Massive crossover elevation via combination of HEI10 and ... · 122 In this work we explore the functional relationship between ZMM pro-crossover and RECQ4 123 anti-crossover meiotic

1

Massive crossover elevation via combination of HEI10 and recq4a 1

recq4b during Arabidopsis meiosis 2

3

Heïdi Serra1, Christophe Lambing1, Catherine H. Griffin1, Stephanie D. Topp1, Mathilde 4

Séguéla-Arnaud2, Joiselle Fernandes2, Raphaël Mercier2 and Ian R. Henderson1* 5 61Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 7

3EA, United Kingdom 82Institute Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, RD10, 9

78000 Versailles, France 10 11

*Correspondence: [email protected] 12

13

Abstract 14

During meiosis homologous chromosomes undergo reciprocal crossovers, which generate 15

genetic diversity and underpin classical crop improvement. Meiotic recombination initiates 16

from DNA double strand breaks, which are processed into single-stranded DNA that can 17

invade a homologous chromosome. The resulting joint molecules can ultimately be resolved 18

as crossovers. In Arabidopsis, competing pathways balance the repair of ~100-200 meiotic 19

DSBs into ~10 crossovers per meiosis, with the excess DSBs repaired as non-crossovers. In 20

order to bias DSB repair towards crossovers, we simultaneously increased dosage of the pro-21

crossover E3 ligase gene HEI10 and introduced mutations in the anti-crossover helicase 22

genes RECQ4A and RECQ4B. As HEI10 and recq4a recq4b increase interfering and non-23

interfering crossover pathways respectively, they combine additively to yield a massive 24

meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage 25

increases crossover coincidence, which indicates an effect of HEI10 on interference. We also 26

show that patterns of interhomolog polymorphism and heterochromatin drive recombination 27

increases towards the sub-telomeres in both HEI10 and recq4a recq4b backgrounds, while 28

the centromeres remain crossover-suppressed. These results provide a genetic framework for 29

engineering meiotic recombination landscapes in plant genomes. 30

31

Key words: Meiosis, recombination, crossover, ZMM, HEI10, RECQ4, polymorphism. 32

33

author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/159764doi: bioRxiv preprint

https://doi.org/10.1101/159764

2

Main text 34

Meiosis is a conserved cell division required for eukaryotic sexual reproduction, during 35

which a single round of DNA replication is coupled to two rounds of chromosome 36

segregation, generating haploid gametes1,2. Homologous chromosomes pair and recombine 37

during prophase of the first meiotic division, which can result in reciprocal exchange, termed 38

crossover1,2. Crossovers have a major effect on sequence variation in populations and create 39

genetic diversity. Meiotic recombination is also an important tool used during crop breeding 40

to combine useful variation. However, crossover numbers are typically low, ~1-2 per 41

chromosome per meiosis, and can show restricted chromosomal distributions, which limit 42

crop improvement. For example, recombination is suppressed in large regions surrounding 43

the centromeres of many crop species3. In this work we sought to use our understanding of 44

meiotic recombination pathways to genetically engineer super-recombining Arabidopsis. 45

46

Meiotic recombination initiates from DNA double strand breaks (DSBs), induced by SPO11 47

transesterases, which act in topoisomerase-VI-like complexes1,4 (Fig. 1a). During catalysis 48

SPO11 becomes covalently bound to target site DNA and is liberated by endonucleolytic 49

cleavage by the MRN (MRE11-RAD50-NBS1) complex and COM14. Simultaneously, 50

exonucleases generate 3′-overhanging single stranded DNA (ssDNA), approximately 100-51

1000s of nucleotides in length4 (Fig. 1a). Resected ssDNA is bound by RAD51 and DMC1 52

RecA-like proteins, which promote invasion of a homologous chromosome and the formation 53

of a displacement loop (D-loop)5 (Fig. 1a). Stabilization of the D-loop can occur by template-54

driven DNA synthesis from the invading 3′-end4,6 (Fig. 1a). Strand invasion intermediates 55

may then progress to second-end capture and formation of a double Holliday junction (dHJ), 56

which can be resolved as a crossover or non-crossover, or undergo dissolution1,4,6 (Fig. 1a). 57

58

The conserved ZMM pathway acts to promote formation of the majority of crossovers in 59

plants, which are known as class I1,6,7 (Fig. 1a). Mutations in ZMM genes severely reduce 60

Arabidopsis crossover frequency, causing univalent chromosome segregation at metaphase I, 61

aneuploid gametes and infertility1,8,9. Importantly, ZMM-dependent crossovers show 62

interference, where double crossover events are spaced out more widely than expected by 63

chance10,11. The ZMM pathway in plants includes the MSH4/MSH5 MutS-related 64

heterodimer, MER3 DNA helicase, SHORTAGE OF CROSSOVERS1 (SHOC1) XPF 65

nuclease, PARTING DANCERS (PTD), ZIP4/SPO22, HEI10 E3 ligase and the 66


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Self-fertilize

192 F2 plants Genotype by Sequencing

HEI10 C2 420

Ler

recq4a recq4b

HEI10/+ recq4a/+ recq4b/+ 420/++

HEI10/+ recq4a recq4b 420

×

×

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191 F2 plants Genotype by Sequencing

×

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Self-fertilize

b

&

a

RESECTION

CROSSOVER[CLASS ONE]

RECQ4A RECQ4BRMI1 TOP3a

NON-CROSSOVERRESOLUTION

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recq4a recq4b

SDSA

STRAND INVASION

MLH1 MLH3

SPO11-1 SPO11-2 MTOPVIB

MRE11 RAD50 NBS1 COM1

DMC1RAD51RPA1

ZMMHEI10 SHOC1MER3 PTDMSH4 ZIP4MSH5

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FANCM MHF1MHF2

Figure 1


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Figure 1. Combining increased HEI10 dosage with recq4a recq4b mutations in order to 67

elevate meiotic crossovers. a. Schematic diagram showing a subset of pathways acting 68

during Arabidopsis meiotic recombination. Homologous chromosomes are indicated as red 69

and blue DNA duplexes. The remaining two sister chromatid duplexes are not shown for 70

simplicity. Recombination pathways (green) and factors acting within them (red) are printed 71

alongside chromosomes. b. Crossing diagram showing the generation of Col/Ler F1 plants 72

that were recq4a recq4b mutant, with and without HEI10 (black triangles). F2 progeny from 73

these plants were analysed by genotyping-by-sequencing to map crossover locations. These 74

populations were compared to wild type F2 progeny and to a previously reported HEI10 F2 75

population12. 76

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MLH1/MLH3 MutL-related heterodimer1,6,7 (Fig. 1A). Within the ZMM pathway the HEI10 101

E3 ligase gene shows dosage sensitivity, with additional copies being sufficient to increase 102

crossovers throughout euchromatin12. A minority of crossovers in plants, known as class II, 103

do not show interference and are formed by a different MUS81-dependent pathway13,14. 104

105

From cytological measurement of Arabidopsis DSB-associated foci (e.g. γH2A.X, RAD51 106

and DMC1) along meiotic chromosomes, it is estimated that between 100-200 breaks initiate 107

per nucleus15–17. However, only ~10 crossovers typically form throughout the genome18–21, 108

indicating that anti-crossover pathways prevent maturation of the majority of initiation events 109

into crossovers. Indeed, genetic analysis has identified at least three distinct anti-crossover 110

pathways in Arabidopsis: (i) the FANCM DNA helicase and MHF1 and MHF2 co-factors22–11124, (ii) the AAA-ATPase FIDGETIN-LIKE125 and (iii) the RTR complex of RECQ4A, 112

RECQ4B DNA helicases, TOPOISOMERASE3α and RMI126–28 (Fig. 1a). For example, 113

recq4a recq4b mutants show highly increased non-interfering crossovers when assayed in 114

specific intervals27 (Fig. 1a). This is thought primarily to result from a failure to dissolve 115

interhomolog strand invasion events, which are alternatively repaired by the non-interfering 116

crossover pathway(s)22,25,27. As combining mutations between these pathways, for example 117

fancm fidgl1, leads to additive crossover increases they reflect parallel mechanisms25. Hence, 118

during meiosis competing pathways act on SPO11-dependent DSBs to balance crossover and 119

non-crossover repair outcomes (Fig. 1a). 120

121

In this work we explore the functional relationship between ZMM pro-crossover and RECQ4 122

anti-crossover meiotic recombination pathways. Using a combination of increased HEI10 123

dosage and recq4a recq4b mutations, we observe a massive, additive increase in crossover 124

frequency throughout the chromosome arms. Surprisingly, we observe that increased HEI10 125

dosage causes increased crossover coincidence, indicating an effect on interference. We show 126

that HEI10 and recq4a recq4b crossover increases are biased towards regions of low 127

interhomolog divergence, distal from centromeric heterochromatin. Hence, both genetic and 128

epigenetic information likely constrain the activity of meiotic recombination pathways. 129

130

Combination of HEI10 and recq4a recq4b massively elevates crossover frequency 131

Crossover increases in HEI10 and recq4a recq4b represent mechanistically distinct effects 132

via class I and class II crossover repair pathways (Fig. 1a). We therefore sought to test 133

whether combining these genetic backgrounds would cause further increases in crossover 134


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frequency. We previously showed that transgenic line ‘C2’ carrying additional HEI10 copies, 135

shows a ~2-fold increase in crossovers genome-wide, compared with wild type12 (Table 1). 136

We therefore crossed HEI10 line C2 to recq4a recq4b double mutants, in the Col genetic 137

background12,27,29 (Fig. 1b). A previous genetic screen isolated an EMS allele of recq4a in 138

Ler27. As Ler carries a natural premature stop codon in recq4b27, this provides a recq4a 139

recq4b double mutant in Ler (Fig. 1b). These lines were crossed and F1 progeny identified 140

that were heterozygous for Col/Ler polymorphisms, recq4a recq4b mutant and with or 141

without HEI10 (Fig. 1b). These F1 plants were then used to generate Col/Ler F2 for crossover 142

analysis (Fig. 1b). 143

144

During crossing we maintained the 420 FTL crossover reporter within our lines, which allows 145

measurement of genetic distance in a ~5.1 Mb sub-telomeric region on chromosome 330,31 146

(Figs. 1b, 2a and Supplementary Table 1). This showed that HEI10, recq4a recq4b and 147

HEI10 recq4a recq4b all significantly increase 420 crossover frequency in Col/Ler 148

backgrounds, by 2.7, 3.3 and 3.7-fold, respectively (X2 P=2.73×10-175, P=4.92×10-212 and 149

P=2.80×10-226) (Fig. 2a and Supplementary Table 1). However, it is notable that 420 genetic 150

distance reached 47 cM in HEI10 recq4a recq4b, which is close to the maximum observable 151

recombination frequency for linked markers (i.e. 50 cM) (Fig. 2a and Supplementary Table 152

1). We next used genotyping-by-sequencing (GBS) to generate genome-wide, high-resolution 153

maps of crossover distributions in these backgrounds. 154

155

We sequenced genomic DNA from 191-245 Col/Ler F2 progeny derived from wild type, 156

recq4a recq4b and HEI10 recq4a recq4b F1 parents (Fig. 1b and Table 1), and compared to a 157

previously described HEI10 F2 population12. We observed that recq4a recq4b caused 3.3-fold 158

more crossovers genome-wide (25 crossovers/F2, 95% confidence interval +/- 0.93), 159

compared with wild type (7.5 crossovers/F2, 95% confidence interval +/- 0.28), which is 160

greater than the 2-fold increase previously seen in HEI10 (15.3 crossovers/F2, 95% 161

confidence interval +/- 0.49)12 (Fig. 2b-2d and Table 1). If the HEI10 and recq4a recq4b 162

crossover increases combined in a purely additive manner then we would expect to see the 163

sum of their crossover differentials in HEI10 recq4a recq4b, equivalent to 7.5 + 7.7 + 17.4 = 164

32.6 crossovers/F2. Indeed, this was similar to the observed value for HEI10 recq4a recq4b 165

of 31 crossovers/F2 (95% confidence interval +/- 0.97) (Fig. 2b-2d and Table 1). For all 166

populations, the physically largest chromosomes had the longest genetic maps 167

168


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812

Figure 2


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Figure 2. Combination of HEI10 and recq4a recq4b massively increases meiotic 169

crossover frequency. a. 420 genetic distance (cM) was measured during breeding of the 170

HEI10 and recq4a recq4b mutant populations. All samples were Col/Ler heterozygous. 171

Replicate measurements are shown as black dots and mean values as red dots. Plotted data is 172

shown in Supplementary Table 1. The HEI10 data was previously reported12. b. 173

Chromosomal genotypes are shown from two representative individuals from the wild type 174

and HEI10 recq4a recq4b F2 populations. The five Arabidopsis chromosomes are depicted 175

and colour-coded according to Col/Col (blue), Col/Ler (green) or Ler/Ler (red) genotypes. c. 176

Histograms showing the frequency of F2 individuals containing different crossover numbers 177

in each population, with the mean value indicated by the horizontal dotted red lines. d. 178

Genetic maps (cM) shown for each chromosome for HEI10 (blue), recq4a recq4b (magenta) 179

and HEI10 recq4a recq4b (red). Each map shown alongside the wild type map (left), and 180

markers between the maps are connected. 181

182

Table 1. Crossovers in wild type, HEI10, recq4a recq4b and HEI10 recq4a recq4b 183

Col/Ler F2 populations. Crossover numbers identified by genotyping-by-sequencing in the 184

indicated F2 populations are listed. The number of individuals analysed per population is 185

indicated in the n row. Absolute crossover (COs) numbers are reported, as well as mean 186

crossovers per F2 individual, by chromosome and in total. The CO StDev row shows the 187

standard deviation in total crossover numbers per F2 for each genotype. 188

GENOTYPE

Col/Ler

HEI10

Col/Ler

recq4a recq4b

Col/Ler

HEI10

recq4a recq4b

Col/Ler

Chromosome COs COs/F2 COs COs/F2 COs COs/F2 COs COs/F2

1 437 1.8 765 4.0 1259 6.6 1641 8.5

2 326 1.3 525 2.7 799 4.2 1001 5.2

3 345 1.4 546 2.8 993 5.2 1195 6.2

4 309 1.3 417 2.2 576 3.0 663 3.5

5 423 1.7 675 3.5 1156 6.1 1448 7.5

Total 1840 7.5 2928 15.3 4783 25.0 5948 31.0

n 245 192 191 192

CO StDev 2.26 3.41 6.52 6.59

189

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(Supplementary Fig. 1). A subset of wild type and HEI10 recq4a recq4b F2 individuals were 191

sequenced to higher depth and crossover patterns found to be robustly identified 192

(Supplementary Fig. 2 and Supplementary Table 2). Together these data show that crossover 193

elevations caused by increased HEI10 dosage and loss of the RECQ4A RECQ4B anti-194

crossover helicases combine in an additive manner, consistent with class I and class II 195

crossover pathways being independent in Arabidopsis. 196

197

Crossover coincidence increases in both HEI10 and recq4a recq4b 198

Underdispersion of crossover numbers per meiosis occurs due to the action of crossover 199

interference and homeostasis6,32, causing an excess of values close to the mean. Consistently, 200

we observe that the distribution of crossovers per wild type F2 individual is significantly non-201

Poisson (Goodness-of-fit test for Poisson distribution, P=0.012) (Fig. 3a). Observed 202

frequencies are displayed as bars (grey) plotted from the fitted frequencies (red line), such 203

that grey bars lying above or below zero on the y-axis represent deviation from the Poisson 204

expectation (Fig. 3a). Crossover distributions per individual in recq4a recq4b and HEI10 205

recq4a recq4b were also non-Poisson (recq4a recq4b P=1.85×10-5 and HEI10 recq4a recq4b 206

P=0.0223). However, the high recombination populations also showed significantly greater 207

variation in crossover numbers compared to wild type (Brown-Levene test, HEI10 208

P=4.17×10-7, recq4a recq4b P=<2.2×10-16, HEI10 recq4a recq4b P=<2.2×10-16) (Fig. 3a 209

and Table 1). We therefore sought to examine the distributions of crossovers within the GBS 210

data in more detail, with respect to inter-crossover spacing. 211

212

Due to F2 individuals being analysed, which result from two independent meioses, it is not 213

possible to distinguish between double crossovers (DCOs) occurring on the same (cis) or 214

different (trans) chromosomes (Fig. 3b). Importantly, only the cis class is informative for 215

estimation of interference. Despite this limitation, we considered each F2 individual 216

separately and identified adjacent DCOs for each chromosome and recorded their distances 217

(Fig. 3b). Simultaneously, for each individual and chromosome the same number of 218

randomly chosen positions were used to generate a matched set of randomized distances (Fig. 219

3b). Consistent with the action of crossover interference, wild type DCO distances were 220

significantly greater than random (mean=8.57 Mb vs 6.99 Mb, Mann-Whitney Wilcoxon test 221

P=1.87×10-16) (Fig. 3b). In HEI10 DCO distances were substantially reduced compared to 222

wild type, although they were still significantly greater than random (mean=6.09 Mb vs 5.08 223

Mb, Mann-Whitney Wilcoxon test P=9.46×10-13) (Fig. 3b). However, in both recq4a recq4b 224


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Figure 3. Crossover coincidence increases in HEI10 and recq4a recq4b. a. Plots of the 225

square root of the frequency of total crossovers per F2 individual in wild type, HEI10, recq4a 226

recq4b and HEI10 recq4a recq4b populations, generated using the R package goodfit. The 227

expected Poisson distribution is plotted in red, from which the observed data is plotted. 228

Deviation from the Poisson expectation is shown by the grey bay (observed data) falling 229

either above or below the zero value on the y-axis. b. A graphical diagram is shown to 230

illustrate cis versus trans double crossovers detected in F2 genotyping-by-sequencing data. 231

Kernel density estimates are plotted for measured DCO distances in the indicated populations 232

(red), versus the same number of matched randomly chosen double events (blue). The 233

vertical dotted lines indicate mean values. c. I3b and I3c genetic distances in wild type and 234

HEI10, and crossover interference (1-CoC) between the I3b and I3c intervals. Replicate 235

measurements are shown by black dots and mean values by red dots. The plotted data in 236

found in Supplementary Tables 3 and 4. 237

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(mean=3.84 Mb vs 3.49 Mb, Mann Whitney Wilcoxon test P=0.268) and HEI10 recq4a 259

recq4b populations (mean=3.30 Mb vs 3.08 Mb, Mann Whitney Wilcoxon test P=0.165), 260

observed DCO distances were not significantly different from random (Fig. 3b). This is 261

expected due to increased class II crossovers caused by recq4a recq4b being randomly 262

distributed26. However, the significant reduction in HEI10 DCO distances was unexpected, 263

due to this gene acting in the interference-sensitive ZMM pathway9,12. 264

265

To further investigate crossover interference in HEI10 we used three-colour FTL analysis, 266

using the adjacent I3b and I3c intervals, which measure crossover frequency in a sub-267

telomeric region of chromosome 331,33 (I3bc is located within the 420 interval described 268

earlier) (Fig. 2a and 3c). We used flow cytometry to measure inheritance of pollen 269

fluorescence in wild type and HEI10 and calculate I3b and I3c genetic distances (Fig. 3c and 270

Supplementary Tables 3-4). Both I3b and I3c showed a significant increase in crossover 271

frequency in HEI10, consistent with our previous 420 measurements (X2 test both 272

P=<2.2×10-16) (Figs. 2b, 3c and Supplementary Tables 1 and 3). I3b and I3c genetic 273

distances were used to estimate the number of DCO pollen expected in the absence of 274

interference, using the formula: Expected DCOs=(I3b cM/100)×(I3c cM/100)×total pollen 275

number. The ratio of ‘observed DCOs’ to ‘expected DCOs’ gives the coefficient of 276

coincidence (CoC), and interference is calculated as 1-CoC, such that zero indicates an 277

absence of interference31,33 (Fig. 3c and Supplementary Tables 2-3). Consistent with the 278

reduction in DCO distances seen in the HEI10 GBS data, I3bc interference (1-CoC) 279

significantly decreased from 0.64 in wild type to 0.34 in HEI10 (X2 test P=<2.2×10-16) (Fig. 280

3c and Supplementary Tables 3-4). These experiments confirm our GBS observations and 281

reveal that although HEI10 functions in the interfering ZMM pathway, higher HEI10 dosage 282

causes increased crossover coincidence compared to wild type, although not to the degree 283

observed in recq4a recq4b26 (Fig. 3b). 284

285

Crossover frequency, interhomolog divergence and DNA methylation landscapes 286

We next sought to analyse crossover distributions along the chromosomes, and relate these 287

patterns to other aspects of genome organization (Fig. 4). On average 7.5 crossovers were 288

observed per wild type F2 individual, 5.6 of which occurred in the chromosome arms and 1.9 289

in the pericentromeric heterochromatin (Supplementary Fig. 3 and Supplementary Table 5). 290

In HEI10, recq4a recq4b and HEI10 recq4a recq4b, crossovers in the arms increased 2.3, 4.1 291

and 5-fold, respectively (5.6 -> 13.1 -> 23 -> 28.2 crossovers), whereas the pericentromeres 292


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Figure 4


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Figure 4. Genomic landscapes of crossover frequency, interhomolog divergence and 293

DNA methylation. a. Plots of normalized crossover frequency measured in wild type 294

(black), HEI10 (blue), recq4a recq4b (purple) and HEI10 recq4a recq4b (red). The five 295

chromosomes are plotted on a continuous x-axis, with the positions of telomeres (TEL) and 296

centromeres (CEN) indicated by vertical lines. The position of NBS-LRR resistance gene 297

homologs are indicated by the x-axis ticks. The putative location of an inversion in the 298

recq4a recq4b derived populations is also indicated and labelled ‘INV?’. b. As for a., but 299

showing wild type crossover frequency plotted against Col/Ler SNPs (green)34 and DNA 300

methylation (blue)35. c. As for b., but showing both wild type (black) and HEI10 recq4a 301

recq4b (red) crossover frequency. 302

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Figure 5. Crossover frequency, interhomolog divergence and DNA methylation along 327

telomere-centromere chromosome axes. Analysis of crossover frequency in wild type 328

(black), HEI10 (blue), recq4a recq4b (purple) and HEI10 recq4a recq4b (red), Col/Ler SNPs 329

(green)34 and DNA methylation (blue)35, analysed along the proportional length of all 330

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increases of 1.1, 1.1 and 1.5-fold, respectively (1.9 -> 2.1 -> 2.0 -> 2.7 crossovers), were 361

considerably lower (Supplementary Fig. 3 and Supplementary Table 5). Consistent with 362

previous observations12,27, we observed that despite massive crossover increases throughout 363

the chromosome arms, HEI10, recq4a recq4b and HEI10 recq4a recq4b maintain 364

suppression of recombination within the centromeric regions (Fig. 4a). We also observed that 365

a sub-telomeric region on the long arm of chromosome four showed relative suppression of 366

crossovers, specifically in the recq4a recq4b and HEI10 recq4a recq4b populations (Fig. 4a). 367

This may reflect a lineage-specific sequence rearrangement, such as an inversion, shared 368

among the recq4a recq4b backgrounds. 369

370

We hypothesized that genetic and epigenetic factors could contribute to the observed biases 371

in crossover increases towards the telomeres. Therefore, we compared recombination to 372

patterns of Col/Ler interhomolog divergence34 (i.e. heterozygosity), and DNA cytosine 373

methylation35. Within the chromosome arms we observed that wild type crossovers showed a 374

positive relationship with divergence (r=0.514), which is similar to correlations previously 375

observed between historical recombination and sequence diversity31 (Figs. 4b and 5). In 376

contrast, an opposite, negative correlation was seen between HEI10 recq4a recq4b crossovers 377

and divergence (r=-0.658) (Figs. 4b and 5). This indicates that the crossover elevations seen 378

in HEI10 recq4a recq4b are biased towards the least polymorphic regions of the 379

chromosomes. Hence, while the class II repair pathway that is active in recq4a recq4b is not 380

completely inhibited by heterozygosity, it shows a preference for regions of low divergence. 381

The densely DNA methylated centromeric regions are also strongly crossover suppressed in 382

all populations, consistent with heterochromatin inhibiting meiotic recombination35 (Figs. 4b 383

and 5). Therefore, although combination of HEI10 and recq4a recq4b causes a massive 384

crossover increase, the localization of recombination is significantly constrained by both 385

interhomolog sequence divergence and chromatin. 386

387

Discussion 388

We show that elevating the ZMM crossover pathway, via increased dosage of the HEI10 389

meiotic E3 ligase gene, while simultaneously increasing the activity of non-interfering repair, 390

via mutation of RECQ4A and RECQ4B anti-recombination helicase genes, is sufficient to 391

cause a massive increase in Arabidopsis meiotic crossovers. This is consistent with class I 392

and class II acting as independent crossover repair pathways in plants. HEI10 is a conserved 393

ubiquitin/SUMO E3 ligase with unknown targets during Arabidopsis meiosis, which may 394


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include other ZMM factors1,6,36. In plants HEI10 associates with paired homologous 395

chromosomes throughout meiotic prophase, showing gradual restriction to a small number of 396

foci that correspond to crossover locations9,37. We propose that HEI10 acts quantitatively to 397

promote ZMM pathway crossover repair at recombination sites via SUMO or ubiquitin 398

transfer. Unexpectedly, we show that increased HEI10 dosage causes higher crossover 399

coincidence and therefore a decrease in genetic interference. Crossover interference has been 400

modelled as a mechanical force, thought to be transmitted via the meiotic chromosome axis 401

and/or synaptonemal complex (SC)38. Therefore, HEI10 may modify recombination factors at 402

repair foci and decrease their sensitivity to the interference signal, thereby increasing the 403

likelihood of ZMM-dependent crossover designation. Alternatively HEI10 may alter 404

transmission of the interference signal per se, for example, if components of the axis or SC 405

are SUMO/ubiquitin targets. 406

407

The RECQ4 helicases have biochemically characterised activities in (i) disassembly of D-408

loops and (ii) decatenation of dHJs39–41, and thus can promote non-crossover outcomes at 409

multiple recombination steps post-strand invasion. In the recq4a recq4b mutant it is likely 410

that unrepaired joint molecules persist, which are instead repaired as non-interfering class II 411

crossovers27 (Fig. 1a). We show that combination of genetic backgrounds that increase class I 412

and class II crossovers is sufficient to cause a massive and additive recombination increase 413

from 7.5 to 31 crossovers per Arabidopsis F2 individual. However, given that ~100-200 DSB 414

foci have been cytogenetically observed in Arabidopsis there likely remains the capacity for 415

further crossover increases15–17. As the Arabidopsis anti-crossover pathways do not show 416

complete redundancy22,25,27,42, combination of mutations in the FANCM, RECQ4A-RECQ4B 417

and FIGL1 pathways has the potential to cause further increases. Furthermore, the 418

Arabidopsis MSH2 MutS homolog acts to suppress crossovers specifically when homologous 419

chromosomes are polymorphic43, and therefore introduction of msh2 mutations may further 420

increase recombination. The use of msh2 is attractive, as it may reduce or lessen the bias 421

against crossovers observed in divergent regions in HEI10 recq4a recq4b. Equally, 422

modification of epigenetic information has the potential to increase crossovers in centromeric 423

regions. However, as plant heterochromatin is maintained by multiple interacting systems of 424

epigenetic marks, including DNA methylation, H3K9me2, H3K27me1 and H2A.W44,45, these 425

marks may have differentiated functions in control of recombination35. In conclusion, 426

advanced tailoring of genetic backgrounds may further bias meiotic DSB repair to crossover 427

fates, which has the potential to accelerate crop breeding and improvement. 428


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Materials and Methods 429

430

Plant Materials 431

Arabidopsis lines used in this study were the Col HEI10 line ‘C2’12, Col recq4a-4 (N419423) 43229, Col recq4b-2 (N511130)29 and Ler recq4a line (W387*)27. Genotyping of recq4a-4 was 433

performed by PCR amplification using recq4a-F and recq4-wt-R oligonucleotides for wild 434

type and recq4a-F and recq4-mut-R for recq4a-4. Genotyping of recq4b-2 was carried out by 435

PCR amplification using recq4b-wt-F and R oligonucleotides for wild type and recq4b-mut-F 436

and R oligonucleotides for recq4b-2. Genotyping of recq4a mutation in Ler was performed 437

by PCR amplification using recq4a-Ler-F and R oligonucleotides and subsequent digestion of 438

the PCR products by ScrFI restriction enzyme, which yields ~160 bp products for wild type 439

and ~180 bp products for recq4a. The presence of HEI10 transgene was tested for by PCR 440

amplification using HEI10-F and HEI10-R oligonucleotides. Oligonucleotide sequences are 441

provided in Supplementary Table 6. 442

443

Measurement of crossover frequency using fluorescent tagged lines 444

420 genetic distance was measured using microscopic analysis of seed fluorescence, as 445

described30,31. I3bc genetic distances and the coefficient of coincidence were measured using 446

fluorescent pollen and flow cytometry, as described31. 447

448

Genotyping-by-sequencing 449

Genomic DNA was extracted and used to generate genotyping-by-sequencing libraries as 450

described42. Sequence data was analysed to identify crossover locations using the TIGER 451

bioinformatics pipeline46. In order to generate genetic maps the GBS genotypes called from 452

each library were used to call ‘marker’ genotypes at 1 Mb intervals. These calls were then 453

used as an input for the R package Rqtl in order to generate genetic maps using the Haldane 454

mapping function47. The R package goodfit was used to compare observed crossover 455

numbers per individual to the Poisson expectation. Statistical analysis of FTL crossover 456

frequency and interference measurements were performed as described12,31. 457

458

459

460

461

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Acknowledgements 578

This work was supported by grants from BBSRC (BB/L006847/1), ERA-CAPS/BBSRC 579

‘DeCOP’ (BB/M004937/1), ERC (CoG), the Gatsby Charitable Foundation (GAT2962) and a 580

Royal Society University Research Fellowship. 581

582

Competing financial interests 583

The use of HEI10 to increase meiotic recombination is claimed in UK patent application 584

number GB1620641.9 filed 5th December 2016 by the University of Cambridge. Patents were 585

deposited by INRA on the use of RECQ4 to manipulate meiotic recombination in plants 586

(EP3149027). 587

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Massive crossover elevation via combination of HEI10 and ... · 122 In this work we explore the functional relationship between ZMM pro-crossover and RECQ4 123 anti-crossover meiotic