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

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

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

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 F2

• To reduce effect of minor loci confounding phenotypic analysis and to increase the number of SFPs (chromosome 7 region)

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

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

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 suggesteda paracentric inversion as a possibility (CT52 & LPT4D21)

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

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

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

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

The making of a bell pepper-shaped tomato fruit: identification of loci controlling fruit morphology

van der Knapp and TanksleyTAG 2003

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

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

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/(2r)

Latitudinal sections (x and z) are 10% distance from top or bottom

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

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?

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

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 ?

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%

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