Regulation of interference-sensitive crossover distribution ensures crossover assurance in Arabidopsis

During meiosis, crossovers (COs) are typically required to ensure faithful chromosomal segregation. Despite the requirement for at least one CO between each pair of chromosomes, closely spaced double COs are usually underrepresented due to a phenomenon called CO interference. Like Mus musculus and Saccharomyces cerevisiae, Arabidopsis thaliana has both interference-sensitive (Class I) and interference-insensitive (Class II) COs. However, the underlying mechanism controlling CO distribution remains largely elusive. Both AtMUS81 and AtFANCD2 promote the formation of Class II CO. Using both AtHEI10 and AtMLH1 immunostaining, two markers of Class I COs, we show that AtFANCD2 but not AtMUS81 is required for normal Class I CO distribution among chromosomes. Depleting AtFANCD2 leads to a CO distribution pattern that is intermediate between that of wild-type and a Poisson distribution. Moreover, in Atfancm, Atfigl1, and Atrmi1 mutants where increased Class II CO frequency has been reported previously, we observe Class I CO distribution patterns that are strikingly similar to Atfancd2. Surprisingly, we found that AtFANCD2 plays opposite roles in regulating CO frequency in Atfancm compared with either in Atfigl1 or Atrmi1. Together, these results reveal that although AtFANCD2, AtFANCM, AtFIGL1, and AtRMI1 regulate Class II CO frequency by distinct mechanisms, they have similar roles in controlling the distribution of Class I COs among chromosomes.

Meiosis is a specialized cell division process that includes two rounds of chromosome segregation following a single round of premeiotic DNA replication and is essential for sexual reproduction in most eukaryotes. During meiosis, homologous recombination (HR) is employed to repair double strand breaks (DSBs) catalyzed by the SPO11 transesterase, yielding crossovers (COs) or noncrossovers (1). In most eukaryotes, meiotic COs are required to ensure the faithful segregation of homologous chromosomes (homologs). COs create new haplotypes, which in turn generate phenotypic diversity among offspring (2). Numerous studies in multiple species have shown that the number and distribution of COs are tightly regulated (3, 4). For instance, the model plant Arabidopsis thaliana has about 10 COs during each meiosis (5) that are nonrandomly distributed along and among chromosomes. Despite the small number of COs, each of the five pairs of Arabidopsis homologs experiences at least one CO—a phenomenon known as CO assurance (6). A second regulatory mechanism, CO interference, inhibits closely spaced double COs (7). However, the molecular mechanisms controlling CO distribution are elusive.Most eukaryotes have two kinds of COs (1): Class I COs that are sensitive to interference and Class II COs which are interference insensitive. In wild-type (WT) Arabidopsis, 85 to 90% of COs belong to Class I and are mediated by the ZMM group of proteins (Zip1-4, Mer3, and Msh4-5) and MLH1/3 (8–13). Class II COs can be generated by at least two parallel pathways in Arabidopsis (14–16), which depend on either the structure-specific endonuclease AtMUS81 (15, 16) or a homolog of Fanconi Anemia Complementation Group D2 (AtFANCD2) (14). The homolog of the Holliday junction resolvase GEN1 is required for the formation of Class II CO in rice (17) but not in Arabidopsis (18), suggesting that Class II CO pathways have diverged between monocots and dicots. In addition to pro–Class II CO factors, Arabidopsis has at least three anti–Class II CO mechanisms: the AtFANCM helicase and its cofactors AtMHF1 and AtMHF2 (19, 20); members of BLM-TOP3-RMI1 (BTR) complex AtRECQ4A, AtRECQ4B, AtTOP3α, and AtRMI1 (21, 22); and AtFIGL1 AAA-ATPase and its interacting protein AtFLIP1 (23, 24). Interestingly, although total CO frequency increases and Class I CO numbers remain unchanged when these anti–Class II CO pathways are perturbed, univalents are still occasionally observed, suggesting that CO assurance has also been weakened (22, 23).In this study, we investigated the influence of AtFANCD2, AtFANCM, AtFIGL1, and AtRMI1 on the distribution of Class I COs among chromosomes. We demonstrate that these factors have a role in promoting a non-Poisson distribution of Class I COs among chromosomes.