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
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
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?
Brassica: U diagram, 1935
B. nigra (L.) Koch
B. nigra
(n=8)
BB
B. carinata
(n=17)
BBCC
B. oleracea L.
B. oleracea
(n=9)
CC
Is found in small isolated areas, truly
wild types are only found around the
European Atlantic
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. juncea
(n=18)
AABB
B. napus
(n=19)
AACC
B. rapa
(n=10)
AA
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.
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)
B. nigra (B) x B. rapa (A)
:
:
B. rapa (A) x B. oleracea (C):
B. oleracea (C) x B. rapa (A):
(AB)
(BA)
(AC)
(CA)
Analogous to B. juncea
Analogous to B. napus
Hybrids doubled with colchicine
AB
BA
AC
CA
F2
AABB
BBAA
AACC
CCAA
x
…..
F5
Compared RFLP patterns between single F2 plants and F5
Included the parental diploid species to verify the donor genome of fragments
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
Patterns, timing and frequency of genome change
Some F5 plants presented fragments observed in diploid parents but not in F2 plants
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
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
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.
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
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
Directional genome change and cytoplasmic effect
AB-A: 0.7
AB-B: 2.4
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
BA-A: 3.9
BA-B: 3.8
A
B
A and C cytoplasmic genomes are
more closely related than A and B
cytoplasmic genomes.
There are more cytoplasmicnuclear genome compatibility in
the AC and CA polyploids.
C
AC-A: 0.31
AC-C: -0.51
CA-A: 0.82
CA-C: 0.29
Summary
Extensive changes in few generations after polyploidization
New genetic variation for selection
Contribution to successful adaptation and diversification
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?
Flowering locus C (FLC): regulates flowering and vernalization
Arabidopsis: 1 copy At FLC
B. rapa:
One unexpected:
B. oleracea:
Some genotypes:
B. napus:
Br FLC1
Br FLC2
Br FLC3
Br FLC5
R10
R2
R3
R3
Bo FLC1
Bo FLC3
Bo FLC5
Bo FLC2
O9
O3
O3
O2
8 mapped
4 in B. rapa portion
4 in B. oleracea portion
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
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
Analyses of Bn FLC 2
pw241
Expression
analysis
consistent with
Southern hyb.
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 1:16 having no FLC2
Segregation analysis in F2 did not show association of BnFLC2 with flowering time
Analyses of Bn FLC 3
Double dose of BrFLC3
Additive
pattern
in late
flowering
Lack of
expression
Change in dosage from 2:2 to 3:1
Non-reciprocal transposition supported
Segregation analyses of BnFLC3
Range of flowering time
S6 ES341
S6 ES342
• Identical results from recyprocal
crosses: no maternal effect
• Segregation ratio: 1:2:1 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
Analyses of BnFLC5
Additive pattern
Silencing
No evidence that BnFLC5 had an effect on divergence of flowering time
Summary
Only six generations of synthetic polyploids allowed to create lineages
with divergence in flowering time…in nature?
Mechanisms: structural (chromosomal rearrangements) and
expression changes
Maybe also another genetic or epigenetic changes arising with or after
polyploid formation