Potato Genomics In Fredericton Dr. Barry Flinn Co-Lead
Investigator - Genome Atlantic CPGP Research Director - Solanum
Genomics International Inc.
Slide 2
Economic Importance Of The Potato Integral part of the diet of
a large proportion of the worlds 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)
Slide 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?
cDNA Collections (Libraries) Various tissues are collected from
the plant, and messages are extracted from each of these Leaf
Messages Tuber Messages
Slide 7
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
Slide 8
cDNA Collections (Libraries) The cDNAs are then read or
sequenced, to give the order of As, Cs, Gs or Ts 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
Slide 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
Slide 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; 2 o bacterial and fungi often infect
through late blight lesions
Slide 11
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
Database Contains all the ESTs sequences Contains useful
annotations Blast Searches Contig Assemblies Transmembrane Spanning
Regions Gel Pictures EST Information
Slide 15
Database
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Database - Sequence Info
Slide 17
Slide 18
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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
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
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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
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Assumptions Overall mean average ratio should be 1 Most genes
are not differentially expressed Total intensity of dyes are
equivalent Microarray Analysis - Data Normalization
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Microarray Analysis - Data Normalization ( LOWESS )
Slide 31
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
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Cluster genes based on expression profiles Gene expression
across several treatments Hypothesis: Genes with similar function
have similar expression profiles Microarray Analysis
-Clustering
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Expression Profile Clustering
Slide 34
Project Database Engine Microarray Analysis - Data
Management
Slide 35
Late Blight Project cDNA Microarray Using SGII Clones
hybridized with Cy3 (resistant) + Cy5 (susceptible) probes
(reciprocal labelling experiments)
Slide 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
Slide 37
Late Blight Project cDNA Microarray Using SGII Clones Top 5
Expression Profiles Clone ID Ratio Of BLAST Homology
Resistant/Susceptible Expression 384 21.8Pathogenesis-related
protein PR-1 1256 19.9Osmotin-like protein 857 11.3Hypothetical
protein 922 10.0Unknown 2345 8.1Class II chitinase RT-PCR Using
PR1-1 Primers MW S R
Slide 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
Slide 39
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
Slide 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
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