Nucleotide-binding Leucine-rich Repeat Genes Research Articles

A high-quality pseudo-phased genome for Melaleuca quinquenervia shows allelic diversity of NLR-type resistance genes.

Melaleuca quinquenervia (broad-leaved paperbark) is a coastal wetland tree species that serves as a foundation species in eastern Australia, Indonesia, Papua New Guinea, and New Caledonia. While extensively cultivated for its ornamental value, it has also become invasive in regions like Florida, USA. Long-lived trees face diverse pest and pathogen pressures, and plant stress responses rely on immune receptors encoded by the nucleotide-binding leucine-rich repeat (NLR) gene family. However, the comprehensive annotation of NLR encoding genes has been challenging due to their clustering arrangement on chromosomes and highly repetitive domain structure; expansion of the NLR gene family is driven largely by tandem duplication. Additionally, the allelic diversity of the NLR gene family remains largely unexplored in outcrossing tree species, as many genomes are presented in their haploid, collapsed state. We assembled a chromosome-level pseudo-phased genome for M. quinquenervia and described the allelic diversity of plant NLRs using the novel FindPlantNLRs pipeline. Analysis reveals variation in the number of NLR genes on each haplotype, distinct clustering patterns, and differences in the types and numbers of novel integrated domains. The high-quality M. quinquenervia genome assembly establishes a new framework for functional and evolutionary studies of this significant tree species. Our findings suggest that maintaining allelic diversity within the NLR gene family is crucial for enabling responses to environmental stress, particularly in long-lived plants.

ANTHRACNOSE RESISTANCE GENE2 confers fungal resistance in sorghum.

Sorghum is an important food and feed crop globally; its production is hampered by anthracnose disease caused by the fungal pathogen Colletotrichum sublineola (Cs). Here, we report identification and characterization of ANTHRACNOSE RESISTANCE GENE 2 (ARG2) encoding a nucleotide-binding leucine-rich repeat (NLR) protein that confers race-specific resistance to Cs strains. ARG2 is one of a cluster of several NLR genes initially identified in the sorghum differential line SC328C that is resistant to some Cs strains. This cluster shows structural and copy number variations in different sorghum genotypes. Different sorghum lines carrying independent ARG2 alleles provided the genetic validation for the identity of the ARG2 gene. ARG2 expression is induced by Cs, and chitin induces ARG2 expression in resistant but not in susceptible lines. ARG2-mediated resistance is accompanied by higher expression of defense and secondary metabolite genes at early stages of infection, and anthocyanin and zeatin metabolisms are upregulated in resistant plants. Interestingly, ARG2 localizes to the plasma membrane when transiently expressed in Nicotiana benthamiana. Importantly, ARG2 plants produced higher shoot dry matter than near-isogenic lines carrying the susceptible allele suggesting an absence of an ARG2 associated growth trade-off. Furthermore, ARG2-mediated resistance is stable at a wide range of temperatures. Our observations open avenues for resistance breeding and for dissecting mechanisms of resistance.

Engineering Resistance against Sclerotinia sclerotiorum Using a Truncated NLR (TNx) and a Defense-Priming Gene.

The association of both cell-surface PRRs (Pattern Recognition Receptors) and intracellular receptor NLRs (Nucleotide-Binding Leucine-Rich Repeat) in engineered plants have the potential to activate strong defenses against a broad range of pathogens. Here, we describe the identification, characterization, and in planta functional analysis of a novel truncated NLR (TNx) gene from the wild species Arachis stenosperma (AsTIR19), with a protein structure lacking the C-terminal LRR (Leucine Rich Repeat) domain involved in pathogen perception. Overexpression of AsTIR19 in tobacco plants led to a significant reduction in infection caused by Sclerotinia sclerotiorum, with a further reduction in pyramid lines containing an expansin-like B gene (AdEXLB8) potentially involved in defense priming. Transcription analysis of tobacco transgenic lines revealed induction of hormone defense pathways (SA; JA-ET) and PRs (Pathogenesis-Related proteins) production. The strong upregulation of the respiratory burst oxidase homolog D (RbohD) gene in the pyramid lines suggests its central role in mediating immune responses in plants co-expressing the two transgenes, with reactive oxygen species (ROS) production enhanced by AdEXLB8 cues leading to stronger defense response. Here, we demonstrate that the association of potential priming elicitors and truncated NLRs can produce a synergistic effect on fungal resistance, constituting a promising strategy for improved, non-specific resistance to plant pathogens.

Genetic Insight into Disease Resistance Gene Clusters by Using Sequencing-Based Fine Mapping in Sunflower (Helianthus annuus L.).

Rust and downy mildew (DM) are two important sunflower diseases that lead to significant yield losses globally. The use of resistant hybrids to control rust and DM in sunflower has a long history. The rust resistance genes, R13a and R16, were previously mapped to a 3.4 Mb region at the lower end of sunflower chromosome 13, while the DM resistance gene, Pl33, was previously mapped to a 4.2 Mb region located at the upper end of chromosome 4. High-resolution fine mapping was conducted using whole genome sequencing of HA-R6 (R13a) and TX16R (R16 and Pl33) and large segregated populations. R13a and R16 were fine mapped to a 0.48 cM region in chromosome 13 corresponding to a 790 kb physical interval on the XRQr1.0 genome assembly. Four disease defense-related genes with nucleotide-binding leucine-rich repeat (NLR) motifs were found in this region from XRQr1.0 gene annotation as candidate genes for R13a and R16. Pl33 was fine mapped to a 0.04 cM region in chromosome 4 corresponding to a 63 kb physical interval. One NLR gene, HanXRQChr04g0095641, was predicted as the candidate gene for Pl33. The diagnostic SNP markers developed for each gene in the current study will facilitate marker-assisted selections of resistance genes in sunflower breeding programs.

A super pan-genomic landscape of rice

Pan-genomes from large natural populations can capture genetic diversity and reveal genomic complexity. Using de novo long-read assembly, we generated a graph-based super pan-genome of rice consisting of a 251-accession panel comprising both cultivated and wild species of Asian and African rice. Our pan-genome reveals extensive structural variations (SVs) and gene presence/absence variations. Additionally, our pan-genome enables the accurate identification of nucleotide-binding leucine-rich repeat genes and characterization of their inter- and intraspecific diversity. Moreover, we uncovered grain weight-associated SVs which specify traits by affecting the expression of their nearby genes. We characterized genetic variants associated with submergence tolerance, seed shattering and plant architecture and found independent selection for a common set of genes that drove adaptation and domestication in Asian and African rice. This super pan-genome facilitates pinpointing of lineage-specific haplotypes for trait-associated genes and provides insights into the evolutionary events that have shaped the genomic architecture of various rice species.

Haplotype-resolved powdery mildew resistance loci reveal the impact of heterozygous structural variation on NLR genes in Muscadinia rotundifolia.

Muscadinia rotundifolia cv. Trayshed is a valuable source of resistance to grape powdery mildew. It carries 2 powdery mildew resistance-associated genetic loci, Run1.2 on chromosome 12 and Run2.2 on chromosome 18. The purpose of this study was to identify candidate resistance genes associated with each haplotype of the 2 loci. Both haplotypes of each resistance-associated locus were identified, phased, and reconstructed. Haplotype phasing allowed the identification of several structural variation events between haplotypes of both loci. Combined with a manual refinement of the gene models, we found that the heterozygous structural variants affected the gene content, with some resulting in duplicated or hemizygous nucleotide-binding leucine-rich repeat genes. Heterozygous structural variations were also found to impact the domain composition of some nucleotide-binding leucine-rich repeat proteins. By comparing the nucleotide-binding leucine-rich repeat proteins at Run1.2 and Run2.2 loci, we discovered that the 2 loci include different numbers and classes of nucleotide-binding leucine-rich repeat genes. To identify powdery mildew resistance-associated genes, we performed a gene expression profiling of the nucleotide-binding leucine-rich repeat genes at Run1.2b and Run2.2 loci with or without powdery mildew present. Several nucleotide-binding leucine-rich repeat genes were constitutively expressed, suggesting a role in powdery mildew resistance. These first complete, haplotype-resolved resistance-associated loci and the candidate nucleotide-binding leucine-rich repeat genes identified by this study are new resources that can aid the development of powdery mildew-resistant grape cultivars.

The Ups and Downs of Plant NLR Expression During Pathogen Infection.

Plant Nucleotide binding-Leucine rich repeat (NLR) proteins play a significant role in pathogen detection and the activation of effector-triggered immunity. NLR regulation has mainly been studied at a protein level, with large knowledge gaps remaining regarding the transcriptional control of NLR genes. The mis-regulation of NLR gene expression may lead to the inability of plants to recognize pathogen infection, lower levels of immune response activation, and ultimately plant susceptibility. This highlights the importance of understanding all aspects of NLR regulation. Three main mechanisms have been shown to control NLR expression: epigenetic modifications, cis elements which bind transcription factors, and post-transcriptional modifications. In this review, we aim to provide an overview of these mechanisms known to control NLR expression, and those which contribute toward successful immune responses. Furthermore, we discuss how pathogens can interfere with NLR expression to increase pathogen virulence. Understanding how these molecular mechanisms control NLR expression would contribute significantly toward building a complete picture of how plant immune responses are activated during pathogen infection—knowledge which can be applied during crop breeding programs aimed to increase resistance toward numerous plant pathogens.

MAP KINASE PHOSPHATASE1 promotes osmotolerance by suppressing PHYTOALEXIN DEFICIENT4-independent immunity.

Initial exposure of plants to osmotic stress caused by drought, cold, or salinity leads to acclimation, termed acquired tolerance, to subsequent severe stresses. Acquired osmotolerance induced by salt stress is widespread across Arabidopsis (Arabidopsis thaliana) accessions and is conferred by disruption of a nucleotide-binding leucine-rich repeat gene, designated ACQUIRED OSMOTOLERANCE. De-repression of this gene under osmotic stress causes detrimental autoimmunity via ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 (PAD4). However, the mechanism underlying acquired osmotolerance remains poorly understood. Here, we isolated an acquired osmotolerance-defective mutant (aod13) by screening 30,000 seedlings of an ion beam-mutagenized M2 population of Bu-5, an accession with acquired osmotolerance. We found that AOD13 encodes the dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1), which negatively regulates MITOGEN-ACTIVATED PROTEIN KINASE3/6 (MPK3/6). Consistently, MPK3/6 activation was greater in aod13 than in the Bu-5 wild-type (WT). The aod13 mutant was sensitive to osmotic stress but tolerant to salt stress. Under osmotic stress, pathogenesis-related genes were strongly induced in aod13 but not in the Bu-5 WT. Loss of PAD4 in pad4 aod13 plants did not restore acquired osmotolerance, implying that activation of immunity independent of PAD4 renders aod13 sensitive to osmotic stress. These findings suggest that AOD13 (i.e. MKP1) promotes osmotolerance by suppressing the PAD4-independent immune response activated by MPK3/6.

Partially Resistant Avocado Rootstock Dusa® Shows Prolonged Upregulation of Nucleotide Binding-Leucine Rich Repeat Genes in Response to Phytophthora cinnamomi Infection.

Avocado is an important agricultural food crop in many countries worldwide. Phytophthora cinnamomi, a hemibiotrophic oomycete, remains one of the most devastating pathogens within the avocado industry, as it is near impossible to eradicate from areas where the pathogen is present. A key aspect to Phytophthora root rot disease management is the use of avocado rootstocks partially resistant to P. cinnamomi, which demonstrates an increased immune response following infection. In plant species, Nucleotide binding-Leucine rich repeat (NLR) proteins form an integral part of pathogen recognition and Effector triggered immune responses (ETI). To date, a comprehensive set of Persea americana NLR genes have yet to be identified, though their discovery is crucial to understanding the molecular mechanisms underlying P. americana-P. cinnamomi interactions. In this study, a total of 161 PaNLR genes were identified in the P. americana West-Indian pure accession genome. These putative resistance genes were characterized using bioinformatic approaches and grouped into 13 distinct PaNLR gene clusters, with phylogenetic analysis revealing high sequence similarity within these clusters. Additionally, PaNLR expression levels were analyzed in both a partially resistant (Dusa®) and a susceptible (R0.12) avocado rootstock infected with P. cinnamomi using an RNA-sequencing approach. The results showed that the partially resistant rootstock has increased expression levels of 84 PaNLRs observed up to 24 h post-inoculation, while the susceptible rootstock only showed increased PaNLR expression during the first 6 h post-inoculation. Results of this study may indicate that the partially resistant avocado rootstock has a stronger, more prolonged ETI response which enables it to suppress P. cinnamomi growth and combat disease caused by this pathogen. Furthermore, the identification of PaNLRs may be used to develop resistant rootstock selection tools, which can be employed in the avocado industry to accelerate rootstock screening programs.

Genome sequences of three Aegilops species of the section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement.

Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for the improvement of wheat. To understand their diversity and advance their utilization, we produced whole-genome assemblies of Aegilops longissima and Aegilops speltoides. Whole-genome comparative analysis, along with the recently sequenced Aegilops sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and are most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides genome is closest to the wheat B subgenome, and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome-wide analysis of nucleotide-binding leucine-rich repeat (NLR) genes revealed species-specific and lineage-specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.

Frequent Gene Duplication/Loss Shapes Distinct Evolutionary Patterns of NLR Genes in Arecaceae Species

Nucleotide-binding leucine-rich repeat (NLR) genes play a key role in plant immune responses and have co-evolved with pathogens since the origin of green plants. Comparative genomic studies on the evolution of NLR genes have been carried out in several angiosperm lineages. However, most of these lineages come from the dicot clade. In this study, comparative analysis was performed on NLR genes from five Arecaceae species to trace the dynamic evolutionary pattern of the gene family during species speciation in this monocot lineage. The results showed that NLR genes from the genomes of Elaeis guineensis (262), Phoenix dactylifera (85), Daemonorops jenkinsiana (536), Cocos nucifera (135) and Calamus simplicifolius (399) are highly variable. Frequent domain loss and alien domain integration have occurred to shape the NLR protein structures. Phylogenetic analysis revealed that NLR genes from the five genomes were derived from dozens of ancestral genes. D. jenkinsiana and E. guineensis genomes have experienced “consistent expansion” of the ancestral NLR lineages, whereas a pattern of “first expansion and then contraction” of NLR genes was observed for P. dactylifera, C. nucifera and C. simplicifolius. The results suggest that rapid and dynamic gene content and structure variation have shaped the NLR profiles of Arecaceae species.

Discovery of a novel powdery mildew (Blumeria graminis) resistance locus in rye (Secale cereale L.)

Powdery mildew is one of the most destructive diseases in the world, causing substantial grain yield losses and quality reduction in cereal crops. At present 23 powdery mildew resistance genes have been identified in rye, of which the majority are in wheat-rye translocation lines developed for wheat improvement. Here, we investigated the genetics underlying powdery mildew resistance in the Gülzow-type elite hybrid rye (Secale cereale L.) breeding germplasm. In total, 180 inbred breeding lines were genotyped using the state-of-the-art 600 K SNP array and phenotyped for infection type against three distinct field populations of B. graminis f. sp. secalis from Northern Germany (2013 and 2018) and Denmark (2020). We observed a moderate level of powdery mildew resistance in the non-restorer germplasm population, and by performing a genome-wide association study using 261,406 informative SNP markers, we identified a powdery mildew resistance locus, provisionally denoted PmNOS1, on the distal tip of chromosome arm 7RL. Using recent advances in rye genomic resources, we investigated whether nucleotide-binding leucine-rich repeat genes residing in the identified 17 Mbp block associated with PmNOS1 on recent reference genomes resembled known Pm genes.

Reduction of the canonical function of a glycolytic enzyme enolase triggers immune responses that further affect metabolism and growth in Arabidopsis.

Primary metabolism provides energy for growth and development as well as secondary metabolites for diverse environmental responses. Here we describe an unexpected consequence of disruption of a glycolytic enzyme enolase named LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 2 (LOS2) in causing constitutive defense responses or autoimmunity in Arabidopsis thaliana. The autoimmunity in the los2 mutant is accompanied by a higher expression of about one-quarter of intracellular immune receptor nucleotide-binding leucine-rich repeat (NLR) genes in the genome and is partially dependent on one of these NLR genes. The LOS2 gene was hypothesized to produce an alternatively translated protein c-Myc Binding Protein (MBP-1) that functions as a transcriptional repressor. Complementation tests show that LOS2 executes its function in growth and immunity regulation through the canonical enolase activity but not the production of MBP-1. In addition, the autoimmunity in the los2 mutants leads to a higher accumulation of sugars and organic acids and a depletion of glycolytic metabolites. These findings indicate that LOS2 does not exert its function in immune responses through an alternatively translated protein MBP-1. Rather, they show that a perturbation of glycolysis from the reduction of the enolase activity results in activation of NLR-involved immune responses which further influences primary metabolism and plant growth, highlighting the complex interaction between primary metabolism and plant immunity.

RXam2, a NLR from cassava (Manihot esculenta) contributes partially to the quantitative resistance to Xanthomonas phaseoli pv. manihotis.

The overexpression of RXam2, a cassava NLR (nucleotide-binding leucine-rich repeat) gene, by stable transformation and gene expression induction mediated by dTALEs, reduce cassava bacterial blight symptoms. Cassava (Manihot esculenta) is a tropical root crop affected by different pathogens including Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of cassava bacterial blight (CBB). Previous studies have reported resistance to CBB as a quantitative and polygenic character. This study sought to validate the functional role of a NLR (nucleotide-binding leucine-rich repeat) associated with a QTL to Xpm strain CIO151 called RXam2. Transgenic cassava plants overexpressing RXam2 were generated and analyzed. Plants overexpressing RXam2 showed a reduction in bacterial growth to Xpm strains CIO151, 232 and 226. In addition, designer TALEs (dTALEs) were developed to specifically bind to the RXam2 promoter region. The Xpm strain transformed with dTALEs allowed the induction of the RXam2 gene expression after inoculation in cassava plants and was associated with a diminution in CBB symptoms. These findings suggest that RXam2 contributes to the understanding of the molecular basis of quantitative disease resistance.

An angiosperm NLR Atlas reveals that NLR gene reduction is associated with ecological specialization and signal transduction component deletion

Nucleotide-binding leucine-rich-repeat (NLR) genes comprise the largest family of plant disease-resistance genes. Angiosperm NLR genes are phylogenetically divided into the TNL, CNL, and RNL subclasses. NLR copy numbers and subclass composition vary tremendously across angiosperm genomes. However, the evolutionary associations between genomic NLR content and ecological adaptation, or between NLR content and signal transduction components, are poorly characterized because of limited genome availability. In this study, we established an angiosperm NLR atlas (ANNA, https://biobigdata.nju.edu.cn/ANNA/) that includes NLR genes from over 300 angiosperm genomes. Using ANNA, we revealed that NLR copy numbers differ up to 66-fold among closely related species owing to rapid gene loss and gain. Interestingly, NLR contraction was associated with adaptations to aquatic, parasitic, and carnivorous lifestyles. The convergent NLR reduction in aquatic plants resembles the lack of NLR expansion during the long-term evolution of green algae before the colonization of land. A co-evolutionary pattern between NLR subclasses and plant immune pathway components was also identified, suggesting that immune pathway deficiencies may drive TNL loss. Finally, we identified a conserved TNL lineage that may function independently of the EDS1–SAG101–NRG1 module. Collectively, these findings provide new insights into the evolution of NLR genes in the context of ecological adaptation and genome content variation.

Major QTL confer race-nonspecific resistance in the co-evolved Cronartium quercuum f. sp. fusiforme-Pinus taeda pathosystem.

Fusiform rust disease, caused by the endemic fungus Cronartium quercuum f. sp. fusiforme, is the most damaging disease affecting economically important pine species in the southeast United States. Unlike the major epidemics of agricultural crops, the co-evolved pine-rust pathosystem is characterized by steady-state dynamics and high levels of genetic diversity within environments. This poses a unique challenge and opportunity for the deployment of large-effect resistance genes. We used trait dissection to study the genetic architecture of disease resistance in two P. taeda parents that showed high resistance across multiple environments. Two mapping populations (full-sib families), each with ~1000 progeny, were challenged with a complex inoculum consisting of 150 pathogen isolates. High-density linkage mapping revealed three major-effect QTL distributed on two linkage groups. All three QTL were validated using a population of 2057 cloned pine genotypes in a 6-year-old multi-environmental field trial. As a complement to the QTL mapping approach, bulked segregant RNAseq analysis revealed a small number of candidate nucleotide binding leucine-rich repeat genes harboring SNP associated with disease resistance. The results of this study show that in P. taeda, a small number of major QTL can provide effective resistance against genetically diverse mixtures of an endemic pathogen. These QTL vary in their impact on disease liability and exhibit additivity in combination.

Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis

Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis

An accumulation of genetic variation and selection across the disease-related genes during apple domestication

Although human-imposed selection is known to have altered plant traits during crop domestication, the effect of selection on host susceptibility or resistance is not well understood. Moreover, the domestication of perennial tree fruit crops, unlike annual crops, was driven by hybridization, clonal propagation, and selection of desirable phenotypes, which could confound the accurate assessment of domestication-associated effects on host resistance or susceptibility genes. We studied the effects of domestication on disease-related nucleotide-binding leucine-rich repeat (NLR) genes by combining phylogenetic, haplotype, and selection signature analysis using sequence data from Golden Delicious double haploid (GDDH13 v.1.1) apple genome and 80 resequenced domesticated and wild Malus accessions. The NLR gene family in the GDDH13 v.1.1 apple genome constituted 546 genes that showed expansion mainly through proximal (39.1%) and dispersed (29.5%) duplications. The genome duplication (WGD) within Rosaceae, affecting the Malus lineage, is also evident in the NLR genes. The NLR genes are found in genomic regions associated with previously detected disease resistance-related quantitative trait loci (QTL) for apple scab, fire blight, powdery mildew, and blue mold. A genomic diversity analysis identified that the NLR genes in domesticated apples (Malus domestica) have more variation (average πdom=4.01 × 10−3) than their main progenitor, wild M. sieversii (average πsie=2.95 × 10−3) and M. sylvestris (average πsyl=2.11 × 10−3), which hybridized with M. domestica during the domestication process. These results suggest evidence of selection on disease-related genes associated with domestication of apple. An increased diversity across NLR genes in the domesticated germplasm may be attributed to their diverse geographical origins and distinct pedigrees, together with selection for disease resistance during domestication. The NLR genes under selection can provide opportunities to explore their role in disease resistance in apples.

Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA.

Genes involved in disease resistance are some of the fastest evolving and most diverse components of genomes. Large numbers of nucleotide-binding, leucine-rich repeat (NLR) genes are found in plant genomes and are required for disease resistance. However, NLRs can trigger autoimmunity, disrupt beneficial microbiota or reduce fitness. It is therefore crucial to understand how NLRs are controlled. Here, we show that the RNA-binding protein FPA mediates widespread premature cleavage and polyadenylation of NLR transcripts, thereby controlling their functional expression and impacting immunity. Using long-read Nanopore direct RNA sequencing, we resolved the complexity of NLR transcript processing and gene annotation. Our results uncover a co-transcriptional layer of NLR control with implications for understanding the regulatory and evolutionary dynamics of NLRs in the immune responses of plants.

A high-quality Brassica napus genome reveals expansion of transposable elements, subgenome evolution and disease resistance.

SummaryRapeseed (Brassica napus L.) is a recent allotetraploid crop, which is well known for its high oil production. Here, we report a high‐quality genome assembly of a typical semi‐winter rapeseed cultivar, 'Zhongshuang11' (hereafter 'ZS11'), using a combination of single‐molecule sequencing and chromosome conformation capture (Hi‐C) techniques. Most of the high‐confidence sequences (93.1%) were anchored to the individual chromosomes with a total of 19 centromeres identified, matching the exact chromosome count of B. napus. The repeat sequences in the A and C subgenomes in B. napus expanded significantly from 500 000 years ago, especially over the last 100 000 years. These young and recently amplified LTR‐RTs showed dispersed chromosomal distribution but significantly preferentially clustered into centromeric regions. We exhaustively annotated the nucleotide‐binding leucine‐rich repeat (NLR) gene repertoire, yielding a total of 597 NLR genes in B. napus genome and 17.4% of which are paired (head‐to‐head arrangement). Based on the resequencing data of 991 B. napus accessions, we have identified 18 759 245 single nucleotide polymorphisms (SNPs) and detected a large number of genomic regions under selective sweep among the three major ecotype groups (winter, semi‐winter and spring) in B. napus. We found 49 NLR genes and five NLR gene pairs colocated in selective sweep regions with different ecotypes, suggesting a rapid diversification of NLR genes during the domestication of B. napus. The high quality of our B. napus 'ZS11' genome assembly could serve as an important resource for the study of rapeseed genomics and reveal the genetic variations associated with important agronomic traits.