1

High-resolution molecular karyotyping uncovers pairing between ancestrally-1

related Brassica chromosomes 2

3

Annaliese S. Mason*1,2, Jacqueline Batley1,2, Philipp Emanuel Bayer1,3, Alice Hayward1,2, 4

Wallace A. Cowling5 and Matthew N. Nelson4,5 5

6

*corresponding author: annaliese.mason@uq.edu.au; Tel: +617 336 59556; Fax: + 617 3365 7

3556; Level 2 John Hines Building, The University of Queensland, Brisbane 4072, Australia 8

9

10 1 School of Agriculture and Food Sciences, 2Centre for Integrative Legume Research and 11 3Australian Centre for Plant Functional Genomics, The University of Queensland, Brisbane 12

4072, Australia 13

4 School of Plant Biology and 5 The UWA Institute of Agriculture, The University of Western 14

Australia, 35 Stirling Highway, Crawley 6009, Australia. 15

16

17

18

Figures and Tables: One Table, Seven Figures (all colour) 19

Total Word Count: 5714 20

Section Word Counts: Summary: 193; Introduction: 582; Materials and Methods: 1514; 21

Results: 1499; Discussion: 1876; Acknowledgements: 50. 22

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Accepted for publication 20th December 2013; published manuscript copyright New Phytologist 2014, vol. 202 24

(3): 964-974; DOI: 10.1111/nph.12706. Available at http://onlinelibrary.wiley.com/doi/10.1111/nph.12706/full 25

2

Summary 26

How do chromosomal regions with differing degrees of homology and homeology 27

interact at meiosis? We provide a novel analytical method based on simple genetics 28

principles which can help to answer this important question. This method interrogates 29

high-throughput molecular marker data in order to infer chromosome behavior at 30

meiosis in interspecific hybrids. 31

We validated this method using high-resolution molecular marker karyotyping in two 32

experimental Brassica populations derived from interspecific crosses between B. 33

juncea, B. napus and B. carinata, using a single nucleotide polymorphism (SNP) chip. 34

This analysis method successfully identified meiotic interactions between 35

chromosomes sharing different degrees of similarity: (1) full-length homologues; (2) 36

full-length homeologues; 3) large sections of primary homeologues; and (4) small 37

sections of secondary homeologues. 38

This analytical method can be applied to any allopolyploid species or fertile 39

interspecific hybrid in order to detect meiotic associations. This genetic information 40

can then be used to identify which genomic regions share functional homeology (that 41

is, retain enough similarity to allow pairing and segregation at meiosis). When applied 42

to interspecific hybrids for which reference genome sequences are available, the 43

question of how differing degrees of homology and homeology affect meiotic 44

interactions may finally be resolved. 45

46

47

Keywords: meiosis, polyploidy, recombination, molecular karyotyping, Brassica 48

49

3

Introduction 50 Successful transfer of genetic information from one generation to the next is essential for all 51

organisms. The mechanism for this transfer in most sexually-reproducing eukaryotic species 52

is meiosis. Chromosome behavior during meiosis must be strictly controlled: in order to 53

ensure correct segregation of chromosomes into daughter cells, each chromosome must pair 54

with its homologous partner. However, homologue recognition is one of the least-well-55

understood meiotic processes (Tiang et al., 2012). In most species, the broader process of 56

meiosis is the same: each homologous chromosome must find its partner, associate with it 57

and undergo the reciprocal “crossing-over” process of genetic exchange, creating one or more 58

physical ties (chiasma) between the chromosomes to ensure correct first division disjunction 59

(Wilson et al., 2005). During homologous chromosome pairing, homologues are roughly 60

aligned before close-range “homology checking” and elimination of associations based on 61

repetitive DNA sequences is then thought to occur (Bozza & Pawlowski, 2008). Although 62

homologous chromosome pairing relies on DNA sequence homology (Bozza & Pawlowski, 63

2008), the exact relationship between DNA sequence similarity and homologue recognition is 64

unknown (Tiang et al., 2012): how similar do genomic sequences have to be to initiate 65

chromosome pairing at meiosis? In particular, how are homologues recognised, and how are 66

meiotic interactions regulated between genomic regions with different levels of sequence 67

homology? 68

69

This question can be addressed in allopolyploid species that are formed when two related 70

diploid species hybridise. Polyploidy is common in many if not most plant and animal 71

species lineages (Otto & Whitton, 2000; Leggatt & Iwama, 2003; Van de Peer et al., 2009; 72

Jiao et al., 2011). Some angiosperm families such as the Brassicaceae have undergone 73

multiple polyploidy events such that the present day species contain homeologous (that is, 74

ancestrally-related) chromosomal regions with a spectrum of sequence similarities (Fig. 1). 75

High-resolution molecular karyotyping is now becoming feasible in non-model species with 76

the increasing availability of high-density genotyping arrays and genotyping-by-sequencing 77

(Elshire et al., 2011; Poland et al., 2012; Cavanagh et al., 2013; Edwards et al., 2013). 78

Therefore, the stage is set for significant advances in our understanding of just how similar 79

DNA sequences have to be for homologue recognition to occur, and of how hybrids and 80

polyploids regulate meiosis when several genomic regions with different levels of sequence 81

4

homology exist. However, the analytical tools for interrogating large molecular genotyping 82

datasets to answer these questions remain under-developed. 83

84

In this study, we address this issue by developing a novel method that is capable of 85

interrogating large molecular karyotyping data sets to rapidly assess chromosome behavior at 86

meiosis in allopolyploid species or interspecific hybrids. This approach uses a set of simple 87

statistical assumptions (Fig. 2) to infer chromosome pairing behavior. A hybrid between two 88

species/genotypes is crossed to a third species/genotype to make a testcross, and then the 89

resulting progeny are assessed for allele inheritance. Segregation of alleles from the two 90

original parent species is inferred to result from chromosome pairing during meiosis in the 91

hybrid. To validate this method we used the well-characterised Brassica system (Fig. 1), for 92

which a high-density single nucleotide polymorphism (SNP) genotyping array has recently 93

been developed (http://www/illumina.com). We show that this SNP array can be applied to 94

detect homologous and homeologous chromosome pairing in segregating progeny derived 95

from hybrids between the Brassica allotetraploid species (Fig. 3). This analysis is supported 96

by a previous cytogenetic study of the same genotypes (Mason et al., 2010) and confirms the 97

validity of our molecular karyotype analysis pipeline, providing novel, interesting data 98

related to chromosome pairing behaviour in Brassica. 99

100

101

Materials and Methods 102 Plant material and growth conditions 103

Two experimental populations were developed by intercrossing homozygous lines of three 104

allotetraploid Brassica species as described previously by Mason et al. (2012) and 105

summarised in Fig. 3. Two to five F1 hybrids per hybrid type (B. napus “N1” × B. juncea 106

“J1”; B. juncea “J1” × B. napus “N1”; B. juncea “J1” × B. carinata “C1” and B. juncea “J1” 107

× B. carinata “C2”;) were used to generate testcross progeny; generation of the F1 hybrids is 108

described in Mason et al. (2011). No significant differences in allele inheritance were 109

observed between “N1J1” and “J1N1” reciprocal hybrids (B. juncea × B. napus (F1: 110

AjAnBjCn) or between the “J1C1” and “J1C2” genotypes (B. juncea × B. carinata (F1: 111

BcBjAjCc): these were subsequently considered as two single populations for the purposes of 112

the analyses. A total of 41 (B. juncea × B. napus (F1: AjAnBjCn)) × B. carinata experimental 113

progeny (out of 42 seeds planted, germination rate 98%) and 62 (B. juncea × B. carinata (F1: 114

5

BcBjAjCc)) × B. napus experimental progeny (out of 73 seeds planted, germination rate 85%), 115

derived from meiotically reduced gametes of AjAnBjCn and BcBjAjCc hybrids (Mason et al., 116

2012), comprised the final experimental populations in this study (Fig. 3). 117

118

Molecular karyotyping using the Illumina 60K Brassica SNP chip 119

Recently, an Illumina Infinium 60 000 SNP array was developed and released for Brassica 120

napus (http://www.illumina.com/). This chip comprised 52 157 SNPs distributed across the A 121

and C genome chromosomes. B genome inheritance could not be characterised in our 122

populations due to lack of available high-throughput molecular tools for this genome. 123

Hybridisation protocols were run according to manufacturer’s instructions for all samples in 124

the population plus controls (parent species and F1 hybrids), and chips were scanned using an 125

Illumina HiScanSQ. Genotyping data were visualised using Genome Studio V2011.1 126

(Illumina, Inc. (San Diego)). 127

128

SNPs were filtered in Genome Studio to retain genome-specific SNPs that were: a) 129

polymorphic between the B. juncea and B. napus A genomes (An and Aj) and did not amplify 130

in B. carinata (2n = BcBcCcCc), or were b) polymorphic between the B. napus and B. carinata 131

C genomes (Cn and Cc) and did not amplify in B. juncea (2n = AjAjBjBj), for the genotypes 132

used in the experimental populations. Filtering for polymorphism between the segregating 133

parent alleles yielded 4 883 SNPs for the A genome, and 7 215 SNPs for the C genome. 134

These 12 098 SNPs were then manually screened in Genome Studio: SNPs which did not 135

show three clear genotype clusters (AA, AB, BB) in the population were removed, leaving 3 136

705 A-genome and 6 489 C-genome SNPs (10 194 SNPs in total). After filtering and quality 137

control measures were implemented, the total number of SNP markers retained for 138

segregation analysis was 3 046 in the B. napus An genome, 4 643 in the B. napus Cn genome, 139

3 055 in the B. juncea Aj genome and 4 687 in the B. carinata Cc genome (Supplementary 140

Table 1). On average, 406 SNP markers were retained per chromosome, with a minimum of 141

124 (B. napus Cn5) and a maximum of 1 279 (B. carinata Cc4) (Supplementary Information). 142

143

Marker genotype calls were imported into Microsoft Excel 2010 (Microsoft Corporation) 144

where SNPs were sorted by chromosome, with reference to the published and available draft 145

reference Brassica diploid genome sequences: B. rapa (Wang et al., 2011) and B. oleracea 146

(http://brassicadb.org/brad/; (Cheng et al., 2011)). Further data cleaning was undertaken to 147

6

remove SNPs that: a) showed allelic segregation patterns inconsistent with the determined 148

genomic locations; b) appeared to detect two or more loci; c) had an excess of no-calls (NCs) 149

or d) were null for one of the parental alleles. 150

151

In the AjAnBjCn population, several blocks of markers (A01_015 – A01_37; A05_300 – 152

A05_305; A10_001 – A10_005; C1_041 – C1_053; C4_1278 – C4_1279; C8_0001 – 153

C8_0004) amplified both an A- and a C-genome allele in B. napus, but only an A-genome or 154

a C-genome allele in B. juncea and B. carinata respectively. These markers were eliminated 155

from the AjAnBjCn (B. juncea × B. napus) population analyses, but retained in the BcBjAjCc 156

(B. juncea × B. carinata) population analyses. For the known B. napus A7 – C6 interstitial 157

homeologous reciprocal translocation in the parental genotype “Surpass 400” (Osborn et al., 158

2003), markers were named according to their physical location, such that “A7” in B. napus 159

comprised what would be part of C6 in B. oleracea and B. carinata, and “C6” in B. napus 160

comprised part of what would be A7 in B. juncea and B. rapa (see Supplementary Table 1). A 161

putative duplication in B. napus covered 17% of An10, from markers A10_284 to A10_341, 162

but was not obviously linked to any other genomic region. 163

164

The SNP data set was highly saturated: many markers were redundant in terms of detecting 165

chromosome segment inheritance and recombination events across each population. Hence, 166

in order to facilitate the segregation analyses and crossing-over analyses, SNP marker data 167

was reduced to one representative marker per haplotype block representing unique 168

information per chromosome across each population (i.e. marker bins). SNPs providing 169

redundant information across the population were removed using an R script that treated 170

missing values (NCs) as wild cards (either 1 or 0) and retained markers with the fewest 171

missing values (Supplementary Information). In the BcBjAjCc testcross population, an 172

average of 19 marker bins per Aj-genome chromosome (range 3 to 33) and 18 marker bins 173

per Cc-genome chromosome (range 3 to 29) were retained after elimination of redundant 174

SNPs. In the AjAnBjCn testcross population, an average of 58 marker bins (range 36 to 93) 175

were retained in the B. juncea Aj and B. napus An genomes and an average of six marker bins 176

(range 2 to 18) were retained in the B. napus Cn genome after elimination of redundant SNPs. 177

178

Data analysis, data visualisation and statistics 179

7

Fisher’s exact test for count data with a significance level of p < 0.05 was used to test if pairs 180

of marker alleles were segregating with one another, as would be expected from e.g. the 181

presence of the two alleles at a single homologous locus (Fig. 2). To test whether any given 182

two marker alleles present anywhere in the genome(s) were segregating with each other, 183

every possible combination of two alleles was concatenated across individuals, such that each 184

individual was scored for presence of both alleles (1/1), presence of only one of the pair of 185

alleles (1/0 or 0/1) or absence of both alleles (0/0). The number of observations in each 186

category was then summed and compared to expected values derived from sums for 2 × 2 187

contingency tables (Fig. 2). Expected values were derived from normal Mendelian 188

expectations for segregation of individuals with both alleles present (1/1), one allele present 189

(1/0 or 0/1) or neither allele present (0/0) for each pair of alleles (1:1:1:1), using contingency 190

tables to adjust for alleles with an excess of 0 or 1 scores. Every marker allele with a known 191

genome location was tested against every other marker allele with a known genome location. 192

193

Deviation from this ratio as a result of linkage was expected to result in an excess of 194

individuals with scores of 0/0 and 1/1 due to co-segregation of pairs of alleles (Fig. 2). 195

Deviation from this ratio as a result of segregation of the two alleles was expected to result in 196

an excess of individuals with scores of 0/1 or 1/0 (only one allele present of the pair) (Fig. 2). 197

A significant deviation which resulted in an excess of individuals with a score of 0/1 or 1/0 198

(one allele present for each pair) was deduced to be the result of segregation of the two alleles 199

(Fig. 2). Significant (p < 0.05) results of the pairwise Fisher’s exact tests for count data are 200

presented in figures only where the sum of 0/1 and 1/0 scores was greater than the sum of 0/0 201

and 1/1 scores, as expected from segregation of alleles. P-values are presented uncorrected 202

for multiple testing, and instead with a range of p-values encompassing rigorous correction 203

cut-offs, as the assumption of independence is invalid for tests of linked alleles. However, the 204

stringent Bonferroni corrections for multiple testing values assuming independent tests were 205

p < 0.00014 for the BcBjAjCc hybrid population and p < 0.000041 for the AjAnBjCn hybrid 206

population for the final set of alleles used for analysis of each population. 207

208

In the absence of meiotic crossing over in AjAnBjCn and BcBjAjCc hybrids, marker alleles on 209

each haploid chromosome in the experimental progeny were expected to be either all present 210

or all absent. Recombination was manifested as changes in the presence or absence of marker 211

8

alleles along haploid chromosomes. Recombination events were recorded for all A- and C-212

genome chromosomes in each population. 213

214

R version 3.0.0 (The R Project for Statistical Computing) was used to carry out the statistical 215

analyses, to reduce duplicate markers to single representatives per block and to generate the 216

associated figures (Supplementary Text S1). Heatmap figures showing allele associations 217

were generated using R software package “gplots”, and linkage disequilibrium was calculated 218

using the “LD” function in software package “genetics”. 219

220

Results 221 Molecular karyotyping 222

A total of 3 046 SNPs polymorphic between the B. napus and B. juncea A genomes (An and 223

Aj), and a total of 4 643 SNPs polymorphic between the B. napus and B. carinata C genomes 224

(Cn and Cc), were selected for final analysis. Linkage disequilibrium values (r, p-value and 225

D') were calculated for every pair of alleles in each of the two populations to provide 226

additional information, and linkage disequilibrium was identified between both adjacent 227

alleles and segregating alleles (Supplementary Tables S4, S5). Using the molecular 228

karyotyping pipeline in R (Supplementary Text S1), every SNP allele in each of the two 229

testcross populations (Fig. 3) was tested for significant segregation (Fig. 2) with every other 230

SNP allele in the same population. This approach allowed discrimination between linkage 231

disequilibrium due to segregation of alleles, and linkage disequilibrium due to co-segregation 232

of alleles (linkage). Increasing significance corresponded to increasing numbers of 233

individuals in the population with either one allele or the other (but not both or neither) for 234

any two alleles. 235

236

A-genome allele segregation in AABC hybrids 237

In testcross population A (Fig. 3), either an Aj or an An allele was present at most homologous 238

loci, as evidenced by the strong diagonal of significant segregation between Aj and An alleles 239

in Fig. 4. In the A genome, which was present as homologous chromosomes from B. juncea 240

(Aj) and B. napus (An), most segregating allele pairs corresponded to homologous loci in the 241

A genome (Fig. 4). Most B. juncea Aj-genome chromosomes segregated with high fidelity 242

with their B. napus An-genome homologues. An exception was part of chromosome A7, 243

9

where strong associations between Aj and An alleles were absent and this region instead 244

showed an Aj7 – Cn6 association. 245

246

Segregation in the BBAC hybrids between alleles sharing primary homeology 247

In testcross population B (Fig. 3), B. juncea A- genome alleles (Aj) segregated with B. 248

carinata C-genome alleles (Cc) with which they shared primary homeology (Fig. 5; see Fig. 1 249

for detailed primary homeologous relationships). These associations between homeologous 250

alleles were of similar significance to those between two alleles at a single homologous locus 251

(p < 0.00000001, Fig. 5). 252

253

Four Aj-Cc chromosome pairs showed significant segregation of homeologous alleles along 254

their entire length: Aj1/Cc1, Aj2/Cc2, Aj3/Cc3 and Aj7/Cc6 (Fig. 5), as indicated by the 255

diagonal red lines across the length of the chromosome blocks. Chromosomes Aj4 and Cc4 256

showed segregation of alleles along the whole length of Aj4, but with decreasing significance 257

(0.00000001 < p < 0.001) towards the end of Cc4 (from ~40 – 55 Mbp). Aj9 and Cc8 alleles 258

segregated along the entire length of Cc8, with the top 8 Mbp of Aj9 segregating instead with 259

the top of Cc9. Clear evidence of trivalent formation was apparent between chromosomes 260

Aj9, Cc8 and Cc9, with Aj9 chromosomes showing matching allele segregation patterns and 261

crossovers with Cc8 at one end and Cc9 at the other end of the chromosome. Likewise, 262

strongly significant segregation was observed between alleles on part of Aj10 and Cc9, with a 263

switch in Cc9 preference to Aj9. 264

265

Other chromosomes showed more fragmentary allele segregation across regions of A-C 266

homeology (Fig. 5, Supplementary Table S4). For example, Aj5 alleles preferentially 267

segregated with Cc5 alleles, but segregation with Cc4 and Cc6 alleles was also observed. Aj8 268

alleles showed little tendency to segregate with Cc alleles, but significant segregation 269

associations were observed with the top of Cc8 and the bottom of Cc9. Similarly, alleles on 270

chromosome Cc7 showed only weak segregation with the alleles in the region of primary 271

homeology at the bottom of chromosome Aj3. 272

273

Segregation between alleles sharing secondary homeology 274

Evidence was also obtained for weakly significant segregation between alleles in regions of 275

secondary homeology (derived from ancient polyploidy events) within each haploid genome 276

10

in both testcross populations, i.e. autosyndesis (Aj-Aj: within the B. juncea A genome; Cc-Cc: 277

within the B. carinata C genome and Cn-Cn: within the B. napus C genome) (Fig. 6, 278

Supplementary Figures S4, S5). 279

280

The largest clusters of segregation between regions of secondary homeology were observed 281

for chromosomes Aj2 and Aj10 and for chromosomes Cc2 and Cc9 (Fig. 6a, 6b). The Aj2 –282

Aj10 association spanned approximately 5 – 10 Mbp based on putative SNP positions, 283

whereas the Cc2 – Cc9 association was significant over 35 – 45 Mbp. Another association was 284

also present between Cc3 and Cc9 over a range of approximately 15 – 35 Mbp. Only two 285

weak regions of segregation between Cn alleles were detected within the haploid B. napus C 286

genome in the AjAnBjCn hybrid type. One of these associations was between Cn2 and Cn9 287

(Fig. 6c), as was observed for Cc2 and Cc9. 288

289

Recombination events: distribution and frequency 290

Chromosome segregation as predicted by the molecular karyotyping analysis using R 291

(Supplementary Text S1) was independently validated by manual inspection of chromosome 292

segregation and recombination events: Brassica Aj and Cc allele segregation patterns between 293

known primary homeologues were assessed (Table 1). In general, predicted associations from 294

molecular karyotyping could be validated by manual assessment of allele segregation and 295

evidence of crossover formation for each chromosome pair (Fig. 5, Table 1). The location of 296

recombination breakpoints along individual chromosomes was assessed for chromosomes 297

that were neither completely absent nor completely present (i.e. recombinant chromosomes). 298

The distribution of breakpoints along each chromosome is shown in Supplementary Figures 299

S1 to S5. 300

301

Aj-An recombination frequencies 302

Homologous Aj and An chromosomes were highly recombined in the AjAnBjCn testcross 303

population, averaging 2.4 crossover breakpoints per chromatid per individual (Fig. 7a). As 304

many as eight breakpoints per chromatid were observed for A3 and A9. A-genome 305

chromatids were transmitted intact without recombination only 8% of the time on average 306

(range 0-15%), except for An7 (unrecombined 22% of the time) which appeared to have the 307

structural An7-Cn6 translocation variant common to many B. napus genotypes. The region on 308

An7 corresponding to the translocation had no recombination breakpoints within it, but many 309

11

recombination breakpoints flanking this region (Supplementary Figure S2). Otherwise, 310

recombination breakpoints were distributed along the lengths of the chromosomes, with a 311

general tendency towards increased frequency approaching distal regions (Supplementary 312

Figure S1, Supplementary Figure S2). 313

314

Cn recombination frequencies 315

Recombination involving secondary homeologues in the haploid B. napus C genome in the 316

AjAnBjCn testcross population occurred at much lower frequencies, averaging 0.26 Cn 317

chromosome breakpoints per plant (Fig. 7b). Cn chromosomes were either wholly present or 318

wholly absent (unrecombined, either lost or transmitted intact as univalents) 59 – 95% of the 319

time, except for 46% of Cn6 chromatids which had undergone recombination with 320

chromosome Aj7 (again as a result of the An7-Cn6 translocation variant). Cn6 had a proximal 321

cluster of recombination breakpoints. However, few recombination breakpoints were 322

observed overall in Cn chromosomes, and these tended to be distally located (Supplementary 323

Figure S3). 324

325

Aj-Cc recombination frequencies 326

Recombination was frequent between primary homeologues in the Aj and Cc genomes in the 327

BcBjAjCc hybrid progeny (Fig. 7c) with an average of 0.46 chromosome breakpoints 328

observed per chromosome per plant. On average, 58% of Aj and Cc chromosomes were 329

transmitted without detectable recombination events. This was not significantly different 330

from the 50% expected to result from a single crossover event per homologous chromosome 331

pair per meiosis, whereby two out of four chromatids would be expected to be transmitted 332

without evidence of recombination (p=0.09, Student’s paired t-test against the mean). 333

However, some chromosomes were far less likely to recombine than others (Fig. 7c): 334

chromosomes Cc7 and Aj8 each had only two detectable recombination events across the 335

whole population. Recombination breakpoints were distributed distally on every 336

chromosome, except in chromosomes Aj7 and Cc6 which had proximal clusters of 337

breakpoints (Supplementary Figures S4, S5). 338

339

Detecting non-random co-inheritance of alleles across genomes 340

In addition to above segregation and recombination analyses, we also sought evidence of 341

positive associations between alleles that would indicate significant co-inheritance of 342

12

chromosomes. In addition to the expected co-inheritance of alleles physically located on the 343

same chromosomes, we found some parts of the genome were significantly associated in this 344

way (Supplementary Table 2, Supplementary Table 3). This generally occurred when two 345

chromosomes both associated with a third chromosome (Supplementary Table 2, 346

Supplementary Table 3). Examples included Cc2 and Cc3 (both paired with Cc9), Cc8 and Cc9 347

(both paired with Aj9), Cc7 and Cc3 (both paired with Aj3) and Aj2 and Cc9 (both paired with 348

Cc2). 349

350 Transmission frequencies of marker alleles from AjAnBjCn and BcBjAjCc hybrids to 351

progeny 352

In the B. juncea × B. napus (AjAnBjCn) hybrids, most Aj, An and Cn genome alleles were 353

transmitted to the testcross population according to normal Mendelian expectations (50% 354

frequency; p > 0.05, Pearson’s χ2 test), (Supplementary Fig. S6). Some bias towards retention 355

of Aj or An alleles was observed over short regions (Supplementary Fig. S6). Alleles from the 356

B. napus chromosome Cn8 and half of chromosome Cn4 were retained more often than 357

expected by chance, and no chromosome was lost more often than the expected 50% 358

frequency (Supplementary Fig. S7). In the B. juncea × B. carinata (BcBjAjCc) testcross 359

population, B. juncea Aj-genome chromosomes were retained more often than B. carinata Cc 360

genome chromosomes for every chromosome except Aj7 and Cc6 (Supplementary Fig. S8). 361

362

363

Discussion 364 In this study, we developed a novel open source analytical pipeline suitable for inferring 365

chromosome segregation in large data sets (see Supplementary Text S1). This molecular 366

karyotyping approach uses a set of simple genetic principles (Fig. 2) to infer chromosome 367

pairing behavior. A hybrid between two species or two genotypes is crossed to another 368

species/genotype to make a testcross (Fig. 3), and then the resulting progeny are assessed for 369

allele inheritance using high-throughput SNP molecular markers. Segregation of alleles from 370

the original two species can then be inferred to result from chromosome pairing during 371

meiosis in the hybrid without prior knowledge of chromosome relationships. This method can 372

provide new insight into homeologous and homologous relationships in species complexes by 373

determining homologous and homeologous relationships between chromosomes and 374

13

estimating stability of chromosome inheritance. By assessing functional chromosome 375

interactions it would also allow empirically-based predictions of transfer of alleles between 376

genomes in interspecific hybrids, a matter of importance for estimating the risk of transgene 377

escape from transgenic crop species to their wild relatives (Chèvre et al., 1997). In future, 378

and with the increasingly availability of genotyping-by-sequencing approaches, this 379

analytical method may provide us with the answer to the question of how similar DNA 380

sequence has to be to pass homology checks and initiate chromosome pairing during meiosis. 381

382

Successful detection of homologous, homeologous and autosyndetic chromosome segregation 383

High-resolution molecular karyotyping of two novel interspecific Brassica populations was 384

carried out to validate this approach. A spectrum of genome interactions was revealed, 385

ranging from almost completely regular pairing and recombination of homologues (A-386

genome chromosomes from B. napus and B. juncea in the AjAnBjCn hybrids; Fig. 4), to 387

mostly regular pairing and reduced recombination frequencies between primary homeologues 388

(A- and C-genome chromosomes in the BcBjAjCc hybrids; Fig. 5), down to low-level 389

autosyndetic chromosomal interactions between secondary homeologues (within the haploid 390

B. napus Cn genome, Fig. 6c). In other words, this molecular karyotyping analysis revealed 391

exactly which regions of both ancient (secondary) and recent (primary) homeology in the 392

Brassica A- and C-genomes have retained enough sequence similarity to form pairing 393

associations at meiosis (Fig. 4, 5 and 6), and this was validated by manual inspection of allele 394

segregation patterns (Table 1, Fig. 7). 395

396

Molecular karyotyping detects segregation between alleles at homologous loci 397

Segregation of homologous alleles belonging to the B. juncea and B. napus Aj and An 398

genomes (Fig. 4) supports the status of these genomes as predominantly un-rearranged 399

relative to each other (Parkin et al., 1995; Axelsson et al., 2000). However, one major 400

discrepancy was observed: an An7-Cn6 translocation in the B. napus line used in this 401

experiment (Osborn et al., 2003) resulted in associations between Aj7 and part of Cn6 (Fig. 4, 402

Supplementary Table S3, Supplementary Figures S1 and S3) rather than between Aj7 – An7 403

over the region of the translocation. Several genomic regions showed amplification of two B. 404

napus alleles at each locus rather than one B. napus allele (An1, Cn1, An5, Cn4, An10 and 405

Cn8), but amplified only one allele per locus in B. juncea and B. carinata. This phenomenon 406

may have been due to non-specificity of SNP primer binding and/or divergence between the 407

14

genomic sequences of B. napus, B. juncea and B. carinata. Therefore, these regions were 408

removed from the analysis except the largest, which comprised 30% of chromosome An10 (5 409

Mb). However, it is more probable that this observation is due to the presence of 410

duplication/deletion events between the An and Cn genomes in the B. napus parent, as similar 411

chromosome rearrangements have been observed in other cultivars of this young 412

allopolyploid species (Udall et al., 2005). 413

414

Molecular karyotyping predicts segregation between regions of primary homeology 415

In the BcBjAjCc hybrids, Aj-Cc allosyndetic (between-genome) recombination between 416

primary homeologous regions approached the high levels observed between homologues in 417

natural Brassica species. Nicolas et al. (2007) observed that only half of the chromosome 418

rearrangements transmitted from B. napus haploids resulted from recombination between 419

regions of primary homeology, with the rest resulting from recombination between other 420

regions of the genome. By contrast, in our BcBjAjCc population the vast majority of crossover 421

events appeared to derive from regions of primary homeology: for chromosome pairs Aj1 – 422

Cc1, Aj2 – Cc2 and Aj3 – Cc3 we predicted 94 – 100% pairing between primary homeologues 423

(Table 1), and in general only a few convincing associations between regions of secondary 424

homeology were detected (Fig. 6). The contrasting results of these two studies are surprising, 425

but may be due to differences in genome structure or genetic factors between the two 426

population types (Leflon et al., 2010; Suay et al., 2013), or to different selection pressures in 427

the generation of the experimental populations. 428

429

Our study suggests that the most frequently formed Aj-Cc bivalents are likely to be collinear 430

pairs Aj1-Cc1, Aj2-Cc2, Aj3-Cc3 (Fig. 5), supporting previous analyses of primary A-C 431

homeology (Parkin et al., 1995). However, the chromosome pair Aj7-Cc6, which lacks whole-432

chromosome collinearity (Parkin et al., 2003), was also very strongly associated across their 433

entire length (Fig. 5). This is surprising, as the B. juncea A genome and the B. carinata C 434

genome lack the interstitial An7 – Cn6 reciprocal translocation found in many genotypes of B. 435

napus (Osborn et al., 2003), including the genotype used in this study to generate the 436

AjAnBjCn hybrid population. In fact, several Aj-Cc chromosome regions that would be 437

predicted to show segregation based on primary homeology (Fig. 1) showed little to no 438

association (Fig. 5, Table 1). For instance, Aj7 and Cc6 also share primary homeology with 439

Aj6 and Cc7 (Fig. 1), but no Aj6 - Cc6 or Aj7 - Cc7 segregation was observed (Fig. 5). Hence, 440

15

factors other than primary sequence homeology must be influencing the Aj7 – Cc6 441

association, and the lack of association between other known homeologues. Concentration of 442

recombination breakpoints around the 8-10 Mbp region of Aj7 and the 15-25 Mbp region of 443

Cc6 in this population (Supplementary Figure S4, S5) suggests an unusual mode of bivalent 444

formation between these two chromosomes: other Aj-Cc chromosome pairs appeared to form 445

mostly distal associations (Supplementary Figure S4, S5). 446

447

Genetic factors present on single chromosomes that affect genome-wide recombination 448

frequency have recently been found in Brassica (Suay et al., 2013). However, genetic factors 449

influencing recombination between particular chromosomes have yet to be identified in the 450

Brassica genus, although chromosome-specific genetic factors have been identified in other 451

species (Jackson et al., 2002). Hence, certain chromosomes or chromosome pairs (e.g. Aj7 – 452

Cc6) may harbour, or be the target of, specific genetic factors encouraging crossover 453

formation involving that chromosome. The “choice” of pairing partner may also be 454

influenced by a combination of availability of other suitable partners (Nicolas et al., 2008), 455

genetic factors and the location of the homeologous sequence in relation to the centromeres 456

as well as degree of sequence similarity (Nicolas et al., 2012). 457

458

Chromosome associations detected for Aj8, Cc8, Aj9, Cc9 and Aj10 were strongly indicative 459

of multivalent formation or partner swapping (Fig. 5), consistent with the status of these 460

linkage groups as the most rearranged between the A and C genomes (Parkin et al., 2003). At 461

least one multivalent association per cell involving A- and C-genome chromosomes only was 462

observed by cytogenetic means by Mason et al. (2010) for this BcBjAjCc hybrid type: 0.5 Aj-463

Aj-Cc (0-2 per cell), 0.3 Cc-Cc-Aj (0-2 per cell) and 0.1 Aj-Aj-Cc-Cc (0-1 per cell). These 464

multivalents are most likely attributable to Cc8, Aj9, Cc9 and Aj10 based on these current 465

findings. 466

467

Molecular karyotyping predicts segregation between regions of secondary homeology 468

Significant autosyndesis (within-genome pairing) in both the A and C genomes was predicted 469

on the basis of segregation between alleles (Fig. 6). These associations are likely due to 470

shared ancestral karyotype blocks resulting from the ancient genomic triplication before the 471

divergence of the Brassica A and C genomes (Parkin et al., 2005; Schranz et al., 2006; Cheng 472

et al., 2013). The three strongest associations observed were between Aj2 and Aj10, between 473

16

Cc2 and Cc9 and between Cc3 and Cc9, all in the BcBjAjCc hybrid population. These 474

chromosomes all share two large conserved ancestral karyotype blocks in the same 475

orientation and terminal to the end of the chromosome (Schranz et al., 2006; Cheng et al., 476

2013). An association between Cn2 and Cn9 was also observed in the AjAnBjCn hybrid 477

population. These autosyndetic associations suggest that associations may form between 478

regions of ancient homeology even in the presence of recent homeologues. 479

480

Recombination frequency and allelic selection pressure 481

Recombination between the B. juncea and B. napus A genomes was greatly enhanced relative 482

to that observed in natural and resynthesised B. napus (Nicolas et al., 2008). This is probably 483

due to the presence of additional univalent chromosomes during meiosis in this hybrid type 484

(Mason et al., 2010) as observed previously in other Brassica hybrids (Leflon et al., 2010). 485

Hence, this hybrid type may offer a useful bridge in Brassica for breeders aiming to break up 486

regions of undesirable linkage disequilibrium, following a trend of interspecific hybridisation 487

for crop improvement in this genus (Rygulla et al., 2007; Navabi et al., 2010; Chen et al., 488

2011; Zou et al., 2011). In the BcBjAjCc hybrid-derived progeny, A-genome loci were 489

retained in preference to C-genome loci (Supplementary Fig. S8). As B. juncea was the 490

maternal parent in the initial crosses, this may indicate a cytoplasmic effect on preferential 491

genome inheritance, as suggested previously in crosses between natural and synthetic B. 492

napus (Szadkowski et al., 2010). Several genomic regions also appeared to be under retentive 493

selection pressure in both hybrid populations (Supplementary Figures S6, S7, S8), and may 494

harbor alleles related to success in interspecific hybridisation or other processes conferring a 495

viability advantage. 496

497

Selection for particular chromosome complements through viability advantage 498

Viability advantage for particular karyotypes could affect the success of the molecular 499

karyotyping approach. For instance, Xiong et al (2011) found strong “dosage compensation” 500

between homeologues, such that presence of four copies of primary homeologue pair A1/C1 501

(e.g. A1/A1/C1/C1, A1/A1/A1/A1, A1/C1/C1/C1 etc.) was selected for in advanced 502

generations of synthetic B. napus. Similar dosage compensation of homeologous 503

chromosome sets has been observed in neo-allopolyploid Tragopogon miscellus (Chester et 504

al., 2012; Chester et al., 2013). Hence, a similar effect could occur in our BcBjAjCc hybrids to 505

eliminate gametes which have not inherited a copy of either chromosome A1 or C1 for 506

17

example, to give the false appearance of chromosome segregation and hence pairing at 507

meiosis. However, recombination events between primary homeologues were manually 508

validated (Table 1), and putative crossover frequency was not significantly different to 509

predictions for one crossover event per chromosome per gamete. Therefore, we predict that 510

the majority of segregation associations identified through the molecular karyotyping analysis 511

are the result of actual chromosome pairing and segregation. In systems with unknown 512

chromosome homeology, even dosage compensation effects will provide information about 513

chromosome similarity, as only primary homeologues are predicted to provide “dosage 514

compensation” (Xiong et al., 2011). 515

516 Conclusions 517

In future, as reference genome sequences for polyploid species (including Brassica crop 518

species) become available, we will have the opportunity to identify at base-pair resolution the 519

chromosomal regions initiating homeologous pairing at meiosis. We provide a simple 520

analytical pipeline that can, once whole genome sequences become available, be interrogated 521

by whole-chromosome comparative sequence analysis to deduce exactly how similar 522

genomic regions need to be to pair and segregate at meiosis. Similar approaches using our 523

analysis pipeline may also be taken in highly complex polyploid species, helping us 524

understand how chromosome pairing is controlled in complex polyploids with homeology 525

from ancient and common ancestors. 526

527

528

Acknowledgements 529 This work was made possible by prior support by the Australian Research Council Linkage 530

Project LP0667805, with industry partners Council of Grain Grower Organisations Ltd and 531

Norddeutsche Pflanzenzucht Hans-Georg Lembke KG. ASM was supported and additional 532

work funded by an Australian Research Council Discovery Early Career Researcher Award 533

(DE120100668). 534

535

536

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- 22 -

Figure Legends Figure 1: Rearrangements between the Brassica A and C genomes (relative to the A

genome). Regions of primary homeology between the A and C genomes are represented by

different colours. Secondary homeology within the A genome is indicated by letters in light,

medium and dark grey font representing the order of ancestral karyotype blocks resulting

from the ancestral Brassiceae genome triplication (most fractionated subgenome 1 = light

grey, most fractionated subgenome 2 = medium grey and least fractionated subgenome = dark

grey, see Cheng et al. (2013)). Approximate centromere positions in the C genome are based

on cytogenetic information (Xiong & Pires, 2011). Data for this figure were synthesised from

Parkin et al. (2003), Parkin et al. (2005), Schranz et al. (2006), Xiong and Pires (2011) and

Cheng et al. (2013).

Figure 2: The relationship between allele presence and absence for two different alleles

at unknown genomic locations transmitted on individual chromatids in an example

population (n = 16) expected as a result of linkage, segregation or no relationship. The

parent of the population is assumed to be a perfect F1 heterozygote, such that its parents have

different alleles for every locus in the population, but no information about the relationship of

any two alleles is assumed. A) Linkage between two alleles, as would be observed if allele A

and B were present at loci close together on the same chromosome. The blue box indicates a

recombination event that has occurred between the locus of allele A and allele B to separate

parent alleles that were on a single chromatid in the F1 heterozygote parent. B) Segregation

between two alleles, as would be observed for two homologous alleles at a single locus, or for

alleles at two homeologous loci that were undergoing meiotic pairing. The orange and purple

boxes indicate the inheritance of neither and both parental alleles for that allele pair,

respectively. If both alleles were present at a single homologous locus, this would represent

homologous pairing failure, such that a multivalent or univalent chromosome association

resulted in transmission of both or neither allele to a resulting daughter cell. C) No

relationship between two alleles, as expected for the majority of alleles randomly tested in a

segregating population.

Figure 3: Schematic diagram of interspecific crossing undertaken to produce two

interspecific Brassica experimental populations, following Mason et al. (2012); (A) (B.

- 23 -

juncea × B. napus) × B. carinata population and (B) (B. juncea × B. carinata) × B. napus

population.

Figure 4: Segregation of A-genome alleles in a B. juncea × B. napus (AjAnBjCn)-derived

testcross population. Statistically significant interactions between homologous regions are

shown (Fisher’s exact test for count data). The Bonferroni correction for multiple testing at

the p < 0.05 significance level in this population is p < 4.12E-05. Only non-redundant SNP

alleles are presented, arranged sequentially according to their genetic location (not drawn to

scale).

Figure 5: Segregation of A- and C-genome alleles in a B. juncea × B. carinata (BjBcAjCc)-

derived testcross population. Statistically significant interactions between regions of primary

homeology are shown (that is, allosyndesis; Fisher’s exact test for count data). The

Bonferroni correction for multiple testing at the p < 0.05 significance level in this population

is p < 0.00014. Only non-redundant SNP alleles are presented, arranged sequentially

according to their genetic location (not drawn to scale).

Figure 6: Segregation of alleles within A) the B. juncea A genome and B) the B. carinata C

genome in a B. juncea × B. carinata (BjBcAjCc)-derived testcross population, and C) the B.

napus genome in a B. juncea × B. napus (AjAnBjCn)-derived testcross population, showing

statistically significant interactions between regions of secondary homeology (that is,

autosyndesis; Fisher’s exact test for count data). The Bonferroni correction for multiple

testing at the p < 0.05 significance level is p < 0.00014 for A) and B) and p < 4.12E-05 for

C). Only non-redundant SNP alleles are presented, arranged sequentially according to their

genetic location (not drawn to scale). Circles indicate strong associations putatively based on

shared ancestral karyotype blocks R and W.

Figure 7: Average number of chromosome breakpoints observed per A- and C-genome

chromosome in testcross individuals derived from A) and B) Brassica juncea × B. napus (2n

= AjAnBjCn) hybrids and C) Brassica juncea × B. carinata (2n = BjBcAjCc) hybrids.

- 24 -

Supporting Information Supplementary Table S1: SNP markers used to characterise the (B. juncea × B. napus) × B.

carinata and the (B. juncea × B. carinata) × B. napus experimental populations, with

information from the Brassica SNP consortium and the two sequenced diploid Brassica

genomes.

Supplementary Table S2: Segregation of all alleles within B. juncea × B. carinata

(BjBcAjCc) hybrids, showing statistical significance of segregation (red) and co-segregation

(blue) interactions (Fisher’s exact test for count data).

Supplementary Table S3: Segregation of all alleles within B. juncea × B. napus (AjAnBjCn)

hybrids, showing statistical significance of segregation (red) and co-segregation (blue)

interactions (Fisher’s exact test for count data).

Supplementary Table S4: Linkage disequilibrium between A- and C-genome markers in a

B. juncea × B. carinata (BjBcAjCc) hybrid testcross population: r correlations, p-values and

D'.

Supplementary Table S5: Linkage disequilibrium between A- and C-genome markers in a

B. juncea × B. napus (AjAnBjCn) hybrid testcross population: r correlations, p-values and D'.

Supplementary Figure S1: Frequency of recombination breakpoint locations distributed

along the length of each An chromosome in a population derived from a B. juncea × B. napus

(AnAjBjCn) hybrid.

Supplementary Figure S2: Frequency of recombination breakpoint locations distributed

along the length of each Aj chromosome in a population derived from a B. juncea × B. napus

(AnAjBjCn) hybrid.

Supplementary Figure S3: Frequency of recombination breakpoint locations distributed

along the length of each Cn chromosome in a population derived from a B. juncea × B. napus

(AnAjBjCn) hybrid.

- 25 -

Supplementary Figure S4: Frequency of recombination breakpoint locations distributed

along the length of each Aj chromosome in a population derived from a B. juncea × B.

carinata (BjBcAjCc) hybrid.

Supplementary Figure S5: Frequency of recombination breakpoint locations distributed

along the length of each Cc chromosome in a population derived from a B. juncea × B.

carinata (BjBcAjCc) hybrid.

Supplementary Figure S6: Allelic inheritance of SNP markers in the B. juncea and B. napus

A genomes in testcross progeny derived from a B. juncea × B. napus (AjAnBjCn) hybrid. Only

non-redundant SNP alleles are presented, arranged sequentially according to their genetic

location (not drawn to scale). Dotted lines indicate approximate cut-offs for significant

deviations (p < 0.05) from 50% inheritance (Pearson’s χ2 test).

Supplementary Figure S7: Allelic inheritance of SNP markers in the B. napus C genome in

testcross progeny derived from a B. juncea × B. napus (AjAnBjCn) hybrid. Only non-

redundant SNP alleles are presented, arranged sequentially according to their genetic location

(not drawn to scale). Dotted lines indicate approximate cut-offs for significant deviations (p <

0.05) from 50% inheritance (Pearson’s χ2 test).

Supplementary Figure S8: Allelic inheritance of SNP markers in testcross progeny derived

from a B. juncea × B. carinata (BcBjAjCc) hybrid. Only non-redundant SNP alleles are

presented, arranged sequentially according to their genetic location (not drawn to scale).

Dotted lines indicate approximate cut-offs for significant (p < 0.05) deviations from 50%

inheritance (Pearson’s χ2 test).

Supplementary Text S1: R scripts used to analyse segregation of alleles from B. juncea × B.

carinata (BjBcAjCc) and B. juncea × B. napus (AjAnBjCn) hybrids.

- 26 -

Table 1: Proportion of chromosomes segregating with primary homeologue(s) based on

matching patterns of allelic inheritance (presence of one chromosome and absence of the

other, with or without recombination between the two) in a population of n = 62 plants

derived from Brassica juncea × B. carinata (BjBcAjCc) hybrids segregating for Brassica

juncea A genome (Aj) and B. carinata C-genome (Cc) alleles.

Cc1 Cc2 Cc3 Cc4 Cc5 Cc6 Cc7 Cc8 Cc9

Unrecombined

chromosomes2

Aj1 94% 39%

Aj2 100% 37%

Aj3 94% 47%

Aj4 65% 81%

Aj5 29% 53% 63%

Aj6 47% 84%

Aj7 97% 50%

Aj8 97%

Aj9 66%1 47%1 52%

Aj10 34% 50%

Unrecombined

chromosomes2 37% 37% 45% 50% 50% 56% 97% 63% 68%

1Percentages that add up to more than 100% are strongly suggestive of multivalent formation involving that

chromosome. 2Proportion of non-recombinant chromosomes (transmitted without recombination breakpoints indicating

crossover formation), regardless of segregation with primary homeologue. Indicates maximum possible

percentage univalent inheritance for each chromosome.

A2 A3 A4 A5 A6 A7 A8 A9 A10

C1 C2 C3 C4 C5 C6 C7 C8 C9

A1

Allele A

Allele D

Allele A 0 1 1 0 0 1 0 0 0 0 0 1 1 0 1 1

Allele D 0 0 1 1 1 0 0 1 0 1 1 0 1 0 1 0

c): Unlinked (no relationship)

Allele B 0 1 1 0 0 1 0 1 0 0 0 1 1 0 1 1

Allele C 1 0 0 1 1 0 1 0 0 1 1 1 0 1 0 0

b): Segregation

Indi

vidu

al 0

1In

divi

dual

02

Indi

vidu

al 0

3In

divi

dual

04

Indi

vidu

al05

Indi

vidu

al 0

6In

divi

dual

07

Indi

vidu

al 0

8In

divi

dual

09

Indi

vidu

al 1

0In

divi

dual

11

Indi

vidu

al 1

2In

divi

dual

13

Indi

vidu

al 1

4In

divi

dual

15

Indi

vidu

al 1

6

Allele A 0 1 1 0 0 1 0 0 0 0 0 1 1 0 1 1

Allele B 0 1 1 0 0 1 0 1 0 0 0 1 1 0 1 1

a): Linkage in coupling/co-segregation Allele AAllele B

Allele B

Allele A 1 0

1 8 0

0 1 7p = 0.0014

Allele C

Allele B 1 0

1 1 7

0 7 1

Allele D

Allele A 1 0

1 4 4

0 5 3

Fisher’s exact test for count data (2 × 2

contingency tables)

p = 0.0101

Allele B Allele C

p = 1

× B. juncea

Aj Aj Bj Bj Bc Bc Cc Cc

×

××

B. carinata

F1 hybrid

AjAnBjCn testcross population BcBjAjCc testcross population

a) b)

Alleles assessed: An, Aj, Cn Alleles assessed: Aj, Cc

B. juncea

Aj Aj Bj Bj

B. napus

An An Cn Cn

B. carinata

Bc Bc Cc Cc

F1 hybrid

Aj An Bj Cn

Homologous chromosomes

Bj Bc Aj Cc

B. napus

An An Cn Cn

A = 10 chromosomesB = 8 chromosomesC = 9 chromosomes

A = 10 chromosomesB = 8 chromosomesC = 9 chromosomes

Homeologouschromosomes

An1 An2 An3 An4 An5 An6 An7 An8 An9 An10

Aj1

Aj2

Aj3

Aj4

Aj5

Aj6

Aj7

Aj8

Aj9

Aj10

B. napus A genome

B. juncea

A genome

p < 0.000000011E-8 ≤ p < 1E-5 1E-5 ≤ p < 1E-3 0.001 ≤ p < 0.05p ≥ 0.05

Cc1 Cc2 Cc3 Cc4 Cc5 Cc6Cc7Cc8 Cc9

Aj1

Aj2

Aj3

Aj4

Aj5

Aj6

Aj7Aj8

Aj9

Aj10

B. carinata C genome

B. j

unce

aA

geno

me

p < 0.000000011E-8 ≤ p < 1E-5 1E-5 ≤ p < 1E-3 0.001 ≤ p < 0.05p ≥ 0.05

a) b)

Aj1 Aj2 Aj3 Aj4Aj5Aj6 Aj7Aj8 Aj9 Aj10

Aj1

Aj2

Aj3

Aj4Aj5Aj6Aj7Aj8Aj9

Aj10

Cc1

Cc2

Cc3

Cc4

Cc5

Cc6Cc7Cc8

Cc9Cc1 Cc2 Cc3 Cc4 Cc5 Cc6Cc7Cc8 Cc9

B. carinata C genome

B. c

arin

ata

C g

enom

e

c)Cn1

Cn2

Cn3

Cn4

Cn5

Cn6

Cn7Cn8Cn9

Cn1 Cn2 Cn3 Cn4 Cn5 Cn6 Cn7Cn8Cn9B. napus C genome

B. n

apus

C g

enom

eB.

junc

eaA

geno

me

B. juncea A genome

p < 0.0010.001 ≤ p < 0.01 0.01 ≤ p < 0.05p ≥ 0.05

Aver

age

num

ber o

f chr

omos

ome

brea

kpoi

nts

dete

cted

per

pla

nt

00,5

11,5

22,5

33,5

44,5

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

B. napusB. juncea

0

0,5

1

1,5

C1 C2 C3 C4 C5 C6 C7 C8 C9

B. napus

0

0,2

0,4

0,6

0,8

1

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 C1 C2 C3 C4 C5 C6 C7 C8 C9

B. juncea

B. carinata

a)

c)

b)

Chromosome

An01 An02

An03 An04

An05 An06

An07 An08

An09 An10

Figure S1: Frequency of recombination breakpoint locations distributed along the length of each An chromosome in a population derived from a B. juncea x B. napus (AnAjBjCn) hybrid.

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

Figure S2: Frequency of recombination breakpoint locations distributed along the length of each Aj chromosome in a population derived from a B. juncea x B. napus (AnAjBjCn) hybrid.

Aj01 Aj02

Aj03 Aj04

Aj05 Aj06

Aj07 Aj08

Aj09 Aj10

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp) Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

Cn1 Cn2

Cn3 Cn4

Cn5 Cn6

Cn7 Cn8

Cn9

Figure S3: Frequency of recombination breakpoint locations distributed along the length of each Cn chromosome in a population derived from a B. juncea x B. napus (AnAjBjCn) hybrid.

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

Aj01 Aj02

Aj03 Aj04

Aj05 Aj06

Aj07 Aj08

Aj09 Aj10

Figure S4: Frequency of recombination breakpoint locations distributed along the length of each Aj chromosome in a population derived from a B. juncea x B. carinata (BjBcAjCc) hybrid.

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp) Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp) N

um

ber

of

reco

mb

inat

ion

bre

akp

oin

ts

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

Figure S5: Frequency of recombination breakpoint locations distributed along the length of each Cc chromosome in a population derived from a B. juncea x B. carinata (BjBcAjCc) hybrid.

Cc1 Cc2

Cc3 Cc4

Cc5 Cc6

Cc7 Cc8

Cc9

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints

SNP position (Mbp) N

um

ber

of

reco

mb

inat

ion

bre

akp

oin

ts

SNP position (Mbp)

Nu

mb

er o

f re

com

bin

atio

n b

reak

po

ints