Frary et al.
Advanced Backcross QTL analysis of a Lycopersicon
esculentum x L. pennellii cross and identification of possible
orthologs in the Solanaceae
LE x PN Advanced Backcross
Population Development
LE x PN
F1 x LE
BC1
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
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.
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
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?
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 !
Table 1. Putative QTL
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.
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?
Causse et al.
A Genetic map of Candidate Genes and QTLs Involved in
Tomato Fruit Size and Composition.
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.
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.
Phenotype Data Collected
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Fruit Weight
Soluble Solids Content (brx)
Reducing Sugar Content (red)
Titratable acidity (ta)
pH
Glucose (glu)
Fructose (fru)
Citric acid (ca)
Malic acid (ma)
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.
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.
Fig. 1
Gene and QTL Location.
QTL / Candidate Gene Co
localization
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?
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.
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
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.
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.
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.
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.
Stigma Exsertion Population
Diagram of Chromosome 2
IL2-5
x M82-1-8
F1 (X)
TG469
TG167
1535 F2 plants screened with
Markers flanking se2.1.
123 recombinant plants identified
and selfed
123 Selected F2 (X)
3 Homozygous recombinant F3 plants
were selected from each of the 123
F2 families.
Homozygous recombinant F3 families
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.
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.
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).
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.
Fig. 2. Fine Mapping se2.1
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.
Fig. 3 A. Fine mapping loci
contributing to the se2.1 QTL
• 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?