Presentation by CTO Eric Johnson on the various applications and features of RAD based sequencing technology to plant sciences.

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