Transcript Slide 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
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 suggested
a 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 l genomic libraries to clone sun
The making of a bell pepper-shaped tomato fruit:
identification of loci controlling fruit morphology
van der Knapp and Tanksley
TAG 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/(2pr)
Latitudinal sections (x and z) are 10% distance from top or bottom
Markers and Statistical Analyses
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96 RFLP makers obtain across 12 chromosomes
• Spanned 1076 cM, average map distance 13 cM
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Skewing of alleles for
• Chromosome 2, 7, 9, 11
• Commonly observed in populations derived from interspecific
crosses
• Self-compatibility, gametophytic &/or hybrid viability
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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
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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