Reducing MSH4 copy number prevents meiotic crossovers between non-homologous chromosomes in Brassica napus

In allopolyploids, correct chromosome segregation requires suppression of non-homologous crossovers while levels of homologous crossovers are ensured. To date, no mechanism able to specifically inhibit non-homologous crossovers has been described in allopolyploids other than in bread wheat. Here, we show that reducing the number of functional copies of MSH4, an essential gene for the main crossover pathway, prevents non-homologous crossovers in allotetraploid Brassica napus. We show that non-homologous crossovers originate almost exclusively from the MSH4-dependent recombination pathway and that their numbers decrease when MSH4 returns to single copy in B. napus; by contrast, homologous crossovers remain unaffected by MSH4 duplicate loss. We also demonstrate that MSH4 systematically returns to single copy following numerous independent polyploidy events, a pattern that is probably not by chance. These results suggest that stabilization of allopolyploid meiosis can be enhanced by loss of a key meiotic recombination gene.


Supplementary Figure 1: Phylogeny of MSH5 in angiosperms
A Maximum Likelihood tree based on amino acid sequences is provided. For the sake of clarity, species names are indicated instead of gene names. Branch support is given as Shimodaira-Hasegawa-like Likelihood Ratio Test (aLRT SH-like). Coloured disks superimposed along the branches of the trees give the age range for past WGDs. Fulllength duplicates and recent duplicated with one fractionated copies are written with the color that corresponds to the age of the WGD (i.e. red: <10,000 years; orange: <20MY; light blue: 50MY< < 85MY).

Supplementary Figure 2: Phylogeny of ZIP4 in angiosperms
A Maximum Likelihood tree based on amino acid sequences is provided. For the sake of clarity, species names are indicated instead of gene names. Branch support is given as Shimodaira-Hasegawa-like Likelihood Ratio Test (aLRT SH-like). Coloured disks superimposed along the branches of the trees give the age range for past WGDs. The extra copy of ZIP4 found in bread wheat (T.aestivum Ph1) is indicated in black because it originated from a Small Scale Duplications. Full-length duplicates and recent duplicated with one fractionated copies are written with the color that corresponds to the age of the WGD (i.e. red: <10,000 years; orange: <20MY; light green: 25MY< < 50MY). The connector superimposed over the phylogeny associates the full-length copy of ZIP4 with a fractionated copy that originated from the same specific WGD (e.g. in Glycine max).

Supplementary Figure 3: Phylogeny of MER3 in angiosperms
A Maximum Likelihood tree based on amino acid sequences is provided. For the sake of clarity, species names are indicated instead of gene names. Branch support is given as Shimodaira-Hasegawa-like Likelihood Ratio Test (aLRT SH-like). Coloured disks superimposed along the branches of the trees give the age range for past WGDs. Fulllength duplicates are written with the color that corresponds to the age of the WGD (i.e. red: <10,000 years; orange: <20MY for L. usitatissimum).

Supplementary Figure 4: Phylogeny of SHOC1 in angiosperms
A Maximum Likelihood tree based on amino acid sequences is provided. For the sake of clarity, species names are indicated instead of gene names. Branch support is given as Shimodaira-Hasegawa-like Likelihood Ratio Test (aLRT SH-like). Coloured disks superimposed along the branches of the trees give the age range for past WGDs. Fulllength duplicates are written with the color that corresponds to the age of the WGD (i.e. red: <10,000 years; orange: <20MY; light green: 20MY< < 50MY; light blue: 50MY< < 85MY).

Supplementary Figure 5: Phylogeny of PTD in angiosperms
A Maximum Likelihood tree based on amino acid sequences is provided. For the sake of clarity, species names are indicated instead of gene names. Branch support is given as Shimodaira-Hasegawa-like Likelihood Ratio Test (aLRT SH-like). Coloured disks superimposed along the branches of the trees give the age range for past WGDs. The pink triangles (and associated copies) represents tandem duplicates. Full-length duplicates are written with the color that corresponds to the age of the WGD (i.e. red: <10,000 years; orange: <20MY). The connectors superimposed over the phylogenies are used to associate the duplicates originating from a specific WGD but that scattered around the phylogeny (G. max).
Connectors associated with open circle along a branch are used to correct the misplacement of some species with respect to some past WGDs; i.e. to show that Zostera maritima and Spirodela polyrhiza did not experience the WGD that is common to all other monocots.

Supplementary Figure 6: Phylogeny of HEI10 in angiosperms
A Maximum Likelihood tree based on amino acid sequences is provided. For the sake of clarity, species names are indicated instead of gene names. Branch support is given as Shimodaira-Hasegawa-like Likelihood Ratio Test (aLRT SH-like). The pink triangle (and associated copies) represents tandem duplicates. Full-length duplicates and recent duplicated with one fractionated copies are written with the color that corresponds to the age of the WGD (i.e. red: <10,000 years; orange: <20MY; light blue: 50MY< < 85MY).
The connectors superimposed over the phylogenies are used to associate the duplicates that originated from a specific WGD but scattered around the phylogeny: e.g. Olea europea, Gossypium raimondii or Brassica rapa. Connectors associated with open circle along a branch are also used to correct the misplacement of some species with respect to some past WGDs; i.e. to show that Ananas comosus share a common WGD with all grasses

Supplementary Figure 7: Phylogenies of MSH4 in animals
Maximum Likelihood trees based on MSH4 amino acid sequences is provided. For the sake of clarity, species names are indicated instead of gene names. Branch support is given as Shimodaira-Hasegawalike Likelihood Ratio Test (aLRT SH-like). Coloured disks superimposed along the branches of the trees give the age range for past WGDs. Note that this Figure does not reflect all of the WGDs that contributed to the evolution of animals. The reason is that many of the additional unrepresented WGD events were detected using somatic transcriptome data 1 from which MSH4 is excluded. The connectors superimposed over the phylogenies are used to associate the duplicates (usually a fulllength copy and a fractionated one) originating from a specific WGD but that scattered around the phylogeny (because the fractionated copy is highly divergent).

Supplementary Figure 8: Phylogenies of MSH4 in animals and fungi
Maximum Likelihood trees based on MSH4 amino acid sequences is provided. For the sake of clarity, species names are indicated instead of gene names. Branch support is given as Shimodaira-Hasegawalike Likelihood Ratio Test (aLRT SH-like). Coloured disks superimposed along the branches of the trees give the age range for past WGDs. The ppen disk indicates a burst of gene duplication that have not been formally associated with a WGD event 2 . The pink triangle and associated Rhizopus delemar copie represents tandem duplicates.
Regarding the most recent WGD (i.e. red: <10,000 years), only allopolyploids are represented because there is no way to account for the presence of MSH4 "duplicates" in extant autopolyploids, which truly correspond to (multiple) real alleles.