Economic Importance Of The Potato

1. Potato Genomics In FrederictonDr. Barry FlinnCo-Lead Investigator - Genome Atlantic CPGPResearch Director - Solanum Genomics International Inc.

2. Economic Importance Of The Potato • Integral part of the diet of a large • proportion of the world’s population • Supplies at least 12 essential vitamins • and minerals • Still much unknown regarding the • control of potato development and • processing/quality traits • (ie. disease resistance, stress tolerance, carbohydrate metabolism, tuber shape)

3. What Does Genomics Mean? • “Genomics” is a science that studies the genetic material • of a species at the molecular level • A scientific approach that seeks to identify and define • the function of genes, as well as uncover when and how • genes work together to produce traits • “Structural Genomics” approaches (mapping) generally • focus on traits controlled by one or a few genes, and • often only provide information regarding the location • of a gene or genes • We can examine the interrelationships and interactions • between thousands of genes How do we do this?

4. Chromosome DNA Leaf Tuber Genome Organization

5. DNA Gene 1Gene 2 Etc. ....TATACAGCAAAATAGAAAGATCTAGTGTCCCATGGCGATGAGTCGTGTAGCTTCT…. Genome Organization Promoter “Switch” Coding ORF “Message”

6. Leaf Messages Tuber Messages cDNA Collections (Libraries) • Various tissues are collected from the plant, • and messages are extracted from each of these

7. Leaf cDNA Tuber cDNA cDNA Collections (Libraries) • The messages are “copied” to form double- • stranded DNA copies (cDNA) of each message • Each copy is “glued” into a piece of bacterial DNA • for easier storage, handling and propagation, resulting • in a collection or “library” of cDNAs for each tissue

8. cDNA Collections (Libraries) • The cDNAs are then read or “sequenced”, to give the • order of A’s, C’s, G’s or T’s for each • We are left with the sequence of each gene that is • active (expressed) in each cell, tissue or organ studies • These are “Expressed Sequence Tags” or ESTs • Using complex computer resources, these ESTs can • be analyzed and compared with known sequences • and proteins • Look for messages associated with specific organs or • characteristic/traits

9. Take Home Points • Messages from various genes are important, as they • dictate which proteins are produced • Promoters are also important, as they dictate where • a specific message and protein is produced • “Genomics” involves the study of all of the • messages produced by the various plant cells • A lot of information which must be organized • and analyzed

10. Project Description Identification Of A Differential Gene Expression Pattern And Genes Related To Resistance In Potato Late Blight • One of the most devastating disease of potato worldwide • If left unmanaged, complete destruction of crops can occur • Attacks leaves and tubers; large necrotic lesions on leaves • and dry rot that spreads through tubers; 2o bacterial and • fungi often infect through late blight lesions

11. Late Blight Project • Collaborative effort withAAFC Potato Research Centre • Population of blight-sensitive and blight-resistant plants • of near isogenicity • cDNA libraries made from leaves of a blight-sensitive and • a blight resistant plant • 2500 messages were sequenced from each library • (5000 total ESTs) • Different ESTs to be profiled for expression • The tremendous amounts of data generated will need to be • managed efficiently

12. Bioinformatics • Intranet Website • Database • Analysis Tools

13. Links (IBM Patent, NCBI, PubMed, etc) Blast Search (on site) Sequence Manipulation Suite ClustalW Modifying Sequences Database Access SGII Intranet Website

14. Database • Contains all the EST’s sequences • Contains useful annotations • Blast Searches • Contig Assemblies • Transmembrane Spanning Regions • Gel Pictures • EST Information

15. Database

16. Database - Sequence Info

17. Database - Sequence Info

18. Database - Sequence Info

19. Data Analysis • Tens of thousands of ESTs available for study • Most methods to study message distributions are • low throughput AND time consuming • “Genomics” necessitates the large scale study of • gene expression How can we do this? Microarray Analysis

20. Microarray Analysis

21. Microarray Analysis

22. Microarray Analysis

23. Microarray Analysis - Processing ImageProcessing Data Normalization Analysis Differential Gene Expression Cluster Analysis Pathway Analysis

24. Microarray Analysis - Processing

25. Microarray Analysis - Processing Signal Background

26. Microarray Analysis - Processing • Irregular size or shape • Irregular placement • Low intensity • Saturation • Spot variance • Background variance miss alignment artifact bad print indistinguishable saturated

27. Microarray Analysis - Processing • Calculate numeric characteristics of each spot • Throw out spots that do not meet minimum requirements for each characteristic • Throw out spots that do not have minimum overall combined quality

28. Microarray Analysis - Data Normalization • Normalize data to correct for variances • Dye bias • Location bias • Intensity bias • Pin bias • Slide bias • Control vs. non-control spots

29. Microarray Analysis - Data Normalization • Assumptions • Overall mean average ratio should be 1 • Most genes are not differentially expressed • Total intensity of dyes are equivalent

30. Microarray Analysis - Data Normalization (LOWESS)

31. Microarray Analysis - Data Normalization Differential Gene Expression: • n-fold change • n typically >/= 2 • May hold no biological relevance • Often too restrictive • 2 expression • Calculate standard deviation  • Genes with expression more than 2 away are differentially expressed

32. Microarray Analysis -Clustering • Cluster genes based on expression profiles • Gene expression across several treatments • Hypothesis: Genes with similar function have similar expression profiles

33. Expression Profile Clustering

34. Microarray Analysis - Data Management Project Database Engine

35. Late Blight Project • cDNA Microarray Using SGII Clones • hybridized with Cy3 (resistant) + Cy5 (susceptible) probes • (reciprocal labelling experiments)

36. Late Blight Project • cDNA Microarray Using SGII Clones • hybridized with Cy3 (resistant) + Cy5 (susceptible) probes • (reciprocal labelling experiments) ANDLBRLF02345HTF.01 - Class II chitinase ANDLBRLF01256HTF.01 - Pathogenesis-related protein P23 precursor ANDLBRLF02041HTF.01 - Unknown protein

37. Top 5 Expression Profiles Clone ID Ratio Of BLAST Homology Resistant/Susceptible Expression 384 21.8 Pathogenesis-related protein PR-1 1256 19.9 Osmotin-like protein 857 11.3 Hypothetical protein 922 10.0 Unknown 2345 8.1 Class II chitinase Late Blight Project cDNA Microarray Using SGII Clones RT-PCR Using PR1-1 Primers MW S R

38. What Use Is All Of This Information? • Transgenics: • - Enhance tuber quality, processing traits, disease • resistance, stress tolerance more rapidly than breeding • Expression Assisted Selection: • - Obtain expression profiles for thousands of genes • associated with specific traits or characteristics • - Use these profiles as a baseline to compare with • the expression profiles of unknown clones; crosses • New Protein Products : • - Identify genes encoding secreted proteins/ligands • - Test these for growth-promoting/other effects • - Express genes in batch cultures and purify proteins

39. Example Of Gene Use GA-20 oxidase in potato: GFP expression in tobacco cells • GA-20 oxidase • knockouts with • enhanced tuber • production • GA-20 oxidase • knockouts with • reduced tuber • sprouting

40. Information Processing and Handling • Assembly and annotation of genomic data • EST analysis and databases • Cluster analysis of microarray data • Comparisons of various transcriptomic methods • Integration of sequence, transcriptomic, proteomic, • metabolomic, transgenic data