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 (n=8) BB B. carinata (n=17) BBCC 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. 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. 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.

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 AB BA AC CA 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 B A C AC-A: 0.31 AC-C: -0.51 CA-A: 0.82 CA-C: 0.29 AB-A: 0.7 AB-B: 2.4 BA-A: 3.9 BA-B: 3.8 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.

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: 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

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 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 1:16 having no FLC2 Segregation analysis in F2 did not show association of BnFLC2 with flowering time

Analyses of Bn FLC 3 Non-reciprocal transposition supported 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 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 S6 ES341 S6 ES342

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