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