ISHS - Molecular Markers In Horticulture Presentation
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Presentation by CTO Eric Johnson on the various applications and features of RAD based sequencing technology to plant sciences.
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ISHS - Molecular Markers In Horticulture Presentation
- 1. Sequenced RAD Markers: A SNP Discovery and Genotyping Platform July 30th, 2009 Floragenex Presenter: Dr. Eric Johnson, Chief Technology Officer © Floragenex, Inc. 2009 www.floragenex.com
- 2. Applications for Sequenced RAD markers • SNP discovery – scalable discovery of markers in any genome • SNP discovery with RAD LongRead markers – identify SNPs and develop flanking sequence • Trait mapping – find linked markers by bulk segregant analysis • Genetic maps – SNP discovery and genotyping of a population © Floragenex, Inc. 2009 www.floragenex.com
- 3. Floragenex Restriction-site Associated DNA (RAD) Technology © Floragenex, Inc. 2009 www.floragenex.com
- 4. RAD Markers Identify a Variety of Polymorphisms Sequences with a single change A B 18 00 CCGGCGAGCACGGCGACAAGTCCGGCGACCACAAGGAAAAGAAGGACAA 38G>A 00 19 CCGGCGAGCACGGCGACAAGTCCGGCGACCACAAGGAGAAGAAGGACAA Sequences with complex changes A B 19 00 CCGGCGCAAGGCCTTCTACTGATCTGGTACGCTATCTCTTAGCATGTCC 20C>T 48A>C 00 17 CCGGCGCAAGGCCTTCTACCGATCTGGTACGCTATCTCTTAGCATGTAC 00 29 CCGGCGCGATCTGCAGGGGCGCCGACGACATTATTCCTTCGGCTCGCCG 33G>A 34+TTC 44 00 CCGGCGCGATCTGCAGGGGCGCCGACGACATTG---CTTCGGCTCGCCGTGG Dominant markers “snip-SNPs” A B 00 42 CCGGCGAAACACGGGGCCACGGCGCAGGCGTTCTCCATCTCCAAAGAAC © Floragenex, Inc. 2009 www.floragenex.com
- 5. Applications for Sequenced RAD markers • SNP discovery – scalable discovery of markers in any genome • SNP discovery with RAD LongRead markers – identify SNPs and develop flanking sequence • Trait mapping – find linked markers by bulk segregant analysis • Genetic maps – SNP discovery and genotyping of a populatio © Floragenex, Inc. 2009 www.floragenex.com
- 6. Paired-end Sequencing for RAD LongReads © Floragenex, Inc. 2009 www.floragenex.com
- 7. RAD LongRead contigs range in size RAD LongRead contig lengths 1500 1125 # of contigs 750 375 0 100 200 300 400 500 600 700 800 900 Contig length © Floragenex, Inc. 2009 www.floragenex.com
- 8. RAD LongReads for Genotyping Assay Development Goal • Identify testcross segregating alleles for translation into Sequenom iPlex assay format RAD LongRead sequencing results • ~43,000 contigs • 9,903 SNPs identified • 1,005 InDels identified Validation test • 280 SNPs were converted into a Sequenom assay (120 bp free of other polymorphisms) Results 1) Nearly 100% conversion rate into a functional assay 2) ~85% of identified SNPs segregated as expected and mapped to LGs © Floragenex, Inc. 2009 www.floragenex.com
- 9. Applications for Sequenced RAD markers • SNP discovery – scalable discovery of markers in any genome • SNP discovery with RAD LongRead markers – identify SNPs and develop flanking sequence • Trait mapping – find linked markers by bulk segregant analysis • Genetic maps – SNP discovery and genotyping of a population © Floragenex, Inc. 2009 www.floragenex.com
- 10. Trait mapping with bulked populations © Floragenex, Inc. 2009 www.floragenex.com
- 11. Applications for Sequenced RAD markers • SNP discovery – scalable discovery of markers in any genome • SNP discovery with RAD LongRead markers – identify SNPs and develop flanking sequence • Trait mapping – find linked markers by bulk segregant analysis • Genetic maps – SNP discovery and genotyping of a population © Floragenex, Inc. 2009 www.floragenex.com
- 12. RAD-based Physical and Genetic Maps can optimize Whole Genome Assembly © Floragenex, Inc. 2009 www.floragenex.com
- 13. RAD Summary RAD markers focus high-depth sequencing on a fraction of the genome • de novo marker discovery • marker density can scale with project goals RAD LongRead markers • develop long contigs of 200-800 bp • good for porting SNPs to other genotyping platforms • discriminates between homeologous genomes Trait mapping • Rapidly identify Mendelian alleles Genetic mapping and physical mapping • RAD marker-based maps can tie together for genome assembly © Floragenex, Inc. 2009 www.floragenex.com
- 14. Questions? Floragenex Plant Genomics Team: Eric Johnson, Chief Technical Officer eric@floragenex.com Rick Nipper, VP of Research rick@floragenex.com Office Phone 541.343.0747 © Floragenex, Inc. 2009 www.floragenex.com