Meiotic chromosome axis remodelling is critical for meiotic recombination in Brassica rapa

Abstract

Meiosis generates genetic variation through homologous recombination (HR) that is harnessed during breeding. HR occurs in the context of meiotic chromosome axes and the synaptonemal complex. To study the role of axis remodelling in crossover (CO) formation in a crop species, we characterized mutants of the axis-associated protein ASY1 and the axis-remodelling protein PCH2 in Brassica rapa. asy1 plants form meiotic chromosome axes that fail to synapse. CO formation is almost abolished, and residual chiasmata are proportionally enriched in terminal chromosome regions, particularly in the nucleolar organizing region (NOR)-carrying chromosome arm. pch2 plants show impaired ASY1 loading and remodelling, consequently achieving only partial synapsis, which leads to reduced CO formation and loss of the obligatory CO. PCH2-independent chiasmata are proportionally enriched towards distal chromosome regions. Similarly, in Arabidopsis pch2, COs are increased towards telomeric regions at the expense of (peri-) centromeric COs compared with the wild type. Taken together, in B. rapa, axis formation and remodelling are critical for meiotic fidelity including synapsis and CO formation, and in asy1 and pch2 CO distributions are altered. While asy1 plants are sterile, pch2 plants are semi-sterile and thus PCH2 could be an interesting target for breeding programmes.

Keywords: Brassica rapa; ASY1; PCH2; crossover; meiosis; meiotic chromosome axis remodelling; meiotic recombination; synaptonemal complex.

Fig. 1.

asy1 and pch2 plants display reduced fertility due to defects during meiosis. (A) Schematic representation of Brassica rapa ASY1 and PCH2 including mutations analysed (arrowheads). (B) B. rapa plant phenotypes (upper panel) including siliques and flowers (lower panel): WT, asy1-13 (a13), asy1-14 (a14), pch2-9 (p9), and pch2-12 (p12). (C) Average, SD, and range of bivalents, univalents, and chiasmata per cell in analysed lines. n=number of cells; a minimum of two independent plants per genotype. (D) B. rapa WT, asy1, and pch2 male meiotic chromosome spreads. In both mutants, synapsis and chiasma formation are impaired, leading to the occurrence of univalents at diakinesis that results in unequal chromosome segregation and unbalanced tetrads. Note, univalents in the mutants could migrate complete to a pole (arrowheads) or separate chromatids (asterisks) during anaphase I. DNA is counterstained with DAPI and shown in grey. Scale bar=10µm.

Fig. 2.

The major 45S rDNA locus-carrying chromosome arm shows increased chiasmata in asy1 but not in pch2 or the WT. (A) Top: B. rapa chromosomes organized by size with 5S and 45S rDNA clusters detected by FISH. Bottom: a representative WT cell after FISH. (B) Percentage of #3 rod bivalents with a chiasma in the 45S-carrying arm (‘45S yes’, example FISH image right) or in the opposite arm (‘45S no’, example FISH image left). Total number of rod bivalents scored: WT 41, pch2 16, asy1 36. FISH images: 5S (red), 45S (green); DNA is counterstained with DAPI and shown in blue.

Fig. 3.

Synapsis is defective in asy1 and pch2. Immunolocalization of ASY1 (red) and ZYP1 (green) in the WT, asy1, and pch2. In asy1, ASY1 is not detected and ZYP1 forms foci in zygotene–pachytene cells and aggregates in late pachytene–diplotene cells. In pch2, different from the WT, ASY1 does not get depleted from the axes and appears highly abundant in limited synapsed regions co-localizing with ZYP1. DNA is counterstained with DAPI and shown in blue. Scale bar=10µm.

Fig. 4.

ASY3 and SMC3 localize to chromosome axes in asy1. Immunolocalization of SMC3 (red, left) and ASY3 (red, right) together with ASY1 (green, WT top) and ZYP1 (green, WT bottom and asy1). ASY3 and SMC3 display similar dynamics in WT and asy1 cells. DNA is counterstained with DAPI and shown in blue. Scale bar=10µm.

Fig. 5.

Chiasma frequency distribution follows a Poisson distribution in asy1 and pch2. Chiasma frequency distribution (blue) and predicted Poisson distribution (red) for the WT, asy1-14, and pch2-9. In the mutants, chiasma frequency distribution does not significantly deviate from a Poisson-predicted distribution.

Fig. 6.

Class I CO formation in asy1 and pch2. Immunolocalization of MLH1 (A, red) and HEI10 (B, red) together with ZYP1 (green) in the WT, asy1, and pch2. In asy1, a subset of MLH1/HEI10 foci co-localize with ZYP1 foci/short stretches (arrows). In pch2, several HEI10 signals appear close to each other in a single ZYP1 stretch (asterisks). Note, to depict HEI10 dynamics in the WT, cells are shown ranging from zygotene (left) to diakinesis (right), whereas in all other cases representative examples of cells used for quantification are shown. DNA is counterstained with DAPI and shown in blue. Scale bars=5µm.

Fig. 7.

Compromised SC formation in pch2. (A) 3D reconstruction of the SC with simple neurite tracer (ImageJ) (pink, left) of representative pachytene cells immunolabelled with ZYP1 (green, right) in the WT and pch2. SC length is indicated in the bottom right corner. (B) SC measurements in individual WT and pch2 cells. Average, SD, and range are indicated.

Fig. 8.

Distalization of chiasmata in pch2 and in asy1. (A) Representative example of B. rapa diakinesis with a majority of chiasmata being interstitial (asterisks) in the WT (left) and distal (arrowheads) in pch2 (right). DNA is counterstained with DAPI and shown in grey. Scale bar=5 µm. (B) Quantification of interstitial versus total chiasmata in B. rapa WT, pch2, and asy1 reveals reduced interstitial CO frequency in the mutants. (C) Recombination frequency in Arabidopsis at genetic intervals defined with genes coding for fluorescent proteins. Genetic intervals are classed based on their locations on the chromosomes. Asterisks represent statistically significant differences (generalized linear model) and ‘n.s.’ means non-significant. (D) Left: staining of MLH1 (red) and DAPI (blue) at diakinesis in Arabidopsis WT and pch2. Scale bars=10 µM. Right: plots showing total MLH1 foci count, MLH1 foci count on euchromatin, and foci count on heterochromatin per cell. Error bars represent the SD. Asterisks represent statistically significant differences (Mann–Whitney–Wilcoxon test) and ‘n.s.’ means non-significant.