h igh -resolution molecular karyotyping uncovers pairing ... · 4 82 homology exist. however, the...
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High-resolution molecular karyotyping uncovers pairing between ancestrally-1
related Brassica chromosomes 2
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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
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*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
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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
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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
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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
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Keywords: meiosis, polyploidy, recombination, molecular karyotyping, Brassica 48
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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
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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
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homology exist. However, the analytical tools for interrogating large molecular genotyping 82
datasets to answer these questions remain under-developed. 83
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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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.
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- 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.
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- 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.
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- 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.
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- 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
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