Mol. Cells 2022; 45(5): 273-283
Published online April 20, 2022
https://doi.org/10.14348/molcells.2022.2054
© The Korean Society for Molecular and Cellular Biology
Correspondence to : kyuha@postech.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
During meiosis, homologous chromosomes (homologs) pair and undergo genetic recombination via assembly and disassembly of the synaptonemal complex. Meiotic recombination is initiated by excess formation of DNA double-strand breaks (DSBs), among which a subset are repaired by reciprocal genetic exchange, called crossovers (COs). COs generate genetic variations across generations, profoundly affecting genetic diversity and breeding. At least one CO between homologs is essential for the first meiotic chromosome segregation, but generally only one and fewer than three inter-homolog COs occur in plants. CO frequency and distribution are biased along chromosomes, suppressed in centromeres, and controlled by pro-CO, anti-CO, and epigenetic factors. Accurate and high-throughput detection of COs is important for our understanding of CO formation and chromosome behavior. Here, we review advanced approaches that enable precise measurement of the location, frequency, and genomic landscapes of COs in plants, with a focus on Arabidopsis thaliana.
Keywords crossover, fluorescence-tagged lines, genotyping-by-sequencing, interference, meiosis, synaptonemal complex
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
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
Cytological analysis is a powerful tool for evaluating meiotic chromosome behavior and COs (Fig. 1) (Sims et al., 2021). In
COs between homologous pairs in
Class I interfering COs have been visualized and quantified by immunostaining in
In plants, COs can be detected using segregation assays that measure co-inheritance or separation of linked, heterozygous genetic markers on homologs during meiosis. Meiotic COs between markers lead to changes in the types of linkage in post-meiotic products such as pollen, seeds, and F2 individuals. Single nucleotide polymorphisms (SNPs), simple sequence length polymorphisms (SSLPs), and transfer DNA (T-DNA) have been used as genetic markers (Copenhaver et al., 1998; Giraut et al., 2011; Salomé et al., 2012). Segregation assays with T-DNAs that express fluorescent proteins in seeds or pollen have been extensively developed to detect CO frequency in a high-throughput manner (Figs. 2C and 2D) (Berchowitz and Copenhaver, 2008; Francis et al., 2007; Melamed-Bessudo et al., 2005). Genome-wide sets of seed and pollen fluorescence-tagged lines (FTLs) are available, which facilitates the detection of CO frequency along chromosomes as well as in a specific region on a chromosome (Berchowitz and Copenhaver, 2008; Wu et al., 2015).
The seed FTL system uses T-DNAs expressing eGFP or dsRed in the seed-coat under a seed-specific napin promoter to measure CO frequency (Melamed-Bessudo et al., 2005; Wu et al., 2015). Each seed FTL contains a pair of homozygous T-DNA markers expressing eGFP (G) and dsRed (R) linked
A sensitive seed FTL, the
The pollen FTL system provides a powerful tool for the detection of COs and tetrad analysis in
In
Recently, a linked sequencing technique was applied to a pool of pollen DNA from F1 hybrids in
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
We thank Choi lab members for their critical reading and helpful comments. This work was funded by the Suh Kyungbae Foundation (SUHF) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education NRF-2020R1A2C2007763.
H.K. and K.C. wrote the manuscript. H.K. generated figures.
The authors have no potential conflicts of interest to disclose.
Comparison of CO measurement methods
Material | Equipment | Time for preparation | Time for data analysis | CO interference measurement | Single-interval DCO measurement | High-throughput analysis | References | ||
---|---|---|---|---|---|---|---|---|---|
Cytology | Chiasmata | FM | 1 day | 1 h | No | No | No | Armstrong, 2013 Kurzbauer et al., 2018 López et al., 2012 Sanchez-Moran et al., 2002 | |
MLH1 foci | MLH1 antibody | CLSM | ~2 days | 1 h | No | No | No | Chelysheva et al., 2010 Lloyd et al., 2018 | |
Seed-based | Seed FTLs | FM, CellProfiler | 1 h | 1 h | No | No | Yes | Melamed-Bessudo et al., 2005 Wu et al., 2015 | |
Pollen-based | Manual counting | Pollen FTLs | FM, graphics software | 2.5 h | 1 day | Yes | Yes | No | Berchowitz and Copenhaver, 2008 Fernandes et al., 2018 Francis et al., 2007 |
FACS | Pollen FTLs | FM, flow cytometer | 1 h | 5 h | Yes | No | Yes | Yelina et al., 2013 Ziolkowski et al., 2017 | |
DeepTetrad | Pollen FTLs | FM, DeepTetrad package | 1 h | 2.5 h | Yes | Yes | Yes | Lim et al., 2020 Nageswaran et al., 2021 | |
GBS | F2 hybrid population GBS library | 2 days | 1 month | Yes | No | No | Nageswaran et al., 2021 Rowan et al., 2015 |
Strengths and weaknesses of CO measurement methods
Strength | Weakness | ||||
---|---|---|---|---|---|
Cytology | Chiasmata | • Quick and simple method to analyze CO numbers per cell | • Difficult to analyze large number of cells • Difficult to measure CO positon and frequency precisely | ||
MLH1 foci | • Visualize class I crossover sites per cell and per chromosome • Able to combine with other cytological analysis | ||||
Seed-based FTLs | • High-throughput analysis of CO frequency is possible • Able to get the average of female and male-specific CO frequency | • CO rate measurement range is limited to 50 • Cannot detect DCOs • Fluorescence can be silenced or unstable | |||
Pollen-based FTLs | Manual counting | • Able to detect DCOs and measure CO interference | • No need to install graphic card and DeepTetrad or flow cytometer equipment | • Silencing of fluorescence can occur in some genetic backgrounds | • Laborious |
FACS | • High-throughput analysis is possible | • Cannot measure double CO in a single interval • Requires flow cytometer equipment | |||
DeepTetrad | • Simple sample preparation • High-throughput analysis is possible | • Requires DeepTetrad pipeline | |||
GBS | • Precisely detect genome-wide CO sites | • High-cost and time-consuming |
Mol. Cells 2022; 45(5): 273-283
Published online May 31, 2022 https://doi.org/10.14348/molcells.2022.2054
Copyright © The Korean Society for Molecular and Cellular Biology.
Department of Life Sciences, Pohang University of Science and Technology, Pohang 37673, Korea
Correspondence to:kyuha@postech.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
During meiosis, homologous chromosomes (homologs) pair and undergo genetic recombination via assembly and disassembly of the synaptonemal complex. Meiotic recombination is initiated by excess formation of DNA double-strand breaks (DSBs), among which a subset are repaired by reciprocal genetic exchange, called crossovers (COs). COs generate genetic variations across generations, profoundly affecting genetic diversity and breeding. At least one CO between homologs is essential for the first meiotic chromosome segregation, but generally only one and fewer than three inter-homolog COs occur in plants. CO frequency and distribution are biased along chromosomes, suppressed in centromeres, and controlled by pro-CO, anti-CO, and epigenetic factors. Accurate and high-throughput detection of COs is important for our understanding of CO formation and chromosome behavior. Here, we review advanced approaches that enable precise measurement of the location, frequency, and genomic landscapes of COs in plants, with a focus on Arabidopsis thaliana.
Keywords: crossover, fluorescence-tagged lines, genotyping-by-sequencing, interference, meiosis, synaptonemal complex
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
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
Cytological analysis is a powerful tool for evaluating meiotic chromosome behavior and COs (Fig. 1) (Sims et al., 2021). In
COs between homologous pairs in
Class I interfering COs have been visualized and quantified by immunostaining in
In plants, COs can be detected using segregation assays that measure co-inheritance or separation of linked, heterozygous genetic markers on homologs during meiosis. Meiotic COs between markers lead to changes in the types of linkage in post-meiotic products such as pollen, seeds, and F2 individuals. Single nucleotide polymorphisms (SNPs), simple sequence length polymorphisms (SSLPs), and transfer DNA (T-DNA) have been used as genetic markers (Copenhaver et al., 1998; Giraut et al., 2011; Salomé et al., 2012). Segregation assays with T-DNAs that express fluorescent proteins in seeds or pollen have been extensively developed to detect CO frequency in a high-throughput manner (Figs. 2C and 2D) (Berchowitz and Copenhaver, 2008; Francis et al., 2007; Melamed-Bessudo et al., 2005). Genome-wide sets of seed and pollen fluorescence-tagged lines (FTLs) are available, which facilitates the detection of CO frequency along chromosomes as well as in a specific region on a chromosome (Berchowitz and Copenhaver, 2008; Wu et al., 2015).
The seed FTL system uses T-DNAs expressing eGFP or dsRed in the seed-coat under a seed-specific napin promoter to measure CO frequency (Melamed-Bessudo et al., 2005; Wu et al., 2015). Each seed FTL contains a pair of homozygous T-DNA markers expressing eGFP (G) and dsRed (R) linked
A sensitive seed FTL, the
The pollen FTL system provides a powerful tool for the detection of COs and tetrad analysis in
In
Recently, a linked sequencing technique was applied to a pool of pollen DNA from F1 hybrids in
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
We thank Choi lab members for their critical reading and helpful comments. This work was funded by the Suh Kyungbae Foundation (SUHF) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education NRF-2020R1A2C2007763.
H.K. and K.C. wrote the manuscript. H.K. generated figures.
The authors have no potential conflicts of interest to disclose.
Comparison of CO measurement methods
Material | Equipment | Time for preparation | Time for data analysis | CO interference measurement | Single-interval DCO measurement | High-throughput analysis | References | ||
---|---|---|---|---|---|---|---|---|---|
Cytology | Chiasmata | FM | 1 day | 1 h | No | No | No | Armstrong, 2013 Kurzbauer et al., 2018 López et al., 2012 Sanchez-Moran et al., 2002 |
|
MLH1 foci | MLH1 antibody | CLSM | ~2 days | 1 h | No | No | No | Chelysheva et al., 2010 Lloyd et al., 2018 |
|
Seed-based | Seed FTLs | FM, CellProfiler | 1 h | 1 h | No | No | Yes | Melamed-Bessudo et al., 2005 Wu et al., 2015 |
|
Pollen-based | Manual counting | Pollen FTLs | FM, graphics software | 2.5 h | 1 day | Yes | Yes | No | Berchowitz and Copenhaver, 2008 Fernandes et al., 2018 Francis et al., 2007 |
FACS | Pollen FTLs | FM, flow cytometer | 1 h | 5 h | Yes | No | Yes | Yelina et al., 2013 Ziolkowski et al., 2017 |
|
DeepTetrad | Pollen FTLs | FM, DeepTetrad package | 1 h | 2.5 h | Yes | Yes | Yes | Lim et al., 2020 Nageswaran et al., 2021 |
|
GBS | F2 hybrid population GBS library | 2 days | 1 month | Yes | No | No | Nageswaran et al., 2021 Rowan et al., 2015 |
Strengths and weaknesses of CO measurement methods
Strength | Weakness | ||||
---|---|---|---|---|---|
Cytology | Chiasmata | • Quick and simple method to analyze CO numbers per cell | • Difficult to analyze large number of cells • Difficult to measure CO positon and frequency precisely |
||
MLH1 foci | • Visualize class I crossover sites per cell and per chromosome • Able to combine with other cytological analysis |
||||
Seed-based FTLs | • High-throughput analysis of CO frequency is possible • Able to get the average of female and male-specific CO frequency |
• CO rate measurement range is limited to 50 • Cannot detect DCOs • Fluorescence can be silenced or unstable |
|||
Pollen-based FTLs | Manual counting | • Able to detect DCOs and measure CO interference | • No need to install graphic card and DeepTetrad or flow cytometer equipment | • Silencing of fluorescence can occur in some genetic backgrounds | • Laborious |
FACS | • High-throughput analysis is possible | • Cannot measure double CO in a single interval • Requires flow cytometer equipment |
|||
DeepTetrad | • Simple sample preparation • High-throughput analysis is possible |
• Requires DeepTetrad pipeline | |||
GBS | • Precisely detect genome-wide CO sites | • High-cost and time-consuming |
. Comparison of CO measurement methods.
Material | Equipment | Time for preparation | Time for data analysis | CO interference measurement | Single-interval DCO measurement | High-throughput analysis | References | ||
---|---|---|---|---|---|---|---|---|---|
Cytology | Chiasmata | FM | 1 day | 1 h | No | No | No | Armstrong, 2013 Kurzbauer et al., 2018 López et al., 2012 Sanchez-Moran et al., 2002 | |
MLH1 foci | MLH1 antibody | CLSM | ~2 days | 1 h | No | No | No | Chelysheva et al., 2010 Lloyd et al., 2018 | |
Seed-based | Seed FTLs | FM, CellProfiler | 1 h | 1 h | No | No | Yes | Melamed-Bessudo et al., 2005 Wu et al., 2015 | |
Pollen-based | Manual counting | Pollen FTLs | FM, graphics software | 2.5 h | 1 day | Yes | Yes | No | Berchowitz and Copenhaver, 2008 Fernandes et al., 2018 Francis et al., 2007 |
FACS | Pollen FTLs | FM, flow cytometer | 1 h | 5 h | Yes | No | Yes | Yelina et al., 2013 Ziolkowski et al., 2017 | |
DeepTetrad | Pollen FTLs | FM, DeepTetrad package | 1 h | 2.5 h | Yes | Yes | Yes | Lim et al., 2020 Nageswaran et al., 2021 | |
GBS | F2 hybrid population GBS library | 2 days | 1 month | Yes | No | No | Nageswaran et al., 2021 Rowan et al., 2015 |
. Strengths and weaknesses of CO measurement methods.
Strength | Weakness | ||||
---|---|---|---|---|---|
Cytology | Chiasmata | • Quick and simple method to analyze CO numbers per cell | • Difficult to analyze large number of cells • Difficult to measure CO positon and frequency precisely | ||
MLH1 foci | • Visualize class I crossover sites per cell and per chromosome • Able to combine with other cytological analysis | ||||
Seed-based FTLs | • High-throughput analysis of CO frequency is possible • Able to get the average of female and male-specific CO frequency | • CO rate measurement range is limited to 50 • Cannot detect DCOs • Fluorescence can be silenced or unstable | |||
Pollen-based FTLs | Manual counting | • Able to detect DCOs and measure CO interference | • No need to install graphic card and DeepTetrad or flow cytometer equipment | • Silencing of fluorescence can occur in some genetic backgrounds | • Laborious |
FACS | • High-throughput analysis is possible | • Cannot measure double CO in a single interval • Requires flow cytometer equipment | |||
DeepTetrad | • Simple sample preparation • High-throughput analysis is possible | • Requires DeepTetrad pipeline | |||
GBS | • Precisely detect genome-wide CO sites | • High-cost and time-consuming |
Kyuha Choi*
Mol. Cells 2017; 40(11): 814-822 https://doi.org/10.14348/molcells.2017.0171Hyoeun Kang, Seok Cheol Hwang, Yong Seok Park, and Jeong Su Oh
Mol. Cells 2013; 35(6): 514-518 https://doi.org/10.1007/s10059-013-0029-6Nameun Kim, Rui Xiao, Hojun Choi, Haiin Jo, Jin-Hoi Kim, Sang-Jun Uhm, and Chankyu Park*
Mol. Cells 2011; 31(1): 39-48 https://doi.org/10.1007/s10059-011-0002-1