1. High-Resolution Fine Mapping and Fluorescence in situ Hybridization Analysis of sun, a Locus Controlling Tomato Fruit Shape, Reveals a Region of the Tomato Genome Prone to DNA Rearrangements Knaap, Sanyal, Jackson, Tanksley Genetics 2004

2. Before we start…..Chromosomal Rearrangements Remarks Between tomato and potato 5 major inversions with chromosomes 5, 9, 10, 11, 12 Between tomato and eggplant 28 rearrangements Between Capsicum and the rest 30 breaks as part of 5 translocations, 10 paracentric inversions, 2 pericentric inversions, and 4 disassociations or associations of genomic regions that differentiate tomato, potato, and pepper Within the genus of Solanum/Lycopersicon Few rearrangements

3. Sun locus implicated in fruit morphology Sun locus Short arm chromosome 7 Controls fruit morphology Alleles WT=round shape, Cultivated=oval shape (Roma) Cloning of genes in fruit morphology Reveal molecular basis of tomato domestication Elucidation of developmental pathways Improvement of fruit quality Mapping populations created for fine mapping purposes

4. Mapping Populations for sun Mapping in tomato Typically with Introgression Lines Nucleotide polymorphisms should be high between two parents Two mapping populations EPM= L. esculentum Sun1642 x L. pimpinellifolium LA1589 These two lines are inbred EPN= L. esculentum Sun1642 x L. esculentum IL7-4 IL7-4 has segment of chromosome 7 of L. pennellii LA716 EPN is nearly isogenic in F 2 To reduce effect of minor loci confounding phenotypic analysis and to increase the number of SFPs (chromosome 7 region)

5. First Round Screening Line thickness denotes alleles derived from either parent =selfing of plant Sun initially mapped in the 100 F2 population High resolution was performed on the F3s Recombinants ID’d and analyzed for precise location of sun

6. A: low resolution map (EPM) B: high resolution map (EPN) C: high resolution map (EPM) Numbers above indicate cM distances, numbers below are number of plants Brackets represent genomic clones

7. Mapping with EPM and EPN Populations Work in 2001 mapped sun to this area EPM population mapped to this area with the 100 F2s EPN population high-res mapping nearly isogenic to reduce effects of minor loci 3509 EPN F2s resulted in 25 recombination events within GP121 and TG576 Lp12L2-F used to screen genomic libraries Resulted in 8 large genomic clones Drop in recombination frequency in EPM suggested a paracentric inversion as a possibility (CT52 & LPT4D21)

8. Fluorescent in situ hybridization, FISH Analysis of Sun Pachytene FISH and Fiber FISH High resolution mapping of physical distances on the chromosome Results indicated that clones mapping near the telomere in the EPN population mapped well below the telomere in EPM population PCR using “telomeric primers” TGR-1 showed that EPN contain subtelomeric repeat TGR-1. EPM lacks this repeat Results support a paracentric inversion hypothesis

9. Mapping in the EPM Lack of recombination events in EPN population made further mapping unsuccessful Proceeded to map in the EPM population Still needed to minimize minor loci effects Use a large F3 population 1320 plants screened 234 recombinants identified between Le76E24 & GP121

10. EPM Mapping continued F4 families analyzed for variation in fruit size Overlap in fruit shape indicates presence of minor loci &/or environmental effects Progeny testing showed sun to be flanked here Unfortunately no genomic clones available for this region

11. Concluding Remarks Sun locus is ~30kb larger in L. esculentum Sun1642 compared to L. pimpinellifolium LA1589 Allelic variation due to insertion/deletion in this region? Gene duplication/deletion responsible for dosage effects? Mapping resolved sun to a 68 kb region Region appears to be prone to rearrangements Breakpoint, inversion, deletion/insertion 15X theoretical coverage Reason for missing genomic clones Intrinsic cloning inefficiency, instability of certain fragments Future work is using phage genomic libraries to clone sun

12. The making of a bell pepper-shaped tomato fruit: identification of loci controlling fruit morphology van der Knapp and Tanksley TAG 2003

13. Overview Significant variation in Lycopersicon esculentum Fruit shape: round, elongated, pear, hear Fruit size: grams to 1000 grams Previous crosses of L. esculentum x wild L. esculentum spp 15 mapping populations—ID’d QTL controlling shape and size (Grandillo et al 1999) Fruit was round and slightly elongated and medium Sought to map the more extreme bell shape phenotype Relate map positions of some morphology QTL to pepper and eggplant

14. Phenotypic Analysis L. esculentum cv Yellow Stuffer x L. pimpinellifolium LA1589 200 F2s, 5 of each parent (F1s) planted in randomized design Measurements Minimum of 20 fruit per plant Bell shaped scored visually 1 (round)- 5 (bell) Fruit mass average of 20 fruit Total seed weight average of 20 fruit Locule number Flower number on three inflorescences per plant Digital Images

15. Digital Imaging and Measurements Stem-end blockiness x/y Blossom-end blockiness y/z Heart shape x/z Elongated shape w/y Fruit bumpiness 10*c/(2  r) Latitudinal sections (x and z) are 10% distance from top or bottom

16. Markers and Statistical Analyses 96 RFLP makers obtain across 12 chromosomes Spanned 1076 cM, average map distance 13 cM Skewing of alleles for Chromosome 2, 7, 9, 11 Commonly observed in populations derived from interspecific crosses Self-compatibility, gametophytic &/or hybrid viability QTL mapping with software, QGENE EE homozygous Yellow Stuffer, PP homozygous LA1589 Additivity A= (EE-PP)/2 Degree of dominance D/A D= EP – (EE+PP)/2

17. Frequency Histograms Fruit size and shape distributed continuously Skewed toward wild parent (LA1589) Phenotypes controlled by several loci Wild type alleles confer semi-dominancy Bell-shape and size correlated r=0.48, p < 0.001 Common QTL controlling both?

18. Various Correlations Fruit size & stem end blockiness, r=0.66 Fruit size & heart shape, r=0.65 Fruit size & seeds per fruit, r=0.63 Bell shape & stem end blockiness, r=0.60 Bell shape & bumpiness, r=0.42 Stem end blockiness and bumpiness, not significant

19. QTL Analyses 10 QTL for bell shape and fruit size 40 QTL for potential components of shape and size Regions affecting bell shape and size also affected one or more components of fruit morphology Close linkage or pleiotropic effects ?

20. Simultaneous Fitting of QTL Explain Phenotypic Variation Bell Shape: bell2.1, 2.2, 8.1 =30% Fruit Size: fw1.2, 1.2, 3.2, 5.2, 6.2, 7.2 =46% 5.2 was only novel QTL Stem end blockiness: sblk1.1, 2.1, 3.1, 7.1, 8.1, 12.1 =34% Heart shape: hrt1.1, 2.1, 3.1, 7.1 =each 5-9% Elongated shape: fs 6.2, 9.2 = each 9 % Bumpiness: bpi 8.1, 9.1, 11.1 = each -9% to 9% Seed number: (10QTL) =36% 5 novel QTL Locule number: lcn2.1 =30% Flowers per inflorescence: (9QTL)= each 6-19%

21. Remarks Most loci controlling shape and size have already been identified Three previously reported QTL for tomato shape and size Allelic in Yellow Stuffer Previous esculentum x pimpinellifolium had different major QTLs controlling fruit size Fw2.2 vs. fw1.1, 3.2 Explained by differences in genetic background Multiple alleles per locus Coinciding QTL between Yellow Stuffer, bell pepper, eggplant Selection pressures lead to mutations in at similar loci