1. Frary et al. Advanced Backcross QTL analysis of a Lycopersicon esculentum x L. pennellii cross and identification of possible orthologs in the Solanaceae

2. LE x PN Advanced Backcross Population Development LE x PN F1 x LE BC1

3. LE x PN Advanced Backcross Population Development LE x PN F1 x LE BC1 320 BC1 plants were genotyped and selected for: ·self fertility ·Fruit weight QTL · Determinate growth 8 plants were selected to parent the BC2

4. LE x PN Advanced Backcross Population Development LE x PN F1 x LE (8) BC1 x LE (175) BC2 175 BC2 plants were genotyped at 110 polymorphic RFLPs for mapping.

5. LE x PN Advanced Backcross Population Development LE x PN F1 x LE (8) BC1 x LE (175) BC2 x TA496 (L. esculentum) (175) BC2 F1 families

6. LE x PN Advanced Backcross Population Development LE x PN F1 x LE (8) BC1 x LE (175) BC2 x TA496 (L. esculentum) (175) BC2 F1 families 30 plants from each of 175 BC2 F1 families were phenotyped for 25 fruit related traits in three locations (not all traits measured at all locations). BC2 F1 plants are basically BC3 plants… Why use a different LE for the last cross?

7. LE x PN Advanced Backcross Population Development LE x PN F1 x LE (8) BC1 x LE (175) BC2 x TA496 (L. esculentum) (175) BC2 F1 families 30 plants from each of 175 BC2 F1 families were phenotyped for 25 fruit related traits in three locations (not all traits measured at all locations). BC2 F1 plants are basically BC3 plants… Why use a different LE for the last cross? SCA with TA496 is a contributing factor in the phenotypes observed in the BC2 F1 !

8. Table 1. Putative QTL

9. Results Segregation distortion was detected due to intentional selection at BC1, and to naturally detrimental allelic combinations in the population. 26% of positive effect QTL were from PN. 45% OF QTL found colocalize with previously found QTL Significant QTL for many of the 25 traits were identified (largely from LE). Potentially valuable QTL from PN were identified for BRIX and Viscosity.

10. Questions Would you detect a recessive QTL in this population structure, how about epistatic effects? How would specific combining ability of BC2 lines with TA496 affect QTL discovered? Could observed segregation distortion be a result of sampling (8 plants) at the BC1 rather than loci affecting gamete transmission?

11. Causse et al. A Genetic map of Candidate Genes and QTLs Involved in Tomato Fruit Size and Composition.

12. Mapping Population 75 Introgression lines, each containing a single L pennellii introgression in a common L. esculentum background (M82). M82 (L. esculentum) was included as a control. The IL population of 75 plants effectively divides the L. Pennellii genome into 107 bins. ILs were characterized with RFLP markers, tying the IL population to the existing high density map of tomato, and locating L. pennellii introgression fragments.

13. Mapping Procedure A phenotypic trial consisting of six plants per line, seventy lines per block, and three blocks was conducted. Phenotypic analysis was performed on 21 ripe fruit from each line.

14. Phenotype Data Collected Fruit Weight Soluble Solids Content (brx) Reducing Sugar Content (red) Titratable acidity (ta) pH Glucose (glu) Fructose (fru) Citric acid (ca) Malic acid (ma)

15. QTL Mapping Fruit quality phenotype data were mapped as QTL. A lines mean for each trait was compared to that of the M82 control. For a QTL to be mapped to a specific bin; all ILs with the bin must have a significant change in phenotype (in the same direction) from the M82 control. 81 significant QTL were identified.

16. Candidate Gene Mapping Candidate genes related to carbon metabolism, cell cycle control, and fruit ripening were mapped in the IL population. The candidate gene map (138 loci) was overlaid on the QTL map to identify candidate genes that co localize with QTL identified. Multiple loci were mapped for several genes known to have family homologues.

17. Fig. 1 Gene and QTL Location.

18. QTL / Candidate Gene Co localization

19. Conclusion Potential candidate genes were identified for many of the significant QTL. Good gene candidates were found for fruit weight, sugar content, and acidity QTL. How would you move forward to more narrowly define a QTL and test potential candidate genes?

20. Chen and Tanksley High Resolution Mapping and Functional Analysis of se2.1: A major Stigma Exsertion Quantitative Trait Locus Associated With the Evolution From Allogamy to Autogamy in the Genus Lycopersicon.

21. Chen and Tanksley High Resolution Mapping and Functional Analysis of se2.1: A major Stigma Exsertion Quantitative Trait Locus Associated With the Evolution From Allogamy to Autogamy in the Genus Lycopersicon. Autogamy= Self fertilization Allogamy = Cross fertilization

22. Chen and Tanksley High Resolution Mapping and Functional Analysis of se2.1: A major Stigma Exsertion Quantitative Trait Locus Associated With the Evolution From Allogamy to Autogamy in the Genus Lycopersicon. Autogamy= Self fertilization Allogamy = Cross fertilization Goals: Fine mapping of se2.1 QTL in order to… 1.Ascertain the number of genes at this QTL contributing to the phenotype. 2. Characterize individual aspects of floral morphology under se2.1 control. 3. Prepare for eventual cloning of genes at se2.1.

23. Stigma exertion in Tomato The degree of stigma exertion from the anther cone influences the rate at which a plant will out cross Vs. self. Great variation for this trait exists in tomato and its wild relatives. A major QTL for stigma exertion (se2.1) has been previously identified on chromosome 2.

24. Stigma Exsertion Population An Existing Near Isogenic Line (IL2-5) with a chromosome 2 introgression fragment from L. pennellii (LA716) in an L. esculentum (M82-1-8) background were selected. IL2-5 was crossed to M82-1-8, and the F1 was self-fertilized to produce F2 seed that would segregate at the chromosome 2 introgression. These F2 lines vary in stigma exsertion, as LA716 has a highly exserted stigma and the L. esculentum parent does not.

25. Stigma Exsertion Population 1535 F2 plants were screened with markers flanking the se2.1 QTL. 123 recombinant F2 plants were identified in the screen. Selected F2 plants were selfed and F3 progeny screened to identify 3 individuals from each family homozygus for the recombination event. F4 seed was generated from each selected F3 individual for later phenotypic evaluations.

26. Stigma Exsertion Population Diagram of Chromosome 2 IL2-5 x M82-1-8 F1 (X) 1535 F2 plants screened with Markers flanking se2.1. 123 recombinant plants identified and selfed 3 Homozygous recombinant F3 plants were selected from each of the 123 F2 families. TG469 TG167 123 Selected F2 (X) Homozygous recombinant F3 families

27. Fine-Mapping the se2.1 Region Homozygus recombinant F3 plants were genotyped with the 40 available markers covering the se2.1 interval. Recombination points were identified for each family allowing for the development of a high resolution map of the se2.1 region.

28. Phenotypic Analysis Homozygous F4 progeny from the 123 recombinant families were grown along with parental controls in a randomized block design. Flower traits were evaluated by measuring 10 flowers from each plant. Stigma exsertion = stamen length – style length + ovary length. Least-squares mean of each trait was calculated for each family.

29. Floral Traits Tied to the se2.1 Region Stigma exsertion, due largely to style length and to a lesser degree to stamen length. Anther dehiscence (occurs in L. pennellii).

30. Recombinant Groups Recombinant families were grouped in to 27 bins spanning se2.1 based on marker genotypes. Family phenotype values were compared within and between groups to further dissect the QTL region.

31. Fig. 2. Fine Mapping se2.1

32. Dissection of se2.1 Four loci influencing stamen length (three closely linked) were identified. A single locus for style length was identified (the long style allele was shown to be dominant using a sub-NIL line in the interval of interest). A single locus for anther dehiscence was also found in the region.

33. Fig. 3 A. Fine mapping loci contributing to the se2.1 QTL

34. Five closely linked genes all contribute to stigma exsertion phenotype. The author suggests that this may be the remnant of a “co adapted gene complex” where linked genes cooperatively contribute to an adaptation, and are inherited as a single unit. How would you theorize such a gene cluster coming about?