspection Service for Horticulture Tomato SNP s X X National Taiwan University Rice SSR CE in ABI genetic analyser, Open Array X X Monsanto - Brazil Maize SNP s TaqMan Assay X Syngenta - 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 - Brasil Soybean and eucalyptus SSR CE in ABI genetic analyser X 35
36 Final considerations: VARCOM future: Handbook of molecular techniques for genetic purity and varietal ID: SSR SNPs Arrays of SNPs for some crops?????????? Database development Elisa Vieira 36
37 Acknowledgements: VARCOM members Embrapa Livestock Matsuda Forage Grasses Seeds Pioneer Coodetec DowAgroscience SNPC Agriculture Ministery Brazil Elisa Vieira 37
38 Thank you for your attention Elisa Vieira 38
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