Quantitative Trait Loci Mapping, Genome-wide Association ...
Genome Structure/Mapping
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Transcript of Genome Structure/Mapping
Genome Structure/Mapping
Genome Structure/Mapping
Lisa Malm05/April/2006
VCR 221
Lisa Malm05/April/2006
VCR 221
Genome Structure/Mapping
Genome Structure/Mapping
Characteristics of the tomato nuclear genome as determined by sequencing undermethylated EcoRI digested fragments Want et al. 2006
Development of a set of PCR-based anchor markers encompassing the tomato genome and evaluation of their usefulness for genetics and breeding experiments Frary et al. 2005
Zooming in on a quantitative trait for tomato yield using interspecific introgressions Fridman et al. 2004
Characteristics of the tomato nuclear genome as determined by sequencing undermethylated EcoRI digested fragments Want et al. 2006
Development of a set of PCR-based anchor markers encompassing the tomato genome and evaluation of their usefulness for genetics and breeding experiments Frary et al. 2005
Zooming in on a quantitative trait for tomato yield using interspecific introgressions Fridman et al. 2004
Characterisitics of the tomato nuclear genome as determined by sequencing
undermethylated EcorRI digested fragments
Characterisitics of the tomato nuclear genome as determined by sequencing
undermethylated EcorRI digested fragments
What is CpG and CpNpG methylation?
MethylcytosinePrevious studies of unmethylated
DNAFocused on monocots
Focus of Study
What is CpG and CpNpG methylation?
MethylcytosinePrevious studies of unmethylated
DNAFocused on monocots
Focus of Study
The Tomato GenomeThe Tomato Genome
950 Mb of DNA 25% in gene-rich
euchromatin @ distal ends of chromosomes
75% in gene-deficient heterochromatin
950 Mb of DNA 25% in gene-rich
euchromatin @ distal ends of chromosomes
75% in gene-deficient heterochromatin
One of the lowest G+C contents of any plant species
An estimated 23% of the cytosine residues are methylated
One of the lowest G+C contents of any plant species
An estimated 23% of the cytosine residues are methylated
Estimating the size of the unmethylated portion of the tomato
genome based on EcoRI digested fragments
Estimating the size of the unmethylated portion of the tomato
genome based on EcoRI digested fragments
Detailed analysis of coding UGIs Undermethylated portion extends 676 bp
upstream and 766 bp downstream of coding regions
59% non-coding sequences, 12% transposons, and 1% organellar sequences
Organellar sequences integrated into the nuclear genome over the past 1 million years
Accounts for majority of unmethylated genes in the genome
Estimated to constitute 61 15 Mb of DNA (~5% of the entire genome)
Indicates a significant portion of euchromatin is methylated in the intergenic spacer regions
Detailed analysis of coding UGIs Undermethylated portion extends 676 bp
upstream and 766 bp downstream of coding regions
59% non-coding sequences, 12% transposons, and 1% organellar sequences
Organellar sequences integrated into the nuclear genome over the past 1 million years
Accounts for majority of unmethylated genes in the genome
Estimated to constitute 61 15 Mb of DNA (~5% of the entire genome)
Indicates a significant portion of euchromatin is methylated in the intergenic spacer regions
Implications for sequencing the genome of tomato and other
solanaceous species
Implications for sequencing the genome of tomato and other
solanaceous species 310,000 sequence
reads estimated to cover 95% of the unmethylated tomato gene space
Solanaceous species have same basic chromosome # as tomato (n=12) Similar chromosome
structure Similar gene content
310,000 sequence reads estimated to cover 95% of the unmethylated tomato gene space
Solanaceous species have same basic chromosome # as tomato (n=12) Similar chromosome
structure Similar gene content
Assume methylation patterns also similar
Possible to apply methylation filitration sequencing to genomes of other solanaceous species Use order of tomato
sequence and synteny maps to determine derived order of UGI genes
Assume methylation patterns also similar
Possible to apply methylation filitration sequencing to genomes of other solanaceous species Use order of tomato
sequence and synteny maps to determine derived order of UGI genes
Development of a set of PCR-based anchor markers encompassing the tomato
genome and evaluation of their usefulness for genetics and breeding
experiments
Development of a set of PCR-based anchor markers encompassing the tomato
genome and evaluation of their usefulness for genetics and breeding
experiments
Genetic mapping of morphological traits in tomato began in 1917
Additional types of molecular markers Alternatives to RFLPs
Cheaper, faster, less labor intensive
Lack of PCR based map Map containing PCR-based markers would benefit
many studies
Goals of this Study
Genetic mapping of morphological traits in tomato began in 1917
Additional types of molecular markers Alternatives to RFLPs
Cheaper, faster, less labor intensive
Lack of PCR based map Map containing PCR-based markers would benefit
many studies
Goals of this Study
PCR-based anchor markers
PCR-based anchor markers
Consist of SSRs and CAPs, based on single-copy/coding regions
Encompass entire genome, placed at regular intervals, anchored in linkage map
Priority given to established polymorphism markers.
Consist of SSRs and CAPs, based on single-copy/coding regions
Encompass entire genome, placed at regular intervals, anchored in linkage map
Priority given to established polymorphism markers.
Criteria: Detection of
polymorphism Visualization of
polymorphism Placement of
markers on map Additional SSR
markers
Criteria: Detection of
polymorphism Visualization of
polymorphism Placement of
markers on map Additional SSR
markers
PCR Based Anchor Map of Tomato
PCR Based Anchor Map of Tomato
76 SSRs placed on S. lycopersicum x S. pennelli high density map
76 CAP markers also mapped152 PCR-based anchor markers
Uniformly distributedEncompass 95% of genomeLocus specific
76 SSRs placed on S. lycopersicum x S. pennelli high density map
76 CAP markers also mapped152 PCR-based anchor markers
Uniformly distributedEncompass 95% of genomeLocus specific
ApplicationsApplications
Useful for mapping in other interspecific populations
Useful resource for:qualitative and quantitative trait
mappingMarker assisted seletionGermplasm identificationGenetic diversity studies in tomato
Useful for mapping in other interspecific populations
Useful resource for:qualitative and quantitative trait
mappingMarker assisted seletionGermplasm identificationGenetic diversity studies in tomato
Zooming In on a Quantitative Trait for Tomato Yield Using Interspecific
Introgressions
Zooming In on a Quantitative Trait for Tomato Yield Using Interspecific
Introgressions Previous QTL Projects Multiple Segregating vs Single Region
Segregating QTLSingle region segregating QTL (ILs)
have higher genetic resolutionIncreased identification power for QTL
analysis
Previous QTL Projects Multiple Segregating vs Single Region
Segregating QTLSingle region segregating QTL (ILs)
have higher genetic resolutionIncreased identification power for QTL
analysis
Exploring Natural Tomato Biodiveristy
Exploring Natural Tomato Biodiveristy
Developed and examined a population of 76 segmented introgression lines
Utilized QTL database Examined total soluble content of
tomato fruit in “ketchup tomatoes” measured in refractometer brix (B) units
Developed and examined a population of 76 segmented introgression lines
Utilized QTL database Examined total soluble content of
tomato fruit in “ketchup tomatoes” measured in refractometer brix (B) units
Characterizing the QTL Brix9-2-5
Characterizing the QTL Brix9-2-5
QTL improves B w/out reducing total yield
Restricted to SNP defined region of 484 bp of cell wall invertase LIN5
3 amino acid differences, Asp366, Val373, and Asp348, are responsible for QTL effects
QTL improves B w/out reducing total yield
Restricted to SNP defined region of 484 bp of cell wall invertase LIN5
3 amino acid differences, Asp366, Val373, and Asp348, are responsible for QTL effects
LIN5 exclusively expressed in conductive tissue of flower reproductive tissues
Supports role of LIN5 as “sink gene”
LIN5 exclusively expressed in conductive tissue of flower reproductive tissues
Supports role of LIN5 as “sink gene”
Characterizing the QTL Brix9-2-5
Characterizing the QTL Brix9-2-5
Maps to middle of short arm of chromosome arm
But not present at this location in any of the 5 populations
All lines share 2 of 3 amino acids
Maps to middle of short arm of chromosome arm
But not present at this location in any of the 5 populations
All lines share 2 of 3 amino acids
Evaluated QTN SNP28378
Responsible for ASP348 substitution
Role of ASP348 and SNP28378
Evaluated QTN SNP28378
Responsible for ASP348 substitution
Role of ASP348 and SNP28378
ConclusionsConclusions
Example of the ability of a diverse IL to provide detail information on a QTL involved in increased sugar yield in tomatoes
Example of the ability of a diverse IL to provide detail information on a QTL involved in increased sugar yield in tomatoes