Rapid genome changes after polyploid formation
www.lib.ksu.edu/.../indianmustard online-media.uni-marburg.de/biologie/botex/ex
B. napusB. 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 (n=8) BB
B. carinata(n=17)BBCC
B. juncea (n=18)AABB
B. oleracea(n=9)CC
B. napus(n=19)AACC
B. rapa(n=10)
AA
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.
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
ABBAACCA
AABBBBAAAACCCCAA
x ….. F5
Compared RFLP patterns between single F2 plants and F5Included the parental diploid species to verify the donor genome of fragments
F2
Rapid genome changes in synthetic polyploids of Brassica and its implications for polyploid evolution (Song et al, 1995)
Patterns, timing and frequency of genome change
cpDNA (6 probes)mtDNA (5 probes)
All F5 plants have the same pattern as F2 progenitorsand matched female diploid parents
Nuclear genome: 19 anonymous, 63 cDNA, 7 genes of known function
Accumulated changes from F2 to F5 generations
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
Patterns, timing and frequency of genome change
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
• 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.
Potential causes of genome changes
Changes in DNA methylation?
Hpa II and Msp I
7 probes detected changes in F5 plantsOnly 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% AB8.2% BA4.1% AC3.7% CA
Average distances among F5 plants: 7.7% AB9.4% BA2.1% AC2.5% CA
Phenotypic variationFertility: 0-24.9 % AB/BA
0-100% AC/CA?Morphological varaition
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.
B
A
C
AC-A: 0.31AC-C: -0.51
CA-A: 0.82CA-C: 0.29
AB-A: 0.7AB-B: 2.4
BA-A: 3.9BA-B: 3.8
Summary
Extensive changes in few generations after polyploidization
New genetic variation for selection
Contribution to successful adaptation and diversification
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
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
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. oleracea: Bo FLC1 O9Bo FLC3 O3Bo FLC5 O3
Some genotypes: Bo FLC2 O2
B. rapa: Br FLC1 R10Br FLC2 R2Br FLC3 R3
One unexpected: Br FLC5 R3
B. napus: 8 mapped 4 in B. rapa portion4 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
Phenotypic analysis (days of flowering when 1st flower open)
Used for reciprocal crosses
41.9 days 54.4 days
Analyses of Bn FLC 1
Additive patterns
Expression analysis by cDNA SSCP
Putative location of Bn FLC1based on RFLP
No evidence that Bn FLC1 contributed to differences in flowering time
Analyses of Bn FLC 2
More transcript?Double dosage?
Expression analysis consistent with Southern hyb.pw241
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
It can be explained by a non-reciprocal transposition
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
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