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Genome wide identification and comparative analysis of NBS-LRR
resistance genes in Brassica napus
Salman AlameryA B, Soodeh TirnazD, Philipp BayerA D, Reece TollenaereA, Boulos ChaloubC,
David EdwardsA D and Jacqueline Batley A D E
A School of Agriculture and Food Sciences, University of Queensland, St Lucia, Queensland, 4072, Australia
B Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia
C URGV (Institut National de la Recherche Agronomique, Université Evry Val d'Essonne), Evry, France
D School of Biological Sciences, University of Western Australia, Crawley, Western Australia, 6009, Australia
E Corresponding author. Email: [email protected]
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Abstract
Plant disease resistance genes play a critical role in providing resistance against pathogens.
The largest family of resistance genes are the nucleotide binding site (NBS) and leucine rich
repeat (LRR) genes (NBS-LRRs). They are classified into two major subfamilies,
toll/interleukin-1 receptor-NBS-LRR (TNL) proteins and coiled-coil CC-NBS-LRR (CNL)
proteins. Here, weWe have identified and characterized the complete set of 641, 249 and 443
NBS-LRR genes in Brassica napus, B. rapa and B. oleracea, respectively. A ratio of 1:2 of
CNL: TNL genes was found in the three species. DFurther domain structure analysis revealed
that 57% of the NBS- LRR genes are typical resistance genes and complete withcontain all
three domainsdomains (TIR/CC, NBS, LRR), whereas the remaining genes are partially
deleted or truncated. Fifty nine percent of the NBS-LRR genes were found to be physically
clustered, and individual genes involved in clusters were more polymorphic than those not
clustered. Fifty percent of the NBS-LRR genes in B. napus were identified as duplicates,
reflecting a high level of genomic duplication and rearrangement. The comparative analysis
between B. napus and its progenitor species indicated that more than 60% of NBS-LRR
genes arewere conserved in B. napus. This study provides a valuable resource for the
identification and characterization of candidate NBS-LRR genes.
Additional keywords:
Brassica, comparative genomics, disease resistance, gene cluster, gene duplication
Running head: Identification of resistance genes in Brassica napus
Conflict of interest: The authors declare no conflicts of interest
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Introduction
Plants have developed Resistance (R) genes as a crucial component in the immune system,
which plays an effective role in the resistance towards a wide spectrum of pathogens, such as
fungi, bacteria, viruses, oomycetes and nematodes (Wan et al. 2012; Dangl et al. 2013). The
understanding of the molecular structure and function of disease resistance genes (R genes)
has been crucial for plant resistance research.(Wan et al. 2012). Till now, over 140 R genes
have been cloned in different plant species (Zhang et al. 2016). According to Spoel and Dong
(2012), in the plant immune system, R genes encode proteins monitoring the perturbations of
self- molecules trigged by pathogen effectors (Spoel and Dong 2012). In this way, a few
hundred R genes could be sufficient to interact with a larger range of pathogen-encoded
effectors. These R genes are diverse in terms of their structure, function and evolution,
however, they can be grouped into five different classes, based on structural similarities of
their predicted protein products (Staskawicz et al. 1995; Liu et al. 2007). The largest class
includes proteins with putative nucleotide binding site (NBS) and leucine-rich repeats (LRR).
The NBS-LRR resistance genes appear to code for intracellular receptors that are composed
of a variable N terminal domain followed by the central NBS domain and C-terminal LRR
domains. The LRRs may be the main determinant in recognition specificity of the avirulence
gene product and as components of a signal transduction pathway (Ellis et al. 2000).
Sequence analyses revealed that NBS domains share a high degree of sequence identity and
have a number of conserved motifs. This characteristic specialty can be used to identify NBS-
LRR genes (Meyers et al. 1999; Wan et al. 2012).
Based on the presence of a toll/interleukin-1 receptor (TIR) domain at the N-terminusal,
NBS-LRR genes can be divided into TIR-NBS-LRR (TNL) and non- TIR-NBS-LRR (nTNL)
(Cannon et al. 2002). As the proteins encoded by nTNL genes always contain a coiled-coil
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domain at the N-terminalterminus, nTNL genes are also named referred to as CC-NBS-LRR
(CNL) (Meyers et al., 2003). These two major subfamilies of plant NBS-LRR proteins are
involved in defence against pathogens in different signalling pathways. They are distinct in
their NBS conserved sequence motifs and are therefore found in separate clusters in
phylogenetic analyses (McHale et al. 2006). In addition, a small group of nTNL genes with
RPW8 (RESISTANCE TO POWDERY MILDEW8) domains at the N-terminal terminus
have been identified (Xiao et al. 2004; Collier et al. 2011). Recently the RPW8-NBS-LRR
(RNL) genes were revealed as sister to all CNL genes, (Shao et al. 2014). According to
Collier et al. (2011) suggested that, the RNL genes do not specifically respond to different
pathogensdidn’t show specific response to different pathogens. They work by assisting TNL
and CNL genes, therefore (Bonardi et al. 2011) described them as ‘Helper NBS-LRRs’. With
this specific function, RNL genes are not directly involved in the recognition of rapidly
changing pathogensdo not evolve rapidly to respond to the quick change of pathogens, while
TNL genes and CNL genes’ function depends on the evolve rapid pathogen recognition. ly in
a pathogen-specific way (Zhang et al. 2016).
Plant genome sequencing has facilitated genome wide analysis of NBS-LRR genes in many
species including rice (Monosi et al. 2004), Medicago (Ameline-Torregrosa et al. 2008),
sorghum (Paterson et al. 2009), Arabidopsis (Meyers et al. 2003; Tan et al. 2007), papaya
(Porter et al. 2009), melon (Garcia-Mas et al. 2012), peach (Verde et al. 2013) and soybean
(Zhou et al. 2016). These studies showed that the NBS-LRR class of genes is abundant and
widely distributed throughout the genome. Moreover, it has been demonstrated that the
majority of NSB-LRR genes are present in gene clusters (Hulbert et al. 2001). The clustered
arrangement of these genes may be a critical attribute for the generation of novel resistance
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specificities (Meyers et al. 2003). With the possibility that NBS genes expanded prior to
familial divergence, the comparative analysis of NBS-LRR genes may help with
understanding the evolution and pathogen resistance mechanism of different species, or the
mining of novel R genes (Shao et al. 2016b; Zhang et al. 2016).
Brassica is one of the most important genera in the Brassicaceae family due to its agricultural
and commercially importantceeconomic importance. B. napus is the most important
commercial Brassica species and is grown primarily for its seed which yields approximately
40% oil. B. napus originated from interspecific hybridisation between the diploids B. rapa (A
genome) and B. oleracea (C genome) resulting in an amphidiploid genome, containing the A
and C sub-genomes A and C genome (AACC, n=19). Sequencing of Brassica genomes over
the last decade has resulted in reference genome sequences for B. napus (Chalhoub et al.
2014), B. rapa (Wang et al. 2011) and B. oleracea (Liu et al. 2014; Parkin et al. 2014)
permitting a comprehensive study of R genes in these Brassica species.
In a previous study of R genes in the incomplete B. rapa genome, a lower number of NBS-
LRR genes were predicted than found in other sequenced crops (Mun et al. 2009). However,
these authorsey estimated the number of NBS-LRR genes in the B. rapa genome should be
higher than in Arabidopsis (Mun et al. 2009). This estimation has been confirmedevidenced
by Hofberger et al. (2014) and Zhang et al. (2016b). Both studies performed the NBS-LRR
identification in five Brassicaceae genomes, in which more NBS-LRR genes were detected in
B. rapa than Arabdopsis. However, the difference was not significant (Fourmann et al. 2001;
Vicente and King 2001; Mun et al. 2009; Hofberger et al. 2014). Yu et al.(2014) has
identified 206, 176 and 157 NBS-LRR genes in B. rapa, B. oleracea and Arabidopsis,
respectively (Yu et al. 2014). These authorsy found that the number of NBS-LRR genes in
these three species was similarclose despite the differences in genome size and complexity.
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This is surprising as B. rapa and B. oleracea would be expected to host morea greater
numbers of NBS-LRR genes than Arabidopsis because the Brassica genomes have
undergone triplication following divergence from their common ancestor with Arabidopsis.
Similarly, wWith the similarclose genome size without genome duplication, the number of
NBS-LRR genes of peach is nearly three times more than that of melon, with 408 and 104
respectively (Garcia-Mas et al. 2012; Verde et al. 2013). As Jacob et al. (20135) concluded,
the number and distribution of NBS-LRR genes varied among plant species the variationety
among different species is dramatic regardless ofno matter with the genome size, and the
Brassicaceae family has moderate rich NBS-LRR genes detected . Furthermore, almost 50%
of NBS family members were detected as tandem arrays within homogenous clusters,
suggesting tandem duplication in combination with polyploidy after the separation from the
same ancestor played an important role in the expansion of NBS-LRR genes in the Brassica
genome (Fourmann et al. 2001; Vicente and King 2001; Mun et al. 2009).
The objectives of this study were to identify and characterize the structure and distribution of
NBS-LRR genes in B. napus and compare these with genes from B. rapa and B. oleracea.
Further association between NBS-LRR genes and resistance QTL can help to accelerate
isolation of disease resistance genestics.
Materials and methods
Brassica reference genomes
The genome sequence and annotation of B. napus Darmor was used as the reference genome
(Chalhoub et al. 2014). The genome sequences of B. rapa v3.0 (Wang et al. 2011) and B.
oleracea To1000 v1.0 (Parkin et al. 2014) were also used for comparative analysis.
Identification of NBS-LRR genes by MEME/ MAST and InterProScan analysis
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The MAST/MEME (Motif Alignment Search Tool/Multiple Em for Motif Elicitation) suite
of software was used to identify predicted genes that contain motif homology to known
disease resistance genes (Bailey et al. 2009). NBS-LRR “positive” and non NBS-LRR
“negative” sequence training sets, a consensus of 20 amino acid motifs derived from MEME
analysis (Jupe et al. 2012) were used as queries in a MAST search against the predicted genes
of the B. napus, B. oleracea and B. rapa genomes. Predicted genes were considered to be
candidate CNLC or TNL NBS -LRRs if the reported MAST E values were ≤ E-24.
InterProSacan (Jones et al. 2014) was used for further analysis to identify RNLs, another
group of nTNLs., Tthe protein sequences were subjected to two databases, Pfam for detecting
TIR, RPW8, NBS-LRR domains and Coils for detecting CC domain.
Identification of NBS-LRR genes by CNLs and TNLs for validation
To further validate the results from the MAST output, consensus sequences of plant CNLs
and TNLs (Cannon et al. 2002; Cannon et al. 2004) were obtained (Ameline-Torregrosa et
al. 2008). Candidate genes containing NBS domains were identified using tBLASTn and
BLASTp (maximum E- value 1E-5) (Altschul et al. 1997) performed using these consensus
sequences against the B. napus, B. oleracea and B. rapa genomes. Candidate NBS-LRR
proteins were provisionally assigned to either the CNL or TNL groups on the basis of
sequence similarity. Then, the results of the two different analyses (BLAST and MAST) were
compared NBS-LRR candidate genes identified from the MAST output were compared with
CNL and TNL BLAST results to validate the NBS- LRR genes. Any candidate NBS-LRR
genes that were not identified by both approaches that did not correlate between the two
approaches was were subject to further searches against the NCBI non-redundant nucleotide
database.
Characterization of candidate NBS-LRR genes
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All predicted NBS-LRR genes were surveyed to further determine their domain structure and
whether they contained TIR, CC, NBS, and/or LRR domains. The predicted genes were
searched for similarity to known proteins using BLASTp (Altschul et al. 1997) with a
threshold of E < -10 against the NCBI non-redundant (nr) protein database.
Multiple alignment and phylogenetic analysis
Alignment and phylogenetic analyses were performed with all NBS-containing proteins. The
NBS region was defined as the region extending from the P-loop to the MHDV motif, which
contains about 300 amino acids, using the corresponding conserved motifs from B. rapa
(Mun et al. 2009).
Genes with ≤ 50% of total NBS domain length, such as genes with partial NBS domains, with
length less than 150 aa or genes lacking NBS domains such as TIR genes, TIR-LRR genes
and linker LRR genes were excluded from phylogenetic analysis.
The NBS sequences were then aligned using CLUSTAL W (Thompson et al. 1994). A
phylogenetic tree was constructed using the neighbour-joining method (Saitou and Nei 1987)
with bootstrap multiple alignment resampling set at 1000. These functions were performed
using Molecular Evolutionary Genetics Analysis (MEGA) software version 6.0 (Tamura et
al. 2013).
NBS-LRR gene cluster and duplication analyses
NBS-LRR gene clusters were determined by their physical position on each chromosome
according to criteria previously published and widely used (Holub 2001; Richly et al. 2002;
Meyers et al. 2003). The parameter to define a cluster was three or more NBS genes that
occurred within a maximum of eight non-NBS genes. Furthermore, the distance between
neighbouring NBS-LRRs was required to be ≤ 200 kb.
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Gene duplication was defined in accordance with the criteria previously published and widely
used (Zhou et al. 2004): (1) the alignment covered more than 70% of the longer gene and (2)
The aligned region had an identity >70%. A block of duplications was defined as having
more than one gene in a gene cluster involved in the duplication if more than one gene in a
gene cluster was involved in the duplication. Homologous or paralogous genes were
confirmed by the BLASTp comparison of all the predicted NBS-LRR proteins against each
other with E-value of E-20.
Reciprocal Best BLAST of NBS-LRRs for comparative analysis between of B. napus, B. rapa
and B. oleracea
A reciprocal best BLAST (RBB) analysis was used to compare which of the NBS-LRR genes
predicted across the three genomes; B. napus, B. rapa and B. oleracea, were conserved. The
NBS- LRR genes from B. rapa (v3.0) were compared to with the identified NBS-LRR genes
identified from the B. napus cv. Darmor (A genome) and vice versa using BLAST. The
identified NBS-LRR genes from B. oleracea (To1000 v1.0) were also compared with
identifiedto NBS-LRR genes identified from the B. napus cv Darmor (C genome), and vice-
versa using BLAST.
Results
Genome wide identification of B. napus NBS-LRR genes
In total, 641 candidate NBS-LRR genes were identified in B. napus, consisting of 615 genes
from the MAST output with reported E value of < -24, plus additional genes identified from
consensus sequences of TNLs and CNLs (Table 1, Supplementary Table 1), with
approximately 80% of the NBS-LRR genes identified by both two methods. Genes that were
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not correlated between the two methods were truncated by either a premature stop codon or a
frameshift mutation causing them to fall under the level of significance of MAST or
CNL/TNL analysis, suggesting that they are pseudogenes. The 20 putative conserved motifs
are presented in Table 2 and Figure 1. These motifs have distinct patterns depending on
whether they are present in the TNL or CNL groups.
Of the 641 candidate NBS-LRR genes, 366 (57%) are typical or regular NBS-LRR genes,
with 124 CNLs and 242 TNLs (Table 2). These 366 genes show highly conserved NBS
regions and complete open reading frames. The remaining 188 genes (29%) were classified
as non–regular genes because of the lack of specific domains. These genes were classified
into three distinct groups for TNLs and CNLs. Non-regular TNLs were classified as TN (74),
N (9) and NL (43) whereas, non-regular CNLs were classified as CN (24), N (8) and NL (23).
These non-regular genes were described as being partial or truncated within the N-terminal
domains and/or have an absence of LRR domains. In addition, 87 TIR genes thatwhich lack
both NBS and LRR domains were also identified in B. napus. Furthermore, 73 NBS-LRR
genes (43 CNL and 30 TNL (21 TIR + 9 TNL) were encodedhad by a single reading frame
without introns (single exon) (Supplementary Table 1). ToFor identifying another group of
nTNL, named RNL, further analysis was performed using InterProScan which revealed 17
genes containing the RPW8 domain; whereby, 10 of them contained RPW8 and NBS-LRR
domains (6 genes on the A genome and 4 genes on the C genome) and the remaining 7 of
them contained CC, RPW8 and NBS-LRR domains.
Alignment and phylogenetic analysis
Phylogenetic analyseis of 176 CNL genes and 366 TNL genes were performed separately to
identify the similarity and relationship between genes (Figures 2 and 3). Genes with more
than 50% deletion in the NBS domain or with a lacking of the NBS domain (such as TIR
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genes) were excluded from the analysis. The trees were divided into clades on the basis of
clade rooting resulting in 16 clades for CNLs and 20 clades for TNLs (Figures 2 and 3).
Analysis of NBS-LRR genes on chromosomes A7 and C6 in B. napus and their homologs in
the diploid progenitor species,: B. rapa and B. oleracea, separates the TNL and CNL genes
into two clades (Figure 4). TNL and CNL groups were further divided into distinct subgroups
represented by conserved and orthologous genes from all three species. Further comparison
showed, TNL and CNL genes on homeologous chromosomes, such as A7 and C6, were
located in the same clade.
NBS-LRR gene clustering
The NBS-LRR genes were found to be distributed across the genome and are present on all
19 chromosomes (Table 34 and Supplementary Table 1), with 59% (378) of genes residing in
clusters. A total of 72 clusters were observed, containing up to 11 genes and with an average
of 5 genes. There were a greater number of TNL clusters (51) than CNL clusters (14), with
eight mixed clusters containing both TNLs and CNLs. Out of the total 72 clusters, 32 were
located in monophyletic clades and shared a recent common ancestor. These were considered
to be homogeneous, while the remaining 40 clusters are heterogeneous as they contained
more distantly related NBS-LRR genes.
NBS-LRR gene duplication
A total of 319 (50%) of the NBS-LRR genes were identified as duplicates (Table 34), with
more TNL (216) than CNL duplicates (103). The number of duplicated genes was consistent
between the A and C two sub-genomes, and 171 (53%) of duplicates were in gene clusters
(Table 34). Two types of genomic duplication were observed (Figure 5), intra-genomic and
inter-genomic. There were 41 duplication events between the A and C genomes, many of
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which showed collinearity between homeologous chromosomes. For example, 14 genes on
chromosome A1 were collinear with chromosome C1 (Figure 5). However, duplications were
also observed between non-homeologous chromosomes. For example, five genes in
chromosome A1 were collinear with genes on chromosome C3 (BnC3-49-CNL-NL), C5
(BnC5-2-TNL-TIR), C6 (BnC6-11-CNL-CN), C7 (BnC7-37-TNL-TNL) and C9 (BnC9-37-
TNL-TIR) (Figure 5).
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Comparative and conservation analysis of B. napus NBS-LRR genes with those ofits related
diploid species B. rapa and B. oleracea
In addition to the 641 NBS-LRR genes identified in B. napus, we identified a total of 249
genes in B. rapa and 443 genes in B. oleracea (Tables 3 and 5). The proportion of CNLs to
TNLs was consistent across the three species, with approximately 70% of R genes
comprising TNLs.
Many of the NBS-LRR genes showed highdemonstrated synteny between the genomes, with
260 and 149 B. napus NBS-LRR genes haveing orthologous genes in B. oleracea and B.
rapa, respectively (Table 45). Compared to the diploids, 100 NBS-LRR genes were missing
in the B. napus A genome and 183 in the C genome. NBS-LRR genes were also identified in
B. napus which wereare absent in B. rapa (103) and B. oleracea (121) respectively.
Approximately 324 (50%) of NBS-LRR in the A and C genomes of B. napus were found to
be syntenic and collinear to their respective progenitor genomes (Table 34). In addition, 72%
of these syntenic and collinear genes in B. napus were located within a gene cluster (Table
34).
Comparative analysis of chromosome A7 and C6 in B. napus and Rlm disease resistance
association
Comparative analysis of gene content of chromosomes A7 and C6 of Analysis of A7 and C6
between B. napus and the diploids showed that gene content was similar, with 19 NBS-LRR
genes on chromosome A7 of both B. rapa and B. napus, and 44 NBS-LRR genes on
chromosome C6 in B. napus compared to 45 genes in B. oleracea. We also found
14Fourteen NBS-LRRs wereinvolved in homeologous exchanges between B. napus
chromosomes A7 and C6 (Figure 6). Moreover, we found four genes were absent in the
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syntenic region of B. rapa and two genes showed substantial differences in sequence identity
(Figure 6).
Discussion
Genome wide identification distribution of B. napus NBS-LRR genes
The sequencing and assembly of Brassica genomes provides a valuable resource to study the
gene structure and evolution. Here we used the three public Brassica genomes for the
identification and characterization of candidate NBS-LRR genes to examine their
distribution, domain structure, clustering, duplication and conservation.
A combination of MAST and CNL/TNL analyses was applied. This enabled 20 NBS-LRR
specific motifs to be distinguished between TNL and CNL subfamilies. The MEME/MAST
analysis is highly dependent on accurate gene prediction and annotation. In contrast,
consensus CNL and TNL analysis using either tBLASTn or BLASTp can identify any gene
or protein sequence with sequence identity with known NBS-LRR motifs.
The 641 NBS-LRR genes in the B. napus was less than the sum of 249 genes in B. rapa and
443 genes in B. oleracea reflecting gene loss that may occur during polyplidization and
interspecific hybridization processes. The polyploidization events occur during divergence of
B. rapa, B. oleracea and B. nigra from a common hexaploid ancestor. Evidence for such
polyploidization events include genome replication and rearrangement (Lukens et al. 2004).
As a result of these events, novel genetic variation yet with high genome similarity between
each of the Brassica diploid species is created, despite difference in chromosome number
(Lagercrantz and Lydiate 1996; Lagercrantz 1998; Babula et al. 2003). Gene loss is reported
to be common during polyploidization event (Town et al. 2006) and interspecific hybridiation
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between B. rapa and B. oleracea (Zhang et al. 2016). The proportion of genes encoding
NBS-LRR in all three species is consistent with estimates for other plant species, which range
between 0.6 and 1.8% (Meyers et al. 2003; Monosi et al. 2004; Ameline-Torregrosa et al.
2008; Kohler et al. 2008; Yang et al. 2008).
The number of NBS-LRR genes identified here is higher than that identified by Chalhoub et
al. (2014) where 425, 211 and 274 genes were identified in B. napus, B. rapa and B.
oleracea, respectively. This difference is likely to be caused by a combination of a less
stringent MAST E value used here and manually checking for genes with partial NBS
domains. Their focus was also on the evolutionary relationship of NBS-LRR genes between
B. napus and its progenitor species, B. rapa and B. oleracea, whereas our focus was to study
the structure of NBS-LRR genes. We aimed to identify more genes with NBS-LRR
characteristics that could be potential candidates for disease resistance gene
candidatesidentification. Other studies, using different approaches: HMMER and Pfam, also
reported fewer number of NBS-LRR genes in B. rapa and B. oleracea (Yu et al. 2014; Zhang
et al. 2016) than in B.napus. Our study also identified linker-LRRs, this linker region lies
between the NB and LRR domains of a TNL and CNL, which is also a functional region for
defence mechanism (Eitas and Dangl 2010). The proportion of genes encoding NBS-LRR in
B. napus, B. rapa and B. oleracea is consistent with estimates for other plant species, which
range between 0.6 and 1.8% (Meyers et al. 2003; Monosi et al. 2004; Ameline-Torregrosa et
al. 2008; Kohler et al. 2008; Yang et al. 2008).
Approximately 70% of the NBS-LRR genes in B. napus, B. rapa and B. oleracea genes are
TNLs. This is consistent with B. rapa and B. oleracea. The greater number and diversity of
TNL compared to CNL and RNL genes has been previously reported in B. rapa (Mun et al.
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2009; Yu et al. 2014), Arabidopsis (Yu et al., 2014, Meyers et al., 2003), M. truncatula
(Ameline-Torregrosa et al. 2008), Populus trichocarpa (poplar) (Kohler et al. 2008) and
Linum usitatissimum (linseed) (Kale et al. 2012). The greater number of TNL is unusualique
but not limited to the Brassicaceae family, as compared to other angiosperm lineages such as
in the Fabaceae, Solanaceae and Poaceae families (Shao et al. 2016b). This distinct pattern of
TNL abundance in the Brassicaceae suggestsimplicated that the TIR domain is more
functionally active than the CC (Zhang et al. 2016). The ration of TNLs and CNLs is variable
among species thatand could reflect the adaptation of the R genes to the predominant
pathogens The predominance of TNLs or CNLs in a genome may be due to the nature of
pathogens that infect the plant species or another evolutionary basis (Leister 2004; Lozano et
al. 2012).
In many studies, nTNL genes are commonly named as CNL genes, because a CC domain is
frequently detected at the N-terminal terminus of the NBS-LRR genes. Recently, another
group of nTNL hasve been revealed with a distinct RPW8 domain, which indicates that
nTNL genes have a heterogeneous domain composition (Bonardi et al. 2011; Collier et al.
2011; Zhang et al. 2016). Phylogenetic analysis of NBS genes in some legume genomes
hasve identified the RNL subclass as a sister to all CNL genes, which indicates that the RNL
could be an accidently diverged NBS subclass (Shao et al. 2014). The phylogenetic analysis
for nTNL genes in five Brassicaceae species located all RNL genes in a clade and all CNL
genes fell into a sister clade (Zhang et al. 2016). In this study, among 17 genes with the
RPW8 domain, 7 genes contained both CC and RPW8 domains suggesting the RNL genes
are derived from the CNL lineage. The function of a few number of RNL genes is still
unclear, it has been suggested that they maintain their role in defence signal transduction
(Shao et al. 2016a) or they may help several or specific subclasses of NBS genes to achieve
their function (Collier et al. 2011). These genes are not directly involved in the response to
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the pathogen and are not necessarily duplicated in large numbers (Zhang et al. 2016). This
could support the finding of fewer numbers of RNL genes in the genome compared towith
the TNL and CNL genes.
The NBSSB-LRR genes were unevenly distributed across the genomes where chromosomes
A9 and C9 harbour the most genes while chromosomes A5 and C4 contain the least in the
respective genomes (Table 34). This uneven distribution pattern appears to be common in
plants (Meyers et al. 2003; Zhou et al. 2004; Kohler et al. 2008; Yang et al. 2008). Many
(59%) NBS-LRR genes are physically clustered in B. napus, which corresponds is a similar
proportion to that found in other plants including Arabidopsis (61%), M. truncatula (50%)
(Meyers et al. 2003; Ameline-Torregrosa et al. 2008) and potato (58%) (Jupe et al. 2012).
The B. napus NBS-LRR genes were divided into regular and non-regular NBS-LRR genes.
The regular NBS-LRR genes have all domains and complete open reading frames, whereas
the non-regular NBS-LRR resistance genes have partial motifs of the NBS domain or lack
CC, TIR or LRR domains. These were considered to be truncated or pseudogenes. Truncated
NBS-LRR genes have been described previously in plant genomes and they often surround
regular NBS-LRR genes (Meyers et al. 2003; Ameline-Torregrosa et al. 2008; Jupe et al.
2012). Non-regular NBS-LRR genes may be arise due to the partial deletion of redundant
NBS-LRR genes following whole genome duplication. Here we reported genes that encode
for only a TIR domain, lacking both NBS and LRR domains. These may be truncated NBS-
LRR genes or represent novel TIR containing proteins (Meyers et al. 2002; Yu et al. 2014).
Phylogenetic analysis
Different evolutionary patterns between TIR-NBS and non-TIR-NBS gene types during the
evolution of angiosperms has been suggested (Yang et al. 2008; Shao et al. 2016b). The
average number of genes per clade was similar for both TNLs and CNLs, despite there being
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a greater number of TNLs. This suggests that the NBS domains in CNLs are more divergent,
consistent with observations in other species (Bai et al. 2002; Cannon et al. 2002; Wan et al.
2013) and supporting the hypothesis that the CNL family is highly diverse and originated
prior to the split between gymnosperms and angiosperms (Wan et al. 2013). A phylogenetic
study of the N-terminus domain (TIR or CC) revealed that both TIR and CC play a role in a
distinct signalling pathway, where the genes evolved independently since the split between
monocots and dicots. The TIR domain was also reported to be more ancient than CC when
the TIR homolog was found in ancient trees, such as Pinus radiata (Pan et al. 2000). Another
study supporting this theory is that both monocots (rice) and dicots (Arabidopsis) share high
sequence homology of TIR-NBS genes in their genome, suggesting that the TIR domain is
was contained in the common ancestor before the both genomesmonocots and dicots split
(Bai et al. 2002). However, there is also evidence that CC evolved even earlier, before
gymnosperms and angisosperms split (Bai et al. 2002; Cannon et al. 2002; Wan et al. 2013).
The higher diversity of the CNL family in monocots is supported by the random number and
position of intron position compared to a more conserved intron position of TNLs in dicots
(Hammond-Kosack and Jones 1997; Meyers et al. 1999). HoweverYet, CNLs are less diverse
than TNLs in legumes (Zheng et al. 2016). This occurrence could be attributed to the lower
or slower evolutionary rate of CNLs less involvement of evolutionary events, such as gene
duplication, on the CNL group or the events happen at a slower rate (Song and Chen 2015;
Zheng et al. 2016). The TNL family is more homogeneous where members within a clade are
more related to each other in terms of gene organization and sequence identity (Lozano et al.
2015), and are only found in dicotyledonous species, reflecting their more recent evolution
(Tarr and Alexander 2009; Wan et al. 2013).
CAs compared to TNL, the CNL proteins are commonly encoded by single exons (Bai et al.
2002; Ameline-Torregrosa et al. 2008). In this study we also found that TNL genes showed
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complex structures, and various domains (TIR, NBS, and LRR) were often separated by
introns, with many short exons in the LRR region., Ssimilar results were also found in
Arabidopsis (Meyers et al. 2003).
Based on the phylogenetic analysis the NBS-LRR genes located on homeologous
chromosomes of B. napus were most likely found in the same clade. For example, in this
study, BnA1-1CNL and BnC1-2CNL, BnA1-5TNL and BnC1-6TNL were found in the same
clade. A comparison between TNL genes on chromosomes A7 and C6 were found them in
the same clade suggesting that these genes have been maintained without substantial
divergence or selection during the formation of B. napus from its progenitor species. These
TNL genes are also highly conserved between A and C genome, suggesting effects of whole
genome duplication and/or tandem duplication from a common ancestral source of NBS-LRR
genes. We also found mixed clades dominated by sequences from one chromosome, and
usually from one or a small number of genomic clusters, but many clades also contained
genes from other chromosomes. This could arise through chromosomal rearrangements or by
large-scale genomic duplication suggesting that gene duplication in B. napus may play a role
in the expansion and diversity of NBS-LRR genes.
NBS-LRR gene clustering
The percentage of NBS-LRR genes appearing in clusters is higher in the C genome (B.
oleracea, Table 34, 62%) compared to the A genome (B. rapa, 54%) in B. napus, which is
consistent in line with the previous comparison of B. oleracea, B. rapa and Arabidopsis. (Yu
et al. 2014). ’s study comparing B. oleracea, B. rapa and Arabidopsis. The TNL genes were
commonly found in clusters whereas most of the CNLs were not in clusters, as was also
reported in the potato genome (Jupe et al. 2012), possibly reflecting the greater abundance of
TNL genes.
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Single A single functional gene locusi denotes a stable gene that is not under high
selectionevolution pressure (Guo et al. 2011). Examples of single-copy genes include RPM1
and Rps2, which are both conserved in B. rapa and B. oleracea (Yu et al. 2014). Whereas,
NBS-LRR genes that form clusters tend to be more polymorphic and more prone to
evolutionary process. In this study, the majority of conserved NBS-LRR genes between B.
napus and its related species are not located in clusters but as a singletons.
Clustering of NBS-LRR genes with members having high sequence homology arising from
tandem gene duplication can confer gain of resistance function (Grant et al. 1998). This
situation is compared to heterogeneous clusters where genes involved in the cluster are
distantly related to R genes. These clusters of mixed R-gene related and unrelated genes areis
commonly found in higher plants such as M. truncatula (Zhu et al. 2002) and could have
arisen as a result of either evolutionary mechanism or random co-localization in the genome
(Richly et al. 2002; Meyers et al. 2003). Moreover, clusters contain genes from non- regular
NBS-LRR genes often located adjacent to regular NBS-LRR genes in gene clusters and this
is consistent with phylogenetic lineages. Therefore, the clustered organisation and
arrangement of these genes may be a critical attribute allowing the generation of novel
resistance specificities via intergenic rearrangement, duplication especially tandem
duplication or gene conversion (Hulbert et al. 2001).
NBS-LRR gene duplication
The level of gene duplication in the B. napus genome is thought to be significant with high
levels of genomic redundancy (Parkin et al. 2003; Rana et al. 2004; Parkin et al. 2005). Thus,
gene duplication, including tandem and whole-genome duplication (WGD) could have
resulted in a substantial increase of the gene number to compensate the gene losses during
formation of B. napus.
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In this study, we found that 50% of B. napus NBS-LRRs were duplicated or exist in pairs of
paralogs. The distribution of duplicate genes was random which could be as a result of
genome rearrangements or gene loss/gain occurring after whole genome duplication events.
Intra-genomic and inter-genomic duplication events between B. napus NBS-LRR genes has
been observed suggesting these genes possibly originated via tandem duplication where
duplicated genes are contiguous with its original copy. There was considerable collinearity
between duplicated genes in homologous chromosomes with the exception of some showing
duplication between non homologous chromosomes due to possible translocation or genome
mis-assembly.
It has been suggested that duplication in combination with divergent selection acting on
duplicated genes, influences the generation of gene diversity within existing gene clusters
(Parniske and Jones 1999; Richly et al. 2002; Wei et al. 2013). We found 38% of duplicated
genes were involved in formation of gene clusters suggesting that gene clusters could be
chromosomal hot spots in which the NBS-LRR genes are duplicated with the gene clusters
also expanded by tandem duplication followed byas a result of selection pressure (Wei et al.
2013). Indeed, previous studies revealed that NBS-LRR genes are often present as tightly
linked genes with high homology and are prone to gene duplication and recombination, and
thus evolve more rapidly than the rest of genome (Grant et al. 1998). Therefore, the clustered
distribution of NBS-LRR genes provides a reservoir of genetic variation by which new
specificities to pathogens can evolve via gene duplication (Michelmore and Meyers 1998;
Yang et al. 2008) . For instance, duplicated genes could be allelic variants that show
specificity for different pathogen strains. Together, these findings suggested that tandem
duplication in combination with recombination and selection pressure played a major role in
NBS-LRR genes expansion and diversity in B. napus.
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The duplicate genes were classified as resulting from polyploidization or post-speciation.
Based on their conservation and synteny, it can be difficult to distinguish among orthologs
and paralogs without extensive genome wide analysis. However, the results indicatedgave
indication that a large number of duplicated genes were originated via polyploidization from
the ancestral species: B. rapa and B. oleracea, while the tandemly duplicated genes were
more likely produced by post speciation duplications.
Comparative analysis of NBS-LRR genes on chromosome A7 and C6 in B. napus and Rlm
disease resistance association
Genetic and genomic studies have identified that a cluster of five linked genes on
chromosome A7 in B. napus are a major source of blackleg resistance genes (Delourme et al.
2004; Mayerhofer et al. 2005). An environmentally stable QTL for resistance against L.
maculans has also been identified on chromosome A7 in DH lines of B. napus (Huang et al.
2016), which confirms that this chromosome contains valuable QTLs for selection against L.
maculans.
Preliminary data suggests that there is duplication and clustering of tightly linked genes in the
QTL region containing NBS-LRR genes on chromosome A7 (Raman et al. 2012) and
suggests that map-based cloning of an Rlm gene may also identify additional blackleg
resistance genes (Raman et al. 2012; Tollenaere et al. 2012). The only resistance genes
which have been cloned in B. napus are LepR3 and Rlm2 (Larkan et al. 2014; Larkan et al.
2015), allelic variants of the same gene, located on chromosome A10. These genes belong
to the leucine-rich repeat receptor-like proteins (LRR-RLPs) family and have an important
role in facilitating defence signalling mechanisms. This family of genes was not analysed
within our study, and therefore the identification and role of RLPs should be included in
future analyses. This study identified several candidate NBS-LRR underlying the QTL
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region of blackleg resistance in B. napus. We found that the majority of candidate NBS-LRR
genes linked to blackleg resistance shared a duplicated copy on both chromosomes A7 and
C6 suggesting chromosomal duplication or rearrangements of this region. Furthermore,
several members of an NBS-LRR cluster on A7 that could confer resistance to blackleg
disease in B. napus had conserved orthologous genes in B. rapa. Therefore, the fully
sequenced B. napus genome has enabled us to identify blackleg candidate resistance genes on
chromosome A7 and targeting these genes for validation is underway.
Acknowledgements
The authors acknowledge funding support from Australian Research Council Projects DP0985953,
FT130100604, DP1601004497, LP140100537, LP160100030
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Tables
Table 1: The total number of predicted NBS-LRR genes identified in the three genomes
of Brassica species. The comparison between methods used; MEME / MAST and CNL
and TNL BLAST.
Species B. napus B. oleracea B. rapa
Genome size (Mbp) 1284 650 552
Gene content 111,479 59,225 41,019
Total candidate NBS-LRR genes 641 443 249
Proportion of NBS-LRR genes 0.58% 0.74% 0.60%
Identified in MAST output 615 437 248
Common to MAST and CNL/TNL
analysis509 379 208
Agreement % 79% 85% 83%
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Table 2: NBS-LRR specific motifs sequence identified with psp-gen MEME (Jupe et al., 2012).
Motif No. Length (bp) Motif domain Group or
class Motif sequence
1 21 P-loop kinase 1 NB-ARC CNL/TNL PIWGMGGVGKTTLARAVYNDP
2 21 GLPL NB-ARC CNL/TNL CGGLPLAIKVWGGMLAGKQKT
3 15 Kinase 2 NB-ARC CNL/TNL YLVVLDDVWDTDQWD
4 15 RNBS-B NB-ARC CNL/TNL GSRIIITTRNKHVAN
5 15 RNBS-D NB-ARC CNL LKPCFLYCAIFPEDY
6 21 MHDV NB-ARC CNL/TNL CRMHDMMHDMCWYKAREQNFV
7 15 RNBS-A NB-ARC CNL HFDCRAWVCVSQQYD
8 21 -- Linker CNL/TNL MEDVGEYYFNELINRSMFQPI
9 21 LRR LRR CNL/TNL LIHLRYLNLSGTNIKHLPASI
10 19 RNBA-C NB-ARC CNL/TNL YHMQFLSHEESWQLFHKHA
11 19 LRR LRR CNL/TNL MPNLETLDIRNCPNLEEIP
12 21 -- Pre-NB CNL IDRNKLIWLWMAEGFVPHENG
13 15 -- NB-ARC CNL/TNL NEIMPILRLSYHHLP
14 25 TIR 3 TIR TNL QIVIPIFYDVDPSDVRHQTGSFGEA
15 17 -- Pre-NB CNL DAAYDAEDVIDSFKYHA
16 50 -- Monocot -- AIKDIQEQLQKVADRRDRNKVFVPHPTRPIAIDPCLRALYAEATELVGIY
17 21` TIR 2 TIR TNL KNYATSRWCLNELVKIMECKE
18 50 EDVID Monocot CNL ETSSFELMDLLGERWVPPVHLREFKSFMPSQLSALRGWIQRDPSHLSNLS
19 27 LRR LRR -- FLDIACFFRGRKKDYVMQILESCDFGA
20 21 TIR 1 TIR TNL KYDVFLSFRGPDTRKTFTSHL
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Table 32: The number of NBS-LRR genes classified into different classes and their
subfamilies and domain compositions
CNL 180 TNL 461 Total 641
CNL-NBS-LRR 124 TIR-NBS-LRR 242Regular NBS-LRR
gene366(57%)
NBS-LRR 23 NBS-LRR 43
Non-regular NBS-
LRR gene188(29%)
NBS 8 NBS 9
CC-NBS 24 TIR-NBS 74
LINKER-LRR 1 LINKER-LRR 3
TIR-LRR 3
TIR 87 87(14%)
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Table 43: The distribution and cComparative analysis of candidate NBS-LRR genes
in B. napusCh
rom
osom
e
Tota
l
TNL
CNL
No.
of g
ene
clus
ters
No.
of N
BS-
LRR
gene
s in
clus
ters
No.
of
dupl
icat
ed
gene
s (T
NL+
CNL)
No.
of
dupl
icat
ed
gene
s in
Cons
erve
d
Synt
enic
ge
nes
Synt
enic
ge
nes i
n cl
uste
r
A ge
nom
e B.
nap
us D
arm
or
A1 28 17 11 2 7 18(10+8) 4 17 13 5A2 44 33 11 5 31 22(20+2) 15 26 20 16A3 24 20 4 5 18 16(13+3) 6 15 13 10A4 13 12 1 1 7 5(5+0) 3 6 3 2A5 10 4 6 1 4 9(3+6) 5 5 4 1A6 21 7 14 2 6 16(5+11) 5 11 7 2A7 19 16 3 2 10 14(14+0) 8 12 10 4A8 24 17 7 3 12 13(9+4) 3 12 10 5A9 58 38 20 10 43 38(23+5) 24 40 35 30
A10 11 7 4 0 0 6(4+2) 0 5 4 1
Total 252 171 81 31 138(54%)
157 (106+51)(62%)
73 (28%) 149 119 76
C ge
nom
e B.
nap
us D
arm
or
C1 37 24 13 3 21 16(9+7) 10 25 21 12C2 51 47 4 5 34 21(18+3) 13 32 15 12C3 56 46 10 7 35 20(14+6) 12 33 26 20C4 18 12 6 1 4 5(3+2) 2 11 8 5C5 19 11 8 4 13 13(8+5) 7 14 14 10C6 44 33 11 4 30 28(20+8) 17 33 29 23C7 38 30 8 5 25 14(10+4) 8 32 31 25C8 39 21 18 4 23 19(9+10) 11 24 19 18C9 79 62 17 8 55 26(19+7) 18 54 42 33
Total 381 286 95 41 240(62%)
162 (110+52)(42%)
98 (25%) 258 205 158
Unassigned 8 4 4 0 0 0 0 0 0 0
Total 641 461 180 72 378(59%)
319(50%)
171(53%)
409(63.8%)
324 (50%)
234(72%)
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3
4
Table 54: Comparative analysis of the number of NBS-LRR genes in the three
Brassica species and the reciprocal best BLAST hits
Genome B. oleracea
C genome
B. napus
Darmor
C genome
B. rapa
A genome
B. napus
Darmor
A genome
Total NBS LRR 443 381 249 252
Total TNL and CNL 319 , 124 286 , 95 168 , 81 171 , 81
Reciprocal BLAST hits conserved
genes “retained”260 (60%) 260 (75%) 149 (60%) 149 (60%)
Conserved TNL and CNL 187 , 73 192 , 68 107 , 42 108 , 41
% Conserved TNL and CNL 58% , 58% 67% , 71% 63% , 52% 63% , 52%
No. of conserved genes in cluster 160 (61%) 75 (50%)
Not conserved 183 121 100 103
missing 183 0 100 0
Gained 0 121 0 103
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Supplementary Table 1: A list of NBS-LRR genes identified in the study, including
genome position, gene type and number of exons
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Figure captions
Figure 1: Example of representation of motif patterns in NBS-LRR genes in B. napus. The
different coloured boxes indicated distinct motifs as shown in boxes below identified by
MEME/MAST program. Refer to Table 2 for the type of each motif.
Figure 2: A phylogenetic tree of NBS domains in 176 CNL- encoding genes of B. napus
for NBS domains based on the neighbour-joining method using MEGA 6.0 software. The
numbers on the branches indicate the percentage of 1000 bootstrap replicates. The tree
only shows values > 50%. Gene names are intended to represent chromosome and domain
configurations. The blue and red coloured branches are to separate the clades. The closed
green triangle represent a homogenous cluster on chromosome C6 where all genes
sequence are related and located within a clade as well as their relationship with genes on
homologous chromosome A7 as represented by unfilled green triangles.
Figure 3: A phylogenetic tree of NBS domains in 366 TNL- encoding genes of B. napus
for NBS domains based on the neighbour-joining method using MEGA 6.0 software. The
numbers on the branches indicate the percentage of 1000 bootstrap replicates. The tree
only shows values > 50%. Gene names are intended to represent chromosome and domain
configurations. The blue and red coloured branches are to separate the clades. The closed
green triangles represent a homogenous cluster on chromosome C6 where all genes
sequence are related and located within a clade as well as their relationship with genes on
homologous chromosome A7, as represented by unfilled green triangles.
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2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Figure 4: A phylogenetic tree of TNL and CNL genes located on chromosomes A7 and C6
in B. napus and the diploid species B. rapa and B. oleracea based on a neighbour-joining
method using MEGA 6.0 software. The numbers on branches indicate the percentage of
1000 bootstrap replicates. The coloured squares indicate the gene origin: red from B.
napus, blue from B. rapa and green from B. oleracea.
Figure 5: Inter-genomic and intra-genomic duplication relationship of NBS-LRR -genes
between the two sub-genomes of B. napus: the A and C genomes. Coloured lines represent
the relationship of paralogous gene pairs between homeologous chromosomes. Red lines
represent the gene duplication between chromosomes within a sub-genome. Black lines
represent the gene duplication between homologous chromosomes with collinearity. Blue
lines represent the gene duplication between non homeologous chromosomes. The green
block represents a duplicated block where there is more than one gene involved in
duplication. Chromosomes are scaled based on the number of NBS-LRR genes
Figure 6: Comparative chromosomal map of NBS-LRR genes on chromosomes A7 and
C6 between B. napus (Bn), B. rapa (Br) and B. oleracea (Bo). The squares and triangles
represent NBS-LRR type: TNL and CNL, respectively. The Rlm QTL refers to Huang et
al. (2016). Genes in syntenic relationships are connected by lines. Red lines show inter-
genomic duplications between B. napus A7 and C6. Closed bars are orthologous genes.
Genes within green squares are in a gene cluster. The numbers represent the amino acid
alignments with alignment coverage in bracket.
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