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doi: 10.1111/pbi.12732
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Received Date : 02-Jan-2017
Revised Date : 10-Mar-2017
Accepted Date : 17-Mar-2017
Article type : Research Article
Mapping of homoeologous chromosome exchanges influencing quantitative trait variation in
Brassica napus
Anna Stein1#, Olivier Coriton2, Mathieu Rousseau-Gueutin2, Birgit Samans1, Sarah V. Schiessl1,
Christian Obermeier1, Isobel A.P. Parkin3, Anne-Marie Chèvre 2, Rod J. Snowdon1
1 Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus
Liebig University, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
2 IGEPP, INRA, Agrocampus Ouest, Université de Rennes 1, 35653 Le Rheu, France
3 Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, S7N0X2, Canada
# Corresponding author:
Anna Stein, Department of Plant Breeding, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
Email: anna.stein@agrar.uni-giessen.de / Phone: +49 641 9937445 / Fax: +49 641 9937429
Running title: Homoeologous chromosome exchanges in B. napus
Key Words: Genome rearrangements, homoeologous exchange, genetic mapping, Quantitative Trait
Loci, Single Nucleotide Polymorphism
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Summary
Genomic rearrangements arising during polyploidization are an important source of genetic and
phenotypic variation in the recent allopolyploid crop Brassica napus. Exchanges among
homoeologous chromosomes, due to inter-homoeologue pairing, and deletions without
compensating homoeologous duplications are observed in both natural B. napus and synthetic B.
napus. Rearrangements of large or small chromosome segments induce gene copy number variation
(CNV) and can potentially cause phenotypic changes. Unfortunately, complex genome restructuring
is difficult to deal with in linkage mapping studies. Here we demonstrate how high-density genetic
mapping with codominant, physically anchored SNP markers can detect segmental homoeologous
exchanges (HE) as well as deletions and accurately link these to QTL. We validated rearrangements
detected in genetic mapping data by whole genome re-sequencing of parental lines along with
cytogenetic analysis using fluorescence in situ hybridization with bacterial artificial chromosome
probes (BAC-FISH) coupled with PCR using primers specific to the rearranged region. Using a well-
known QTL region influencing seed quality traits as an example, we confirmed that HE underlies the
trait variation in a DH population involving a synthetic B. napus trait donor, and succeeded in
narrowing the QTL to a small defined interval that enables delineation of key candidate genes.
Introduction
Brassica napus (rapeseed, oilseed rape, canola) is a very recent allopolyploid species that since its
origin has become one of the world’s most important crops. The species was formed by
hybridization and genome doubling from the diploid donor genomes of Brassica oleracea and
Brassica rapa, respectively. Because this cross can be readily reproduced with the help of embryo
rescue techniques, B. napus has become a popular model for studying the genetic and genomic
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consequences of de novo allopolyploidization and how these have shaped natural and agricultural
selection in a modern crop (Mason and Chévre, 2016).
Segmental exchanges between homoeologous chromosomes are frequent throughout the B. napus
genome. Numerous small-scale homoeologous exchanges (HE) are observed throughout the
genomes of natural B. napus accessions, whereas large-scale HE are common in synthetic accessions
(Chalhoub et al., 2014; Rousseau-Gueutin et al., 2017). In fact, resynthesized lines are specifically
prone to homoeologous rearrangements, including deletions, duplications and translocations (Gaeta
et al., 2007; Szadkowski et al., 2010; Xiong et al., 2011). Other than the assumption that
resynthesized rapeseed displays some kind of accelerated oilseed rape evolution, it is unknown
whether different mechanisms take place in natural and resynthesized oilseed rape.
Implementation of synthetic B. napus in breeding is an interesting, yet challenging strategy to
overcome the extreme narrow genetic diversity in modern rapeseed breeding pools. Causes of the
genetic bottlenecks are the small number of founder allopolyploidization events during the origin of
the B. napus species (Allender and King, 2010), strong adaptive selection in strict eco-geographic
gene pools and intensive agronomic selection during recent breeding for essential seed quality traits
(Snowdon et al., 2015). The diploid progenitor species harbor important variation particularly for
disease resistance (e.g. Rygulla et al., 2007b; Rygulla et al., 2007a; Mei et al., 2015; Werner et al.,
2008) and improvement of the heterotic potential (Snowdon et al., 2015). Unfortunately, the rich
genetic diversity available through de novo resynthesis of B. napus carries the price of a heavy
genetic load, with poor fertility and agronomic performance. Although marker-assisted backcrossing
can accelerate incorporation of such exotic materials, the complex genome restructuring common in
synthetic B. napus makes such germplasm particularly difficult to deal with in linkage mapping
studies.
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Furthermore, because most marker systems commonly used for genetic mapping do not assay
presence-absence variation (PAV) or copy number variation (CNV) automatically, the extent to which
this kind of variation underlies QTL for important agronomic traits is still completely unknown in B.
napus. Most genetic linkage maps constructed using high-density SNP array data do not consider
CNV or PAV (e.g. Raman et al., 2014; Delourme et al., 2013; Fopa et al., 2014; Liu et al., 2013)
although both are inherent phenomena in chromosome regions shaped by HEs or deletions. The
consequence may be that genome regions affected by genomic rearrangements including deletions,
which sometimes span entire chromosomes in synthetic B. napus (Chalhoub et al., 2014), may not
be incorporated into genetic maps and result in large gaps in linkage groups.
Accurate mapping of genomic rearrangement events is essential for mapping of associated QTL and
evaluation of their impact in allopolyploid crops. Mason et al. (2017) proposed guidelines for scoring
of SNP calling results that suggest presence-absence variation (PAV), corresponding with the
expected segregation ratio, in a segregating mapping population. Today, high-density SNP data
array, like that generated using the Brassica 60K Illumina Infinium genotyping array (Clarke et al.,
2016), enable rapid, high-resolution mapping of large B. napus mapping populations at low cost. Due
to the large numbers of polymorphic markers that can be assayed using this array, it is easy for users
to generate highly dense genetic linkage maps, even when markers that show unexpected
segregation or excessive quantities of failed SNP calls are excluded.
Similarly, hemi-SNPs (Trick et al., 2009) are frequently encountered between homoeologous loci in
B. napus. A hemi-SNP results from either (i) a simultaneous hybridization of the marker to two
homoeologous loci, or (ii) a simultaneous hybridization of the marker to duplicated fragments
containing a SNP mutation, where only one locus in each instance is polymorphic. These variants
generally exceed common threshold limits for tolerated heterozygosity in segregating mapping
populations, meaning that hemi-SNP data tend to be automatically discarded from genetic mapping
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datasets. On the other hand, inclusion of expected segregation ratios between heterozygous and
homozygous individuals enables genotypes to be resolved at a single-locus level and used for map
calculation in a mapping population.
Physical anchoring of SNP loci provides positional information that can improve confidence in calling
of deletion and duplication events based on missing SNP calls or hemi-SNPs, respectively. Although
missing calls due to technical failures are reasonably common in array-based genotyping systems,
and repeated failure of the same SNP may point to technical problems with the assay, it is unlikely
that two or more physically adjacent SNPs will by chance show the same segregation patterns of
technical failures vs. successful amplifications of one or the other SNP allele. Similarly, the calling of
a hemi-SNP does not necessarily indicate presence of a duplication, but may also occur as the
consequence of unspecific SNP probe hybridization. Nevertheless, using positionally anchored
markers in genetic mapping indicates putatively rearranged genomic regions. Those regions may be
validated by genomic re-sequencing.
Mapping of short-read genomic re-sequencing data to the recently published B. napus reference
genome sequence (Chalhoub et al., 2014) enables comprehensive analysis regarding nucleotide
polymorphisms and gene content, but also and importantly identification of presence-absence
variation (PAV) in B. napus samples. However, neither reciprocal nor nonreciprocal rearrangements
between the homoeologous subgenomes can be detected from re-sequencing data per se.
Nevertheless, segmental deletions that show corresponding duplications of the homoeologous
chromosome segment imply that this incidence is a homoeologous exchange (HE).
Fluorescence in situ hybridization using bacterial artificial chromosome probes (BAC-FISH) in B.
napus enables chromosome painting using BAC probes containing sub-genome specific repeat
sequences (Leflon et al., 2006), or even molecular karyotyping based on chromosome-specific
probes (Xiong and Pires, 2011). Multicolour combination of chromosome-specific BACs and
subgenome-specific BACs enables unambiguous chromosome identification in situ. Thus, comparing
the combination of FISH signals from specific BAC clones within putative HE regions can distinguish
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individuals, carrying single copies of the exchanged region, from individuals with duplicated copies of
one homoeologous locus and/or deleted copies of the other homoeologue. BAC-FISH therefore
represents an interesting method for independent validation of HE events imputed from genomic
sequencing reads or molecular marker segregation data. Since homoeologous reciprocal
translocations cannot be validated by genome resequencing, however in principle they can be
detected by genetic mapping and BAC-FISH.
Results
Natural and synthetic B. napus parental genotypes exhibit widespread genomic rearrangements
The aligned resequencing data yielded a generally uniform coverage over the lengths of each
chromosome in the respective mapping parents. This facilitated calling of putatively deleted and
duplicated segments, which showed consistent patterns of coverage either lower or higher than the
chromosome-wide average and a minimum length of 50kb. As described by (Chalhoub et al., 2014),
we found widespread evidence for deletions, duplications and HEs among homoeologous A-
subgenome and C-subgenome chromosomes. Also, as expected, the natural B. napus accession
Express 617 showed the lowest degree of segmentation (685 deleted or duplicated segments),
whereas the two synthetic B. napus parents exhibited a considerably higher segmentation degree
(821 and 1,630 deletions or duplications, for 1012-98 and R53, respectively). The semi-synthetic
parent V8, derived from backcrossing of a synthetic B. napus to natural B. napus, showed the
expected intermediate degree of segmentation (795 segments), between that of the synthetic and
natural accessions. In summary, Express 617 showed genomic rearrangement events affecting 8.0%
of the genome, 1012-98 16.2% of the genome, V8 12.3% of the genome and R53 41.5% of the
genome. Although these figures may be biased by normalization based on chromosome mean
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coverage values and alignment to a European winter oilseed rape reference sequence (Darmor-bzh),
the overall scale of rearrangements is clearly greater in synthetic B. napus.
As an overview, Supplementary Figure S1 displays locations of segmental deletions or duplications
larger than 500kb in the four mapping parents. Full details of all detected segmental deletions and
duplications, also including those events between 50kb and 500kb in size, are provided in
Supplementary Table S1. Coverage plots for the 19 chromosomes of the four parental genotypes are
given in Supplementary Figures S2, S3, S4 and S5.
In Express 617 only five deletions larger than 500kb were detected, three on chromosome C01, one
on chromosome C02 and one on C08. In contrast, duplications and deletions larger than 500kb were
numerous and widespread across both subgenomes in the synthetic and semi-synthetic accessions:
22 events in V8, 40 events in 1012-98 and 107 events in R53. Interestingly, deletions in five genomic
regions on chromosomes A03, C02, C05, C07 and C09 were consistently detected in all three
parental accessions with synthetic B. napus background.
Chromosome C02 in the genotype R53 was not represented by sequence reads and is therefore
assumed to be missing (completely or almost completely). On the other hand chromosome number
of R53 was confirmed cytogenetically to be 38 (data not shown). Due to the elevated coverage of
sequence reads from chromosome A02 in R53, we assume that chromosome C02 has been replaced
by a duplication of A02. Deletions and duplications affecting whole or nearly whole chromosomes
has also been observed by Rousseau-Gueutin et al. (2017) in other synthetic B. napus.
Genetic mapping
The total amount of SNP markers used to calculate the genetic bin maps was reduced based on allele
frequency and co-segregation from 35,170 pre-selected SNPs, with putative unique positions in the
reference sequence, to sets of 2,204, 3,135 and 2,029 markers for the populations ExV8-DH, ExR53-
DH and Ex1012-98-DH, respectively. The resulting genetic linkage maps comprised 1,733, 2,186 and
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1,631 markers, respectively, covering 2,512 cM, 3,780 cM, and 2,358 cM over 20, 21 and 22 linkage
groups,. A total of 273 consensus markers were found in all three populations, with 704 consensus
markers between ExV8-DH and Ex1012-98-DH, 729 between ExV8-DH and ExR53-DH, and 677
between ExV8-DH and ExR53-DH, respectively.
High collinearity was achieved among the three linkage maps, whereby the linkage groups in the
ExR53-DH map were generally larger than those in the other two populations, reflecting the
considerably greater number of markers. Lengths of individual linkage groups vary among the three
populations, indicating differential degrees of diversity and recombination across the chromosomes
of the synthetic parents 1012-98, V8 and R53. All linkage maps are displayed in Supplementary
Figure S6, the map texts can be found in Supplementary Table S2,
Notably, some linkage groups were found to be considerably longer in a single population than in
the other two. Because all populations had mapping parent Express 617 in common, this could
indicate a propensity for higher recombination frequency on these specific chromosomes conferred
by the particular synthetic/semi-synthetic mapping parent.
We also observed frequent incidents where multiple markers assigned to the same chromosome in
the Darmor-bzh reference were genetically mapped onto two linkage groups that could not be
combined into a single linkage group. The data suggest that this phenomenon, which is common in
B. napus genetic maps, corresponds with the presence of either large or numerous genomic
rearrangements in one of the synthetic or semi-synthetic mapping parents and a consequent
disruption of pairing and linkage disequilibrium in the DH-lines. “Non-contiguous linkage groups are
distinguishable from translocated genomic segments, because the latter map to their new position.
In this study, Ex1012-98DH exhibits two non-continuous linkage groups representing chromosome
A01. Additionally, a duplicated and translocated A01-fragment of 1012-98 maps to its new position
in linkage group C01.
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Genomic rearrangements can be localized by genetic mapping
Calling of “het” and “PA” marker types as outlined above proved extremely helpful in detecting
genomic rearrangements. Segments of the genetic map carrying three or more adjacent “het”
markers or “PA” markers were validated by comparing read densities in the corresponding regions of
the genomic sequence data. For single mapped “PA” or “het” markers validation by resequencing
data is very difficult. These markers were regarded with care, as their annotation may be false due to
sequences similarity. Still, some examples in this study prove them to be useful rearrangement
markers.
Two examples for independent validation of prominent HE and deletion events by plotting genomic
resequencing coverage and genetic mapping in DH mapping populations are shown in Figure 2 and
3. In Figure 2, four homoeologous non-reciprocal translocations (HNRTs) were identified in linkage
group C03 in population ExR53-DH using genomic re-sequencing data, each could be correlated to
some extent with SNP mapping data. The first of them was mapped by a block of C03-PA-markers
spanning the region 0-6.1 cM on top of linkage group C03. This corresponds to a deletion in R53
chromosome C03 of 1.16 Mb. A putatively translocated homoeologous A03 duplicate fragment was
not mapped. The second was a A03-duplication and translocation to C03 mapped by a block of A03-
markers spanning the region 53-108.1 cM. This corresponds to the positions 3.9-6.8 Mb on
chromosome A03 and 5.5-9.0 Mb on chromosome C03. The third was a C03-deletion at the position
15.4-18.8 Mb, which was mapped by a single C03-PA-marker at 189.2 cM. The deletion corresponds
to a duplication of an A03-fragment. Although the genomic segments here are large in size, the
corresponding SNP markers did not meet the expected 1:1 segregation ratio and could not be
mapped. The forth HE event, a C03-deletion at the position 47.9-48.8 Mb and a homoeologous A08-
duplication of 0.8 Mb was mapped by a single A08-marker at 268.7cM.
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In Figure 3, several smaller and larger segmental deletions detected by resequencing coverage
analysis, spanning the same region of chromosome A05 in both V8 and R53, were genetically
mapped by a block of PA-markers in both the ExV8-DH and ExR53-DH genetic maps. The
corresponding regions span 20.5 cM (1.750 Mb) in ExV8-DH and 13.9 cM (4.651 Mb) in ExR53-DH.
Some PA-markers are common between the two populations, whereas others are unique to one of
the populations.
QTL mapping
In this study 15 QTL for 15 traits were identified in the Ex1012-98-DH population, 61 QTL for 37 traits
in the ExV8-DH population and 25 QTL for 14 traits in the ExR53-DH population. All QTL share LOD-
scores higher than 5, allowing an alpha-error not larger than 0.05. Table 1 gives a list of detected QTL
in chromosome regions with putative genomic rearrangements for all measured traits in the three
mapping populations. The list includes their genetic and inferred physical positions (unique BLAST
hits) in the B. napus Darmor-bzh reference genome, R² values indicating the proportion of
phenotypic variation explained by the QTL, and the LOD scores at QTL peaks. The co-localisation of
QTL with regions involved in genomic rearrangements is consistent with the hypothesis that
genomic rearrangements generate significant phenotypic variation with considerable selective and
evolutionary potential.
A homoeologous rearrangement on A09 causes variation in seed fibre content
In a previous study involving the same plant material we concluded that a major QTL for seed colour
and seed coat fibre content, co-localising on chromosome A09 in the mapping populations Ex1012-
98-DH and ExV8-DH, might have derived from an HE causing deletion or conversion of important
candidate genes on chromosome A09 (Stein et al., 2013). Because of the comparatively low marker
density in that study it was not possible to precisely map the HE borders and correctly localize the
physical position of the QTL. In the current study we confirmed the presence of this HE by sequence
coverage, and validated the genetic position using a considerably larger set of molecular markers
than before.
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The coverage plots of the homoeologous chromosomes of the synthetic parent 1012-98 (Figure 4)
clearly show coincidence of a segmental deletion in A09, corresponding to a duplication of the
homoeologous segment in C08. SNP markers derived from chromosome C08 map as “het” markers
on linkage group A09, marking the position of a duplicated block. A09 markers representing a
deletion (“PA”) flank this block. Using the new, high-density genetic map of Ex1012-98-DH
chromosome A09, including the HE-tracing markers, we narrowed the QTL to a very small interval
with an extremely high LOD peak for the seed fiber QTL (Figure 4). The 173 kb interval harbors one
gene from the monolignol biosynthesis pathway, BnaCAD2/3 (BnaA09g42930/BnaC08g35540). The
whole translocation region includes also BnaCCR1 (BnaA09g56490/BnaC08g38580), and BnaAHA10
(BnaA09g41670/BnaC08g34260), confirming the previously hypothesized involvement of these
genes in the seed fibre and seed color QTL (Stein et al., 2013). Cinnamoyl-CoA-reductase (CCR1)
confers reduction of p-coumaroyl-CoA to p-coumaraldehyde, which is subsequently reduced to the
monolignol p-coumarylalcohol by cinnammylalcoholdehydrogenase (CAD2/3). While CCR1 acts
substrate-specific, CAD2/3 has a potentially wider substrate spectrum, which allows a certain
plasticity in monolignol metabolism (Bonawitz and Chapple, 2010). Impaired activity of either CCR1
or CAD2/3 does not necessarily reduce the lignin content in the plant organ, but can affect the lignin
composition. Double knockout of both genes, however, has been shown to decrease lignin content
(Chabannes et al., 2001). Autoinhibited H+-ATPase Isoform 10 (AHA10) is a seed-expressed
transcription factor involved in proanthocyanidin formation in the seed coat endothelium, which has
been shown to influence seed coat pigment accumulation (Baxter et al., 2005). The physical
annotation of these genes within a deleted segment of chromosome A09 in the genotype 1012-98
likely explains the co-localization of QTL for both seed acid detergent lignin (ADL) and seed colour
(Figure 4).
A targeted sequence-capture experiment carried out for BnaCCR1 in the mapping parents Express
617 and 1012-98 underpins the hypothesis that the large deletion described above has led to loss of
an A-genome copy of BnaCCR1 in 1012-98. Details of the experimental setup and data analysis are
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described by Schiessl et al. (2017). Figure 1 presents sequence capture results of the two BnaCCR1
copies BnaA09g56490 and BnaC08g385809 at very high average sequencing depth (approximately
1200x), allowing highly accurate estimations of copy number. Coverage for BnaA09g56490 is
considerably lower and less consistent over the length of the gene copy in 1012-98. In comparison to
the normalized gene coverage across the whole experimental panel of 280 genotypes described by
Schiessl et al. (2017), the gene coverage is reduced to 51%.
PCR and BAC-FISH validation of the QTL-associated HE between chromosomes C08 and A09
We validated the deletion of a 900 kb region (29,274Mb – 30,174Mb) in 1012-98 using three primer
pairs specific to this region. As expected, no amplification was observed in 1012-98, whereas the
expected PCR product, indicating presence of the chromosome segment, was observed in Express
617, R53, V8 and Darmor-bzh.
The BAC KbrB043F18 and BoB014O06 were hybridized to identify the A09 and C08 chromosomes
and all C genome chromosomes, respectively, to detect the homoeologous rearrangement (green)
(Figure 5b and 5c). To further validate the A09 chromosome fragment losses, a BAC-FISH experiment
was performed using BAC clone 54 probe (isolated from Express 617) present in the rearranged
region of 1012-98. Due to the high sequence similarity between A and C genomes, two or four
signals are expected in the case of deletion or a HNRT, respectively. In 1012-98, four signals were
observed with BAC clone 54 (Figure 5c), indicating the presence of an HNRT. Since the limit of GISH-
like resolution is 5Mb to clearly observe rearranged chromosomes by harbouring a dual colour
signal, it is not easy to observe smaller rearrangements by hybridization of the BoB014O06 BAC.
However, in Figure 5b (and enlarged in Figure 5h) the C08-to-A09 translocation in 1012-98 can be
observed by a faint green signal in the otherwise unstained A09 chromosome.
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Discussion
Genomic rearrangements are widespread in the recent allopolyploid species B. napus, making it an
interesting model for studying the cause and effect of de novo polyploidization. The presence of two
additive genomes with a high degree of homoeology in the same nucleus leads to meiotic
chromosome pairing between homoeologous chromosomes during the first generations after
allopolyploidization (Szadkowski et al., 2010), causing considerable HE occurrence and gene
conversion (Chalhoub et al., 2014). Implementation of novel variation caused by genomic
rearrangement events, such as HE events or pure deletions in synthetic B. napus is of considerable
interest for rapeseed and canola breeders. However, skewed marker segregation patterns, which
occur as consequence of genomic rearrangements, prevent standard mapping procedures from
accurately localizing QTL, tightly linked markers and causal genes in these regions.
Here, we present a method which enabled us to accurately map agronomic QTL to a number of
genomic rearrangement events. The 60K Illumina SNP array marker data were used to generate high
density genetic maps in half-sib DH populations from three synthetic B. napus accessions, carrying
interesting disease resistance (Obermeier et al., 2013) agronomic and yield-related traits (Radoev et
al., 2008; Basunanda et al., 2007; Basunanda et al., 2010) and seed quality characters (Badani et al.,
2006; Stein et al., 2013). Although the putative co-localisation of HE events with QTL in B. napus has
been suggested in previous studies (Chalhoub et al., 2014; Liu et al., 2012; Stein et al., 2013) to our
knowledge this is the first study providing independent validation of QTL-HE co-localisation.
Alongside genetic mapping and evidence from genomic coverage sequencing, we provide cytological
indications from BAC-FISH that a chromosome fragment spanning an HE-associated QTL is indeed
involved in the suspected HE. This supports our use of SNP marker data from loci spanning deletions
or duplications, which normally would be discarded in mapping procedures. The results provide an
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interesting reminder about the importance of structural chromosome variation in genome mapping
and QTL analysis, and suggest implementation of routine screening for presence-absence variation
as a resource for breeding diversity.
Using the 60K Infinium SNP array allows genetic mapping of deletions and translocations
Homoeologous translocations were first detected in mapping populations using codominant RFLP
markers (Parkin et al., 1995; Sharpe et al., 1995), which also gave initial insights into the frequency
and extent of HE events in natural and synthetic B. napus (Song et al., 1995). In contrast to RFLP
assays, rapid and cost-effective analysis with high-density SNP arrays provides considerably higher
resolution of genetic recombination in mapping populations. For analyses of homoeologous
rearrangements, the ability to anchor SNP markers to a reference genome sequence and to compare
patterns for multiple SNPs per homoeologous chromosome segment allows a more accurate and
detailed assessment of genome-wide HE events. Genetic mapping of homoeologous reciprocal
translocations is more challenging unless duplicated markers can be assigned to a unique locus.
SNPS with multiple physical BLAST positions were discarded. This may mean that markers from
highly similar homoeologous regions are neglected in the mapping process.
Applicability of HE mapping approaches
Our method to derive HE information from high-density SNP array data in segregating mapping
populations provides an opportunity to include structural genome variation in genetic maps and QTL
studies. Revisiting historical QTL datasets using SNP array data analyzed with this technique will give
a more comprehensive picture of HE associations to QTL. We are currently developing thresholds
and techniques to apply HE calling from SNPs in non-related populations for genome-wide
association studies. In multi-parent mapping populations, for example those described by Snowdon
et al., (2015) for nested-association mapping in B. napus, we expect that capturing of HE variants will
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greatly increase the power to detect QTL in genome regions that were previously very difficult to
accurately access by genetic mapping techniques.
Access to multiple genome assemblies per species, using new assembly methods that greatly
improve genome coverage and accuracy, will further improve our ability to trace the impact of
chromosome-level structural variation on quantitative trait expression.
Experimental procedures
Plant material
Three half-sib doubled haploid (DH) winter-oilseed rape populations were used in this study,
developed from crosses of the black-seeded inbred winter oilseed rape line ‘Express 617’ to the
synthetic B. napus lines 1012-98 and R53, and to the semi-synthetic line V8. The origins of the
parental lines, along with the DH mapping populations Express 617 x 1012-98 (Ex1012-98-DH,
n=164), Express 617 x R53 (ExR53-DH, n=248) and Express 617 x V8 (ExV8-DH, n=248) have been
described previously in detail (Badani et al., 2006; Basunanda et al., 2007; Basunanda et al., 2010;
Radoev et al., 2008).
Detection of genomic rearrangements from genomic resequencing data
Genomic resequencing data were collected from the parental genotypes Express 617, 1012-98, V8
and R53 using the Illumina MiSeq and HiSeq 2000 systems. The MiSeq system delivered 250 bp
paired-end reads, while 100 bp paired-end reads were collected using the HiSeq system. All reads
were aligned to the B. napus reference sequence version 4.1 (Chalhoub et al., 2014) using the short
oligonucleotide alignment program (SOAP2 v2.21) (Li et al., 2008), and read depths were calculated
using the command genomecov in the bedtools package v.2.20.1 for every nucleotide position
genome-wide. Median coverage over 1,000bp blocks was calculated using an R script, and adjacent
blocks with the same coverage value were aggregated to consecutive segments using a circular
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binary segmentation algorithm implemented in R package PSCBS (Bengtsson et al., 2010; Olshen et
al., 2011). Adjacent segments over 50kb in length and with the same mean coverage value were
merged unless separated by gaps larger than 50kb.
Mean read coverage and standard deviation were calculated for each individual and chromosome.
Segments with a coverage value exceeding the chromosome mean by 1 standard deviation were
defined as segmental duplications, while segments with a coverage value of 1 standard deviation
lower than the chromosome mean were defined as deletions. Accordingly, segments not deviating
by more than 1 standard deviation from the chromosome-wide average were assumed to have
“normal” coverage. The script used is provided in Supplementary File S1.
DNA extraction and high-density genetic mapping including HE markers
Total genomic DNA was extracted from 200 mg leaf material of young leaves of mapping parents and
DH lines, using the Qiagen BioSprint 96 DNA Plant Kit (Qiagen GmbH, Hilden, Germany). All samples
were subjected to high-density, genome-wide SNP genotyping using the Brassica Illumina 60K SNP
genotyping array (Clarke et al., 2016). Genotyping was outsourced to TraitGenetics GmbH
(Gatersleben, Germany).
Genomic positions in the B. napus reference genome sequence were assigned to all tested SNP
markers showing a BLAST hit with the SNP flanking sequences, as described previously (Qian et al.,
2014); (Mason and Snowdon, 2016). In brief, SNP markers were physically localized on the Brassica
napus Darmor-bzh reference genome sequence assembly (version 4.1), (Chalhoub et al., 2014) using
the following criteria: minimum overlap of 50 bp length, minimum identity of 95%, no sequence
gaps. SNPs with only one BLAST hit were regarded as informative in terms of physical position and
only these markers were included in the genetic mapping. Linkage mapping was conducted using the
software Joinmap 4 (van Ooijen, 2006), after assignment of co-segregating markers into bins with
Perl in order to reduce the locus number. Markers were subsequently scored according to three
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different genotype patterns (Mason et al., 2017): (i) polymorphic simple SNP calls; (ii) polymorphic
hemi-SNP calls; and (iii) polymorphic presence-absence SNP calls. The latter two allow recording of
markers potentially affected by genomic rearrangement events. All of these scoring types were
required to fit the expected 1:1 segregation for DH populations.
All SNP markers that were included in the mapping file were named with their predetermined
physical position and a suffix indicating markers showing dominant presence-absence segregation
(“PA”) on the one hand, or codominantly segregating heterozygous hemi-SNPs (“het”) on the other
hand. The map was calculated using the maximum-likelihood algorithm and groups were formed
with a cut-off at recombination frequency 0.2. Maps were subsequently joined using Mapchart 2.3
to link bins containing SNP markers shared across the three maps.
QTL mapping
Quantitative trait loci were calculated using Qgene4.3.10 (Joehanes and Nelson, 2008) based on the
calculated genetic maps. Seed trait phenotyping for all populations was performed by near-infrared
reflectance spectrometry (NIRS) analysis on seed samples produced in field trials at four different
locations in Germany, over multiple years from 2003 until 2015, following the seed analysis
procedures (Wittkop et al., 2012). The seed colour and quality data from the first two years of
experiments correspond with those reported by (Badani et al., 2006). A fully randomized complete
block design was used, with plots sizes of 10 to 13 m2 depending on the standard practice at each
location. A high number of locations and years were preferred rather than multiple replications of
genotypes per location, as is standard practice when testing large rapeseed populations, because the
large plot size reduces field homogeneity when too many test plots are included per location. Selfed
seeds from 3-5 representative plants per line were hand-harvested at maturity and subjected to
NIRS as above, with two technical replicates per sample. Germination traits in the ExV8-DH
population were phenotyped at the French national seed testing laboratory (GEVES, Angers, France),
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with 100 seeds per genotype and repetition with seed lots from two different production
environments (Hatzig et al., 2015). Morphological traits in the ExV8-DH population were collected
from two locations in Germany in the years 2006 and 2007.
HE validation using specific primers and BAC-FISH
To validate the deletion of a 900 kb fragment on A09 (29,274Mb – 30,174Mb) in 1012-98, we
extracted this 900 kb region from B. napus ‘Darmor-bzh’ genome (Chalhoub et al., 2014) and blasted
it against the whole ‘Darmor-bzh’ genome sequence, enabling us to identify fragments of at least
500 bp that were specific to this A09 chromosome region (i.e. absent from the homoeologous
region). We then designed primer pairs from these specific A09 regions using Primer 3.0 (Rozen and
Skaletsky, 2000). Only the primers (3 pairs) that gave a single band in different B. napus varieties
tested and no amplification in B. oleracea were retained (primer details are given in Table S3). The
PCR products obtained using B. napus ‘Darmor’ DNA were also directly sequenced, enabling further
validation of the specificity of these primer pairs to A09. Subsequently, these primer pairs were
tested using DNA from synthetic line 1012-98. Each PCR amplification was performed in a total
volume of 50 μl containing 10 μl of 5× buffer (Promega), 4 μl of 25 mM MgCl2, 0.5 μl of 25 mM dNTP
mix, 2.5 μl of each primer (10 mM), 0.2 μl of GoTaq® G2 Hot Start polymerase (5 U μl−1) and 50 ng of
DNA. For PCR reactions, genomic DNA was denatured at 94°C for 2 min, followed by 30 cycles of
94°C for 30 s, 58°C for 30 s and 72°C for 1min.
The proximal 0.5 - 1.5 cm of young seedling roots were excised, treated in the dark with 0.04% 8-
hydroxiquinoline for 2 hours at 4°C, then transferred to room temperature for 2 h to accumulate
cells at metaphase. Root tips were then fixed in 3:1 ethanol-glacial acetic acid for 48 hours at 4°C
and stored in 70 % ethanol at -20 °C until required. After washing in 0.01 M citric acid-sodium citrate
(pH 4.5) for 15 min, the tips were digested in 5% Onozuka R-10 cellulase (Sigma-Aldrich, St. Louis,
MO, USA) containing 1% Y23 pectolyase (Sigma) at 37 °C for 30 min, then washed carefully with
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distilled water for 30 min. Single root tips were transferred to cleaned microscope slides and
macerated with a drop of 3:1 fixation solution using a preparation needle. After air-drying, slides
with good metaphase chromosome spreads were stored at -20 °C until further use.
The B. napus BAC clone 54 from the parental genotype Express 617 was used to probe a
chromosome spread of synthetic B. napus parental genotype 1012-98, in which a putative HE was
suspected between homoeologous chromosomes C08 and A09. The BAC clone was previously
identified by PCR-screens with markers from a QTL region on chromosome A09 (see below). Sanger
sequencing confirmed alignment of the BAC clone to B. napus Darmor-bzh chromosome A09.
BAC clone 54 was labelled by random priming with biotin-14-dUTP (Invitrogen, Life Technologies
Waltham, MA, USA). The BAC clones KBrB043F18 (from B. rapa chromosome A09, homoeologous to
B. oleracea chromosome C08 (Xiong and Pires, 2011) and B. oleracea BoB014O06 (Howell et al.
2002) were labelled by random priming with Alexa 594-5-dUTP and Alexa 488-5-dUTP respectively.
BoB014O06 was used as genomic in situ hybridization (GISH)-like probe to specifically stain all C-
genome chromosomes in B. napus (Suay et al., 2014; Szadkowski et al., 2010).
Chromosome preparations were incubated in RNAse A (100ng/µL) and pepsin (100 µg/ml) in 0.01M
HCl, and fixed with paraformaldehyde (4%). Chromosomes were denatured in a solution of 70%
formamide in 2 x saline-sodium citrate buffer (SSC) at 70°C for 2 min, dehydrated in an ethanol
series (70%, 90% and 100%) and air-dried. The hybridization mixture consisted of 50% deionized
formamide, 10% dextran sulfate, 2xSSC and 1% SDS. Labelled probes (200ng per slide) were
denatured at 92°C for 6 min and transferred to ice. The denatured probe was placed on the slide and
in situ hybridization was carried out overnight in a moist chamber at 37°C. After hybridization, slides
were washed for 5 min in 50% formamide in 2xSSC at 42°C, followed by several washes in 4xSSC-
Tween to remove non-conjugated probe. Biotinylated probe was immunodetected by Texas Red
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Avidin DCS (Vector Laboratories, Burlingame, CA, USA) and the signal was amplified with biotinylated
anti-avidin D (Vector Laboratories). The chromosomes were mounted and counterstained in
Vectashield (Vector Laboratories) containing 2.5µg/mL 4’,6-diamidino-2-phenylindole (DAPI).
Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, AZ, USA)
on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analysed using MetaVueTM
(Universal Imaging Corporation, Downington, PA, USA).
Acknowledgements
This work was financed by grant SN 14/17-1 for the ERA-CAPS project Evo-Genapus “Evolution of
genomes: Structure-function relationships in the polyploid crop species Brassica napus”. The authors
declare no conflict of interest.
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Figure legends
Figure 1. Mapping results from targeted sequence capture of the two BnaCCR1 copies
BnaA09g56490 and BnaC08g385809 in the B. napus genotypes Express617 and 1012-98. Sequence
read coverages were mapped against the B. napus reference sequence Darmor-bzh v. 4.1 and are
displayed along the gene length. SNPs (indicated by coloured bars) in the homozygous doubled-
haploid genotype 1012-98 suggest two variants of the BnaC08g385809 gene copy. This may have
also led to mis-alignment of some reads to the homeologous gene BnaA09g56490. Both the
coverage landscape for BnaA09g56490 in 1012-98 and the according relative coverage values given
in the table indicate a deletion of this gene copy (highlighted in green boxes). Relative coverage
values were calculated as the ratio of the normalized mean coverage of the genotype over
normalized mean coverage over 280 genotypes for each specific gene copy. Sequence capture data
was obtained from (Schiessl et al., 2017).
Figure 2. A03 to C03 Translocation in the synthetic B. napus genotype R53 identified by re-
sequencing and validated by genetic mapping.
The plots show re-sequencing read coverage across the lengths of the respective chromosomes,
calculated for segments of 1kb. The genetic linkage maps on the right of the read maps show genetic
mapping including SNPs with normally segregating, bi-allelic calls with locus names in black text.
SNPs called as deletions (presence-absence markers, with suffix “–PA”) are indicated by bold red
marker names, whereas SNPs with heterozygous-homozygous segregation due to polymorphism in
one of two duplicated copies (with suffix “–het”) are indicated by bold blue marker names.
Polymorphic markers in bold magenta text indicate duplicated markers mapping to their
homoeologous position. Opaque red blocks link putative deletions detected in coverage blocks with
the corresponding regions in the genetic maps. Centromere regions are indicated by black triangles
according to (Mason et al., 2013).
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Figure 3. A05 Deletion in the synthetic B. napus genotypes V8 und R53 identified by re-sequencing
and validated by genetic mapping.
The plots show re-sequencing read coverage across the lengths of the respective chromosomes,
calculated for segments of 1kb. The genetic linkage maps on the right of the read plots show genetic
mapping including SNPs with normally segregating, bi-allelic calls with locus names in black text.
SNPs called as deletions (presence-absence markers, with suffix “–PA”) are indicated by bold red
marker names, whereas SNPs with heterozygous-homozygous segregation due to polymorphism in
one of two duplicated copies (with suffix “–het”) are indicated by bold blue marker names.
Polymorphic markers in bold magenta text indicate duplicated markers mapping to their
homoeologous position. Opaque red blocks link putative deletions detected in coverage blocks with
the corresponding regions in the genetic maps. Centromere regions are indicated by black triangles
according to (Mason et al., 2013).
Figure 4. Localisation of a major QTL on A09 influencing numerous seed quality traits in the
mapping population Ex1012-98-DH, within a prominent HE between the distal ends of
homoeologous chromosomes A09 and C08.
The two plots on the left hand side show median read coverage across the lengths of the respective
homoeologous chromosomes, calculated for segments of 1kb. The two genetic linkage maps on the
right show genetic mapping including SNPs with normally segregating, bi-allelic calls with locus
names in black text. SNPs called as deletions (presence-absence markers, with suffix “–PA”) are
indicated by bold red marker names, whereas SNPs with heterozygous-homozygous segregation due
to polymorphism in one of two duplicate copies (with suffix “–het”) are indicated by bold blue
marker names. Opaque red blocks link putative deletions, detected based on sequence coverage
blocks, with the corresponding regions in the genetic maps, while opaque blue blocks indicate
putative duplications, respectively. Centromere regions are indicated by black triangles position
according to (Mason et al., 2013).
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Figure 5. Fluorescence in situ hybridization using bacterial artificial chromosome probes to identify
a putative HNRT between chromosomes A09 and C08 in the synthetic B. napus genotype 1012-98
(a, e) DAPI background staining of somatic metaphase chromosomes of the synthetic B. napus
genotype 1012-98, carrying a putative HNRT between chromosomes A09 and C08. (b, f) Green FISH
signals from BAC BoB014O06, which identifies all Brassica C-genome chromosomes. (c) Blue FISH
signals and red signals (g) from BAC54, specific for chromosomes A09 and C08. (d) Red FISH signals
from KbrB043F18, also specific for chromosomes A09 and C08. Arrows indicate chromosomes with
putative rearrangements. (h) Schematic representation of translocation of enlarged A09 chromatids.
Green arrows indicate green FISH signal, representing the presence of a C-subgenome fragment on
chromosome A09.
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Tables:
Table 1. Major QTL for seed quality and agronomic traits located in genomic regions that are
rearranged in respect to the B. napus Darmor-bzh reference sequence. Calculation was performed
by composite interval mapping, considering only QTL with a LOD score of >5; QTL in bold text could
also be identified in genetic mapping.
Population
Trait
Linkage
group
Genetic
position
(Marker
interval)
[cM]
Inferred
physical
position
[kb]
LOD
score
R²
Structural
rearrangement
ExV8-DH
Seedling
volume increase
A05
161-165
A05
17,065-17,605
8.0
0.14
Deletion
Seed sulphur
C03
222-227.5
C03
51,659-52,628
5.2
0.09
Deletion
Seed sulphur
C09
22.5-27.5
C09
2,814-3,592
29.7
0.42
Deletion
Seed glucosinolate
C09
22.5-27.5
C09
2,814-3,592
35.0
0.48
Deletion
Ex1012-98-DH
Seed ADL
(acid detergent lignin)
A09
152
C08
33,479
28.9
0.56
HE
Seed NDF
(neutral detergent fibre)
A09
152
C08
33,479
31.6
0.59
HE
Seed colour
A09
152
C08
33,479
7.7
0.20
HE
Seed C18:3
A09
176-177
A09
31,557
8.4
0.21
HE
Days to flowering
A09
193-194.5
A09r
3,826
(~A09 30,000)
7.4
0.20
HE
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Flowering duration
A09
207.5-208
C08
36,510
6.3
0.18
HE
Seeds per silique
A09
208
C08
36,510
5.1
0.15
HE
ExR53-DH
Seed ADL
(acid detergent lignin)
A05
15-17.5
A05
1,464
5.4
0.10
Deletion
Seed ADF
(acid detergent fibre)
C01
193.5-194
C01
34,145-38,105
8.6
0.16
Deletion
Seed NDF
(neutral detergent fibre)
C01
193.5-194
C01
34,145-38,105
10.4
0.19
Deletion
Electronic Supplementary Material:
Figure S1. Genome-wide coverage segmentation derived from resequencing data from four mapping
parents across the 19 chromosomes of the B. napus A and C subgenomes.
Figure S2. Resequencing read coverage plots of genotype Express617.
Figure S3. Resequencing read coverage plots of genotype 1012-98.
Figure S4. Resequencing read coverage plots of genotype R53.
Figure S5. Resequencing read coverage plots of genotype V8.
Figure S6. Genetic linkage maps of three half-sib DH populations.
Table S1. Coverage segments of equal copy number.
Table S2. Map texts for genetic maps of three half-sib DH populations.
Table S3. List of primer pairs designed to validate a 900 kb deletion on the A09 chromosome in the
synthetic B. napus line 1012-98.
Files S1. Code for calculation of genomic coverage and detection of duplication and deletion events.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
Accepted Article
This article is protected by copyright. All rights reserved.
