Potato Genomics In Fredericton Dr. Barry Flinn Co-Lead Investigator - Genome Atlantic CPGP Research Director - Solanum Genomics International Inc.

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)

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?

Genome Organization Leaf Tuber Chromosome DNA

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

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

cDNA Collections (Libraries) The messages are “copied” to form double- stranded DNA copies (cDNA) of each message Leaf cDNATuber cDNA 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

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

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

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; 2 o bacterial and fungi often infect through late blight lesions

Late Blight Project Collaborative effort with AAFC 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

Bioinformatics Intranet Website Database Analysis Tools

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

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

Database

Database - Sequence Info

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

Microarray Analysis - Processing Image Processing Data Normalization Differential Gene Expression Cluster Analysis Pathway Analysis Analysis

Microarray Analysis - Processing

Signal Background Microarray Analysis - Processing

Irregular size or shape Irregular placement Low intensity Saturation Spot variance Background variance indistinguishablesaturated bad print artifactmiss alignment 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 Microarray Analysis - Processing

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

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

Microarray Analysis - Data Normalization ( LOWESS )

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 Microarray Analysis - Data Normalization

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

Expression Profile Clustering

Project Database Engine Microarray Analysis - Data Management

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

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

Late Blight Project cDNA Microarray Using SGII Clones Top 5 Expression Profiles Clone ID Ratio Of BLAST Homology Resistant/Susceptible Expression Pathogenesis-related protein PR Osmotin-like protein Hypothetical protein Unknown Class II chitinase RT-PCR Using PR1-1 Primers MW S R

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

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

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