Fast and Precise: How to Measure Meiotic Crossovers in Arabidopsis

Abstract

INTRODUCTION

Meiosis refers to specialized cell division in sexually reproducing eukaryotes (Villeneuve and Hillers, 2001). The process involves a single round of DNA replication and two successive rounds of cell division, with the resulting cells having half the number of chromosomes as the parent cell. During meiosis in most diploid eukaryotes, homologous chromosomes (homologs) pair to form bivalents and undergo reciprocal exchange of genetic material, called crossover (CO). The presence of at least one CO per bivalent is essential for the accurate segregation of homologs and ensures the generation of viable gametes because the absence of CO results in unbalanced chromosome segregation at meiosis I and aneuploid cells. COs also contribute to genetic diversity in populations, which facilitates local adaptation and breeding in animals and plants (Barton and Charlesworth, 1998).

Meiotic COs are formed by the repair of DNA double-strand breaks (DSBs) induced by topoisomerase-like SPO11 and its associated proteins (Kim and Choi, 2019; Lam and Keeney, 2014). The progression of meiotic recombination is tightly connected to the dynamics of chromosome behavior, including chromosome axis-loop formation, homolog alignment, and synaptonemal complex (SC) assembly and disassembly (Fig. 1A) (Ur and Corbett, 2021; Zickler and Kleckner, 1999). At DSB sites, the 5′ end is bidirectionally resected to produce a 3′ single-strand DNA. Subsequently, the 3′ end undergoes a search for homologs or sister chromatids with the assistance of recombinases such as DMC1 and/or RAD51. The inter-homolog invasion forms a recombination intermediate, called a displacement (D) loop or joint molecule. DNA synthesis extends the D-loop to generate a double Holliday junction (dHJ) intermediate that is resolved to generate CO or non-CO products. COs are formed by two conserved CO pathways in most eukaryotes, named class I and class II (Fig. 1B) (Mercier et al., 2015). In most plants, the class I pathway depends on a group of pro-CO proteins, called ZMMs (ZIP4, MSH4, MSH5, MER3, HEI10, PTD, SHOC1) and MLH1/MLH3 heterodimeric endonucleases (Mercier et al., 2015). Class I COs account for approximately 80%-85% of COs in plants and are sensitive to CO interference. The remaining 10%-15% of COs are interference-insensitive and depend on MUS81 in the class II pathway. Class II COs are restricted by anti-CO factors that promote the generation of non-COs, such as FANCM (Crismani et al., 2012; Mercier et al., 2015; Séguéla-Arnaud et al., 2015; Taagen et al., 2020).

CO frequency and distribution are tightly regulated, which is manifested in phenomena such as CO homeostasis, assurance, and interference. However, the underlying mechanisms remain elusive. COs are homeostatically controlled in many organisms, maintaining consistent CO frequencies despite variations in the number of DSBs in yeast, mice, and the nematode Caenorhabditis elegans (Cole et al., 2012; Martini et al., 2006; Rosu et al., 2011; Yokoo et al., 2012). However, COs are somewhat less strictly controlled in plants (Sidhu et al., 2015; Xue et al., 2018). CO interference refers to non-random spacing of COs along the chromosome, with the occurrence of one CO inhibiting the formation of adjacent COs (Zickler and Kleckner, 1999). The occurrence of more than two COs per pair of homologs is rare among most species (Fernandes et al., 2018) and CO patterns not following Poisson distribution show the effect of interference (Berchowitz and Copenhaver, 2010). Specifically, COs are favored in narrow regions of approximately 1-2 kb at gene promoters, terminators and specific DNA transposons in plants, called recombination hotspots, where nucleosome density and DNA methylation levels are low (Choi, 2017; Choi and Henderson, 2015).

Precise and high-throughput measurements of COs are important for understanding the mechanisms that control meiotic recombination. Cytological analyses, immunostaining, segregation assays of genetic markers, next-generation sequencing, and long-read sequencing methods have been extensively developed to measure CO patterns (Fig. 2, Tables 1 and 2). Here, we provide an overview of the methods used to visualize and detect CO events in Arabidopsis thaliana and highlight novel techniques.

VISULALIZATION AND QUANTIFICATION OF CROSSOVERS

HIGH-THROUGHPUT MEASUREMENT OF CROSSOVER FREQUENCY

GENOME-WIDE MAPPING OF CROSSOVERS

In Arabidopsis, greater CO frequency occurs near both chromosome ends in male meiosis than in female meiosis (Giraut et al., 2011). The number of COs and their distribution along chromosomes in male and female meiosis were determined using BeadXpress technology with a genome-wide set of SNP markers (Drouaud et al., 2006; Giraut et al., 2011). Genomic SNP typing-based CO maps indicated that a large number of COs occurred in F₂ individuals and backcrossed plants of the recq4 figl1 hybrid in Arabidopsis, and the recq4 mutation led to a 3-fold increase in CO frequency in rice, peas, and tomato (Fernandes et al., 2018; Mieulet et al., 2018). In parallel, the genotyping-by-sequencing (GBS) method cost-effectively generated high-resolution genomic CO maps (Fig. 2E) (Rowan et al., 2015). The GBS method involves rapid extraction of F₂ individual genomic DNA, sequencing and indexed library construction, and running trained individual genome reconstruction (TIGER), a bioinformatics pipeline used to reconstruct mosaic genomes of F₂ individuals from sparse sequence coverage. Combining GBS with TIGER enabled a precise comparison of the positioning and number of COs in wild-type hybrids with those of diverse genetic backgrounds such as zyp1, asy1, msh2, hcr1, and HEI10 mutants (Blackwell et al., 2020; Capilla-Pérez et al., 2021; Kim et al., 2021; Lambing et al., 2020; Nageswaran et al., 2021; Serra et al., 2018a; Zhu et al., 2021). GBS-based CO maps have provided the DNA sequence and chromatin features of CO hotspots in Arabidopsis (Shilo et al., 2015; Wijnker et al., 2013).

Recently, a linked sequencing technique was applied to a pool of pollen DNA from F₁ hybrids in Arabidopsis, generating a high-resolution CO landscape that was comparable to single pollen sequencing but more cost-effective (Luo et al., 2019; Sun et al., 2019). In addition, long-read sequencing technology has been adapted to directly sequence a pool of pollen DNA from F₁ hybrids to map genomic COs and altered structures (Naish et al., 2021; Wang et al., 2021). Genomic CO maps have led to the discovery of CO hotspots, including single CO hotspots that have been analyzed using the pollen typing method (Choi et al., 2013; 2016; 2017; Drouaud et al., 2013; Serra et al., 2018b; Yelina et al., 2012; 2015). At a single CO hotspot, DNA methylation or arp6 mutation was shown to induce fewer COs (Choi et al., 2013; Yelina et al., 2015). As promoting COs in specific regions is of great interest in plant breeding, the CRISPR/dCas9 targeting approach was applied to increase CO frequency at a single CO hotspot in Arabidopsis using MTOPVIB (meiotic topoisomerase VIB)-dCas9 fusion, but the moderate effect reflected the complexity of meiotic recombination control (Yelina et al., 2021). Two MTOPVIB proteins interact with SPO11-1 and SPO11-2 topoisomerase-like proteins that catalyze meiotic DSB formation. The MTOPVIB-dCas9 was expressed during meiosis and targeted to 3a CO hotspot via a guide RNA, which was expected to recruit SPO11-1 and SPO11-2, and subsequently induce DSBs and COs.

CONCLUSION AND FUTURE PERSPECTIVES

Recent advances in cytological, genetic, and genomic approaches for detecting COs have contributed significantly to our understanding of CO formation and meiosis in the model plant Arabidopsis. We here summarized the main methods of immnocytology, FTL systems and GBS methods for fast and accurate measurement of COs. The advanced approaches have allowed us to elucidate the roles of pro-CO, anti-CO and epigenetic factors in controlling of CO frequency and distribution. Along with super-resolution microscopy techniques, live imaging systems have been recently established to visualize the dynamics of chromosome behavior during meiosis (Prusicki et al., 2019). In the future, advanced genomic approaches, including meiosis-specific transcriptomics, single-cell RNA sequencing, and epigenome maps using INTACT (isolation of nuclei tagged in specific cell types), will provide important genomic information on CO formation that will help elucidate the molecular mechanisms controlling meiotic recombination in plants (Barra et al., 2021). As an efficient biochemical method for identifying new CO modulators, proximity labeling techniques can be adapted to generate protein interactomes for meiotic proteins in Arabidopsis (Mair and Bergmann, 2022). Moreover, genetic screening and identification of CO modifiers using FTLs and natural accessions will lead to the discovery of new pro-and anti-CO factors in Arabidopsis, further illuminating the CO pathways in plants. These noteworthy technical advances in detecting COs and isolating new CO modifiers will have profound implications for plant breeding and our understanding of meiotic recombination. Furthermore, manipulating the rate and positions of COs using CRISPR/Cas9-based genome editing methods will accelerate plant breeding and QTL (quantitative trait locus) mapping (Gao, 2021; Oh and Kim, 2021; Taagen et al., 2020).

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