Rapid genome changes after polyploid formation

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Rapid genome changes after polyploid formation
online-media.uni-marburg.de/biologie/botex/ex
www.lib.ksu.edu/.../indianmustard
B. napus
B. juncea
Mercedes Ames
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Introduction
Success of polyploid species - ability to
colonize a wider range of habitats - survive in
unstable climates compared to their diploid
progenitors - increased heterozygosity and
flexibility - Genome multiplicity genetic buffer
Genome changes are accelerated in new polyploids
derived from interspecies hybrids due to
instabilities created by the interactions of
diverse genomes. Rapid genetic divergence of
newly formed polyploids Contribution to their
evolutionary success
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How polyploid genomes have evolved after their
formation?

• Studies in B. juncea, B. napus, B. carinata
  proved to be different from diploid progenitors
  B. rapa, B. nigra, and B. oleracea through RFLP
  patterns and linkage order of RFLP loci.
• These studies compared natural polyploids (100s
  to 1000s years) to present forms of hypothesized
  progenitors.
• Does not answer questions about how quickly
  newly formed polyploid genomes evolved.

Synthetic polyploids good model system to study
early events in the evolution of polyploid
genomes.

• Do extensive genome changes occur after
  polyploidization?
• How fast do these genome changes occur?
• How exactly do they happen?

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Brassica U diagram, 1935
B. nigra (L.) Koch
Is found growing as a weed in cultivated fields
in the mediterranean region, In Morocco and
semi-cultivated in Rhodes, Crete, Sicily, Turkey
and Ethiopia
B. oleracea L.
Is found in small isolated areas, truly wild
types are only found around the European Atlantic
B. rapa L. (syn. B. campestris)
Seems to have grown naturally from the West
Mediterranean region to Central Asia, maybe it
was the first domesticated.
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Rapid genome changes in synthetic polyploids of
Brassica and its implications for polyploid
evolution (Song et al, 1995)
Crosses B. rapa (A) x B. nigra (B) (AB)
B. nigra (B) x B. rapa (A) (BA) B. rapa (A)
x B. oleracea (C) (AC) B. oleracea (C) x B.
rapa (A) (CA)
Analogous to B. juncea
Analogous to B. napus
Hybrids doubled with colchicine
F2
..
F5
Compared RFLP patterns between single F2 plants
and F5 Included the parental diploid species to
verify the donor genome of fragments
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Patterns, timing and frequency of genome change
cpDNA (6 probes) mtDNA (5 probes)
All F5 plants have the same pattern as F2
progenitors and matched female diploid parents
Nuclear genome 19 anonymous, 63 cDNA, 7 genes of
known function
Accumulated changes from F2 to F5 generations
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Patterns, timing and frequency of genome change
Some F5 plants presented fragments observed in
diploid parents but not in F2 plants
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Patterns, timing and frequency of genome change
A fragment from C observed in BA plants
Some changes resulted in restriction fragments
that were pre-existing in a parent or in a
related genome
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• Frequencies of genome change
• Different between the 2 polyploid species
• Twice as many genome changes detected in AB and
  BA than in AC and CA

B. rapa genome (A) more closely related to B.
oleracea (C) than to B. nigra (B) Higher degree
of changes related to degree of divergence
Potential causes of genome changes
Genetic instabilities in new polyploids not due
to inbreeding
Processes involved

• Chromosome rearrangements
• Point mutations
• Gene conversions
• DNA methylation

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Potential causes of genome changes

• Not loss of chromosomes (except 1 F5 plant)
• Intergenomic (non-homologous) recombination
  could be a major factor contributing to genomic
  change
• In F2, F3 and F5 generations observed aberrant
  meiosis with chromosome bridges, chromosome
  lagging and multivalents
• Intergenomic chromosome associations resulting
  in loss of RFLP fragments through subsequent
  segregation of recombined or broken chromosomes.
• Small frequency of these events could result in
  gain of novel fragments due to recombination
  with the probed regions.
• Intergenomic associations could provide
  opportunity for gene-conversion like events,
  loss/gain of parental restriction fragment is
  evidence for that.

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Changes in DNA methylation?
Hpa II and Msp I
7 probes detected changes in F5 plants Only 2
seemed to be due to methylation
Methylation not a major factor
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Genetic consequences of genome change
Genome changes resulted in rapid genomic
divergence from each other and from original F2
plant Average pairwise genetic distances between
F5 plants and F2 parents 9.6 AB 8.2 BA 4.1
AC 3.7 CA Average distances among F5 plants
7.7 AB 9.4 BA 2.1 AC 2.5 CA
Phenotypic variation Fertility 0-24.9
AB/BA 0-100 AC/CA? Morphological varaition
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Directional genome change and cytoplasmic effect

• Genetic distances of F2 and F5 plants to their
  diploid parents
• AB A maternal non-significant directional
  change, B paternal significant change.
• BA A paternal significant directional change
• AC and CA non-significant directional changes
• A and C cytoplasmic genomes are more closely
  related than A and B cytoplasmic genomes.
• There are more cytoplasmic-nuclear genome
  compatibility in the AC and CA polyploids.

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Summary
Extensive changes in few generations after
polyploidization New genetic variation for
selection Contribution to successful adaptation
and diversification
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Flowering time divergence and genomic
rearrangements in resynthesized Brassica
polyploids (Brassicaceae) (Pires et al, 2004)
Life history traits variation in flowering time
and flower size are known to differ between
diploids and polyploids and to contribute to
their ecological separation
Schranz and Osborn, 2004 studied de novo life
history trait variation in early generation of
resinthesized B. napus lines and their diploid
parents in 4 different environments They found
that de novo variation and changes in phenotypic
plasticity can occur rapidly for several life
history traits
What exactly are the molecular genetic mechanisms
by which polyploidization contributes to novel
phenotypic variation?
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Flowering locus C (FLC) regulates flowering and
vernalization
Arabidopsis 1 copy At FLC
B. rapa Br FLC1 R10 Br FLC2 R2 Br
FLC3 R3 One unexpected Br FLC5 R3
B. oleracea Bo FLC1 O9 Bo
FLC3 O3 Bo FLC5 O3 Some genotypes Bo
FLC2 O2
B. napus 8 mapped 4 in B. rapa
portion 4 in B. oleracea portion
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Strategy

• Molecular genetic basis for flowering time
  variation in early and late flowering lineages
  derived from resynthesized B. napus
• Measure divergence in flowering time, and
  find patterns of rapid genome structural
  changes as well as expression patterns

Measures for flowering time
Used for reciprocal crosses
Phenotypic analysis (days of flowering when 1st
flower open)
41.9 days
54.4 days
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Analyses of Bn FLC 1
Additive patterns
Expression analysis by cDNA SSCP
Putative location of Bn FLC1 based on RFLP
No evidence that Bn FLC1 contributed to
differences in flowering time
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Analyses of Bn FLC 2
Expression analysis consistent with Southern hyb.
pw241
More transcript? Double dosage?
It can be explained by a non-reciprocal
transposition
If early flowering parent had 2 copies of BrFLC2
and late flowering parent 0 copies digenic
segregation 116 having no FLC2
Segregation analysis in F2 did not show
association of BnFLC2 with flowering time
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Analyses of Bn FLC 3
Double dose of BrFLC3
Additive pattern in late flowering
Lack of expression
Change in dosage from 22 to 31
Non-reciprocal transposition supported
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Segregation analyses of BnFLC3
Range of flowering time

• Identical results from recyprocal crosses no
  maternal effect
• Segregation ratio 121 for BrFLC3 and BoFLC3
  alleles
• Segregation of BnFLC3 associated with
  flowering time
• Plants with 2 rapa alleles early
• Plants with 2 oleracea alleles late (4 days)
• 29 of phenotypic variation for days of
  flowering explained by segregation of BnFLC3

S6 ES341
S6 ES342
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Analyses of BnFLC5
Additive pattern
Silencing
No evidence that BnFLC5 had an effect on
divergence of flowering time
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Summary
Only six generations of synthetic polyploids
allowed to create lineages with divergence in
flowering timein nature? Mechanisms structural
(chromosomal rearrangements) and expression
changes Maybe also another genetic or epigenetic
changes arising with or after polyploid
formation