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