1. Genome Structure/Mapping Lisa Malm 05/April/2006 VCR 221 Lisa Malm 05/April/2006 VCR 221

2. 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

3. Characterisitics of the tomato nuclear genome as determined by sequencing undermethylated EcorRI digested fragments  What is CpG and CpNpG methylation?  Methylcytosine  Previous studies of unmethylated DNA  Focused on monocots  Focus of Study  What is CpG and CpNpG methylation?  Methylcytosine  Previous studies of unmethylated DNA  Focused on monocots  Focus of Study

4. The 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

5. 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

6. 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

7. 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

8. 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

9. PCR Based Anchor Map of Tomato  76 SSRs placed on S. lycopersicum x S. pennelli high density map  76 CAP markers also mapped  152 PCR-based anchor markers  Uniformly distributed  Encompass 95% of genome  Locus specific  76 SSRs placed on S. lycopersicum x S. pennelli high density map  76 CAP markers also mapped  152 PCR-based anchor markers  Uniformly distributed  Encompass 95% of genome  Locus specific

10. Applications  Useful for mapping in other interspecific populations  Useful resource for:  qualitative and quantitative trait mapping  Marker assisted seletion  Germplasm identification  Genetic diversity studies in tomato  Useful for mapping in other interspecific populations  Useful resource for:  qualitative and quantitative trait mapping  Marker assisted seletion  Germplasm identification  Genetic diversity studies in tomato

11. Zooming In on a Quantitative Trait for Tomato Yield Using Interspecific Introgressions  Previous QTL Projects  Multiple Segregating vs Single Region Segregating QTL  Single region segregating QTL (ILs) have higher genetic resolution  Increased identification power for QTL analysis  Previous QTL Projects  Multiple Segregating vs Single Region Segregating QTL  Single region segregating QTL (ILs) have higher genetic resolution  Increased identification power for QTL analysis

12. 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

13. 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, Asp 366, Val 373, and Asp 348, 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, Asp 366, Val 373, and Asp 348, 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”

14. 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 SNP 28378  Responsible for ASP 348 substitution  Role of ASP 348 and SNP 28378  Evaluated QTN SNP 28378  Responsible for ASP 348 substitution  Role of ASP 348 and SNP 28378

15. Conclusions  Example of the ability of a diverse IL to provide detail information on a QTL involved in increased sugar yield in tomatoes