FANCM Limits Meiotic Crossovers in Brassica Crops

Abstract

Meiotic crossovers (COs) are essential for proper chromosome segregation and the reshuffling of alleles during meiosis. In WT plants, the number of COs is usually small, which limits the genetic variation that can be captured by plant breeding programs. Part of this limitation is imposed by proteins like FANCM, the inactivation of which results in a 3-fold increase in COs in Arabidopsis thaliana. Whether the same holds true in crops needed to be established. In this study, we identified EMS induced mutations in FANCM in two species of economic relevance within the genus Brassica. We showed that CO frequencies were increased in fancm mutants in both diploid and tetraploid Brassicas, Brassica rapa and Brassica napus respectively. In B. rapa, we observed a 3-fold increase in the number of COs, equal to the increase observed previously in Arabidopsis. In B. napus we observed a lesser but consistent increase (1.3-fold) in both euploid (AACC) and allohaploid (AC) plants. Complementation tests in A. thaliana suggest that the smaller increase in crossover frequency observed in B. napus reflects residual activity of the mutant C copy of FANCM. Altogether our results indicate that the anti-CO activity of FANCM is conserved across the Brassica, opening new avenues to make a wider range of genetic diversity accessible to crop improvement.

Keywords: Brassica; FANCM; TILLING; Translational biology; meiotic crossover; plant breeding; polyploidy.

Figure 1

One copy of FANCM per genome is present and expressed in Brassica napus. (A) Schematic representation of the relationships between FANCM homologs in Brassica rapa, B. napus, B. oleracea, and Arabidopsis thaliana. Dotted lines represent the fractionated copies; i.e., copies originating from the Brassica specific whole genome triplication (WGT) and subsequently lost. (B) Relative contribution of BnaA.FANCM and BnaC.FANCM to total expression of FANCM in Brassica napus. Pyrosequencing data for BnaA.FANCM (blue) and BnaC.FANCM (red) in three varieties of B. napus. Genomic DNA (gDNA) was used as a control for biased PCR amplification between the two copies. Error bars = 1 SD from 3 biological replicates.

Figure 2

Restoration of bivalent formation in the double mutant braA.msh4-1^−/− braA.fancm-^−/− (A) During metaphase I in WT B. rapa, 10 bivalents and no univalent are formed. They are all aligned on the metaphase plate. (B) In the single braA.msh4-1^−/− mutant, only a few bivalents are formed, most of the chromosomes remain as univalents. (C) Metaphase I in the double mutant braA.msh4-1^−/− braA.fancm-^−/− is reminiscent of metaphase I in WT B. rapa, mostly bivalents are formed, only ~0.5 univalent pair is found on average per cell. Scale bar = 10 μm.

Figure 3

Homoeologous crossovers in fancm allohaploids plants. Boxplot for the number of univalents between mutants and WT allohaploids derived from five heterozygous F1 plants combining bnaA.fancm-1 with bnaC.fancm-2 (h2–h6). At least 2 mutants and 2 WT allohaploids plants were derived per F1 hybrids and were used as replicates. Around 20 meiocytes have been observed per replicate and the counts per replicate were pooled together. ***P < 0.001, Wilcoxon Signed-Rank Test.

Figure 4

Bivalent formation in A. thaliana msh5 fancm double-mutant transformed with different version of FANCM. During metaphase I, 5 bivalents were observed in msh5 fancm double-mutant meiocytes (A). When complementing msh5 fancm with the WT allele of At_FANCM (B), or with a modified copy of FANCM (AtfancmG317R) mimicking BnaC.fancm-2 (C,D), mainly univalents were observed. Scale bar = 10 μm.