Phylogenetic and evolutionary analysis of NBS-encoding genes in Rutaceae fruit crops

The nucleotide-binding site leucine-rich repeat (NBS-LRR) genes are the largest class of disease resistance genes in plants. However, our understanding of the evolution of NBS-LRR genes in Rutaceae fruit crops is rather limited. We report an evolutionary study of 103 NBS-encoding genes isolated from Poncirus trifoliata (trifoliate orange), Citrus reticulata (tangerine) and their F₁ progeny. In all, 58 of the sequences contained a continuous open reading frame. Phylogenetic analysis classified the 58 NBS genes into nine clades, eight of which were genus specific. This was taken to imply that most of the ancestors of these NBS genes evolved after the genus split. The motif pattern of the 58 NBS-encoding genes was consistent with their phylogenetic profile. An extended phylogenetic analysis, incorporating citrus NBS genes from the public database, classified 95 citrus NBS genes into six clades, half of which were genus specific. RFLP analysis showed that citrus NBS-encoding genes have been evolving rapidly, and that they are unstable when passed through an intergeneric cross. Of 32 NBS-encoding genes tracked by gene-specific PCR, 24 showed segregation distortion among a set of 94 F₁ individuals. This study provides new insight into the evolution of Rutaceae NBS genes and their behaviour following an intergeneric cross.     

Genome-Wide Analysis in Wild and Cultivated <Emphasis Type="Italic">Oryza</Emphasis> Species Reveals Abundance of NBS Genes in Progenitors of Cultivated Rice

NBS-encoding genes play a critical role in the plant defense system. Wild relatives of crop plants are rich reservoirs of plant defense genes. Here, we performed a stringent genome-wide identification of NBS-encoding genes in three cultivated and eight wild Oryza species, representing three different genomes (AA, BB, and FF) from four continents. A total of 2688 NBS-encoding genes were identified from 11 Oryza genomes. All the three progenitor species of cultivated rice, namely O. barthii, O. rufipogon, and O. nivara, were the richest reservoir of NBS-encoding genes (214, 313, and 307 respectively). Interestingly, the two Asian cultivated species showed a contrasting pattern in the number of NBS-encoding genes. While indica subspecies maintained nearly equal number of NBS genes as its progenitor (309 and 313), the japonica subspecies had retained only two third in the course of evolution (213 and 307). Other major sources for NBS-encoding genes could be (i) O. longistaminata since it had the highest proportion of NBS-encoding genes and (ii) O. glumaepatula as it clustered distinctly away from the rest of the AA genome species. The present study thus revealed that NBS-encoding genes can be exploited from the primary gene pool for disease resistance breeding in rice.     

Genome-Wide Analysis of Nucleotide-Binding Site (NBS) Disease Resistance (R) Genes in Sacred Lotus (Nelumbo nucifera Gaertn.) Reveals Their Transition Role During Early Evolution of Land Plants

Nucleotide-binding site (NBS) containing genes comprise the largest class in identified plant resistance genes. A total of 137 NBS class resistance genes were identified from the newly sequenced sacred lotus genome (Nelumbo nucifera Gaertn.) through a reiterative computational sequence analysis. Three distinct groups of NBS-encoding genes were classified: 5 with Toll/interleukin-1 receptor homology (TIR) domain at N-terminal (TIR-NBS [-LRR (leucine-rich repeat)]), 37 with CC (coiled coil) domain (CC-NBS [-LRR]), and 95 with neither TIR nor CC at N-terminal (NBS [-LRR]). Sequence analysis revealed high divergence of NBS-LRR genes in sacred lotus. The result of cluster and syntenic analysis of NBS genes suggested a duplication and recombination event, which is consistent with the correspondent result of whole genome analysis. In addition, we also identified 52 NBS genes which have a putative NACHT domain embedded in the NBS domains. This characteristic has only been reported in animals, fungi and bacteria, suggesting that NACHT and NBS domains shared a similar ancient origin; and sacred lotus NBS (NACHT) genes may represent a transition role during the early evolution of disease resistance in land plants.     

Identification and characterization of NBS-encoding disease resistance genes in Lotus japonicus

Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a range of pathogens and insect pests. Consequently, NBS-encoding genes have been the focus of a number of recent studies in molecular disease resistance breeding programs. However, little is known about NBS-encoding genes in Lotus japonicus. In this study, a full set of disease resistance (R) candidate genes encoding NBS from the complete genome of L. japonicus was identified and characterized using structural diversity, chromosomal locations, conserved protein motifs, gene duplications, and phylogenetic relationships. Distinguished by N-terminal motifs and leucine-rich repeat motifs (LRRs), 92 regular NBS genes of 158 NBS-coding sequences were classified into seven types: CC-NBS-LRR, TIR-NBS-LRR, NBS-LRR, CC-NBS, TIR-NBS, NBS, and NBS-TIR. Phylogenetic reconstruction of NBS-coding sequences revealed many NBS gene lineages, dissimilar from results for Arabidopsis but similar to results from research on rice. Conserved motif structures were also analyzed to clarify their distribution in NBS-encoding gene sequences. Moreover, analysis of the physical locations and duplications of NBS genes showed that gene duplication events of disease resistance genes were lower in L. japonicus than in rice and Arabidopsis, which may contribute to the relatively fewer NBS genes in L. japonicus. Sixty-three NBS-encoding genes with clear conserved domain character were selected to check their gene expression levels by semi-quantitative RT-PCR. The results indicated that 53 of the genes were most likely to be acting as the active genes, and exogenous application of salicylic acid improved expression of most of the R genes.     

玉米与其他六种植物NBS类抗病基因的进化比较分析

具有核苷酸结合位点(nucleotide binding site,NBS)的抗病基因在植物抵抗各种病原菌侵染中起关键作用。对玉米全基因组中具有NBS结构的基因进行鉴定和分析,并结合水稻、高粱、拟南芥、百脉根、苜蓿和杨树的NBS类基因比较其在数量、复制、染色体定位和亲缘关系上的进化差异。发现玉米NBS类基因数量、复制数和成簇基因数均明显少于其他植物。低复制频率可能导致玉米NBS类基因较少,并推测可能导致其功能具有多样性。在基因染色体定位上,除高梁外,玉米与其他五种植物相似,呈不均衡分布。此外,进化树分析表明玉米NBS类基因与高粱的亲缘关系最近,与拟南芥的最远,在物种间表现出较高的保守性。结果对掲示玉米NBS基因的进化特点与发掘有益的NBS类抗病基因提供了重要的理论依据。     

Identification of NBS-encoding genes linked to black rot resistance in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis>)

Heading cabbage is a nutritionally rich and economically important cruciferous vegetable. Black rot disease, caused by the bacterium Xanthomonas campestris pv. campestris, reduces both the yield and quality of the cabbage head. Nucleotide binding site (NBS)-encoding resistance (R) genes play a vital role in the plant immune response to various pathogens. In this study, we analyzed the expression and DNA sequence variation of 31 NBS-encoding genes in cabbage (Brassica oleracea var. capitata). These genes encoded TIR, NBS, LRR and RPW8 protein domains, all of which are known to be involved in disease resistance. RNA-seq revealed that these 31 genes were differentially expressed in leaf, root, silique, and stem tissues. Furthermore, qPCR analyses revealed that several of these genes were more highly expressed in resistant compared to susceptible cabbage lines, including Bol003711, Bol010135, Bol010559, Bol022784, Bol029866, Bol042121, Bol031422, Bol040045 and Bol042095. Further analysis of these genes promises to yield both practical benefits, such as molecular markers for marker-assisted breeding, and fundamental insights to the mechanisms of resistance to black rot in cabbage.     

Systematic analysis and comparison of nucleotide-binding site disease resistance genes in maize

Nucleotide-binding site (NBS) disease resistance genes play an integral role in defending plants from a range of pathogens and insect pests. Consequently, a number of recent studies have focused on NBS-encoding genes in molecular disease resistance breeding programmes for several important plant species. Little information, however, has been reported with an emphasis on systematic analysis and a comparison of NBS-encoding genes in maize. In the present study, 109 NBS-encoding genes were identified based on the complete genome sequence of maize (Zea mays cv. B73), classified as four different subgroups, and then characterized according to chromosomal locations, gene duplications, structural diversity and conserved protein motifs. Subsequent phylogenetic comparisons indicated that several maize NBS-encoding genes possessed high similarity to function-known NBS-encoding genes, and revealed the evolutionary relationships of NBS-encoding genes in maize comparede to those in other model plants. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication events of disease resistance genes were lower in maize than in other model plants, which may have led to an increase in the functional diversity of the maize NBS-encoding genes. Various expression patterns of maize NBS-encoding genes in different tissues were observed using an expressed-sequence tags database and, alternatively, after southern leaf blight infection or the application of exogenous salicylic acid. The results reported in the present study contribute to an improved understanding of the NBS-encoding gene family in maize.     

Genome-wide identification and mapping of NBS-encoding resistance genes in Solanum tuberosum group phureja

The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ～41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes.     

Resistance gene analogues identified through the NBS-profiling method map close to major genes and QTL for disease resistance in apple

We used a new method called nucleotide-binding site (NBS) profiling to identify and map resistance gene analogues (RGAs) in apple. This method simultaneously allows the amplification and the mapping of genetic markers anchored in the conserved NBS-encoding domain of plant disease resistance genes. Ninety-four individuals belonging to an F₁ progeny derived from a cross between the apple cultivars Discovery and TN10-8 were studied. Two degenerate primers designed from the highly conserved P-loop motif within the NBS domain were used together with adapter primers. Forty-three markers generated with NBS profiling could be mapped in this progeny. After sequencing, 23 markers were identified as RGAs, based on their homologies with known resistance genes or NBS/leucine-rich-repeat-like genes. Markers were mapped on 10 of the 17 linkage groups of the apple genetic map used. Most of these markers were organized in clusters. Twenty-five markers mapped close to major genes or quantitative trait loci for resistance to scab and mildew previously identified in different apple progenies. Several markers could become efficient tools for marker-assisted selection once converted into breeder-friendly markers. This study demonstrates the efficiency of the NBS-profiling method for generating RGA markers for resistance loci in apple.     

Chromosomal deletion in isolates of Phytophthora infestans correlates with virulence on R3, R10, and R11 potato lines.

In Phytophthora infestans, a cluster of three dominant avirulence genes is located on the distal part of linkage group VIII. In a mapping population from a cross between two Dutch field isolates, probe M5.1, derived from an amplified fragment length polymorphism (AFLP) marker linked to the Avr3-Avr10-Avr11 cluster, hybridized only to DNA from the parent and F1 progeny that is avirulent on potato lines carrying the R3, R10, and R11 resistance gene. In the virulent parent and the virulent progeny, no M5.1 homologue was detected, demonstrating a deletion on that part of linkage group VIII. P. infestans is diploid, so the avirulent strains must be hemizygous for the region concerned. A similar situation was found in another mapping population from two Mexican strains. The deletion was also found to occur in many field isolates. In a large set of unique isolates collected in The Netherlands from 1980 to 1991, 37% had no M5.1 homologue and the deletion correlated strongly with gain of virulence on potato lines carrying R3, R10, and R11. Also, in some old isolates that belong to a single clonal lineage (US-1) and are thus highly homogenous, deletions at the M5.1 locus were detected, indicating that this region is unstable.     

Genome-Wide Identification and Characterization of Nucleotide-Binding Site (NBS) Resistance Genes in Pineapple

Pineapple is a major tropical fruit and the most important crop processing CAM photosynthesis. It originated in southwest Brazil and northeast Paraguay and survived the harsh, semi-arid environment. Disease resistance genes have contributed to the survival and thriving of this species. The largest class of disease resistance (R) genes in plants consists of genes encoding nucleotide-binding site (NBS) domains. The sequenced genome of pineapple (Ananas comosus (L.) Merr.) provides a resource for analyzing the NBS-encoding genes in this species. A total of 177 NBS-encoding genes were identified using automated and manual analysis criteria, and these represent about 0.6 % of the total number of predicted pineapple genes. Five genes identified here contained the N-terminal Toll/Interleukin-l receptor (TIR) domain, and 46 genes carried the N-terminal Coiled-Coil (CC) motif. A majority of these NBS-encoding genes (84 %) contained a leucine-rich repeat (LRR) domain. A total of 130 of 177 (73 %) of these NBS-encoding genes were distributed across 20 pineapple linkage groups. The identification and characterization of NBS genes in pineapple yielded a valuable genomic resource and improved understanding of R genes in pineapple, which will facilitate the development of disease resistant pineapple cultivars.     

Systematic Analysis and Comparison of Nucleotide-Binding Site Disease Resistance Genes in a Diploid Cotton Gossypium raimondii

Plant disease resistance genes are a key component of defending plants from a range of pathogens. The majority of these resistance genes belong to the super-family that harbors a Nucleotide-binding site (NBS). A number of studies have focused on NBS-encoding genes in disease resistant breeding programs for diverse plants. However, little information has been reported with an emphasis on systematic analysis and comparison of NBS-encoding genes in cotton. To fill this gap of knowledge, in this study, we identified and investigated the NBS-encoding resistance genes in cotton using the whole genome sequence information of Gossypium raimondii. Totally, 355 NBS-encoding resistance genes were identified. Analyses of the conserved motifs and structural diversity showed that the most two distinct features for these genes are the high proportion of non-regular NBS genes and the high diversity of N-termini domains. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication of disease resistance genes could play an important role in cotton by leading to an increase in the functional diversity of the cotton NBS-encoding genes. Analyses of phylogenetic comparisons indicated that, in cotton, the NBS-encoding genes with TIR domain not only have their own evolution pattern different from those of genes without TIR domain, but also have their own species-specific pattern that differs from those of TIR genes in other plants. Analyses of the correlation between disease resistance QTL and NBS-encoding resistance genes showed that there could be more than half of the disease resistance QTL associated to the NBS-encoding genes in cotton, which agrees with previous studies establishing that more than half of plant resistance genes are NBS-encoding genes.     

Evidence of Multiple Disease Resistance (MDR) and Implication of Meta-Analysis in Marker Assisted Selection

Meta-analysis was performed for three major foliar diseases with the aim to find out the total number of QTL responsible for these diseases and depict some real QTL for molecular breeding and marker assisted selection (MAS) in maize. Furthermore, we confirmed our results with some major known disease resistance genes and most well-known gene family of nucleotide binding site (NBS) encoding genes. Our analysis revealed that disease resistance QTL were randomly distributed in maize genome, but were clustered at different regions of the chromosomes. Totally 389 QTL were observed for these three major diseases in diverse maize germplasm, out of which 63 QTL were controlling more than one disease revealing the presence of multiple disease resistance (MDR). 44 real-QTLs were observed based on 4 QTL as standard in a specific region of genome. We also confirmed the Ht1 and Ht2 genes within the region of real QTL and 14 NBS-encoding genes. On chromosome 8 two NBS genes in one QTL were observed and on chromosome 3, several cluster and maximum MDR QTL were observed indicating that the apparent clustering could be due to genes exhibiting pleiotropic effect. Significant relationship was observed between the number of disease QTL and total genes per chromosome based on the reference genome B73. Therefore, we concluded that disease resistance genes are abundant in maize genome and these results can unleash the phenomenon of MDR. Furthermore, these results could be very handy to focus on hot spot on different chromosome for fine mapping of disease resistance genes and MAS.     

The leucine-rich repeat domain can determine effective interaction between RPS2 and other host factors in arabidopsis RPS2-mediated disease resistance

Like many other plant disease resistance genes, Arabidopsis thaliana RPS2 encodes a product with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. This study explored the hypothesized interaction of RPS2 with other host factors that may be required for perception of Pseudomonas syringae pathogens that express avrRpt2 and/or for the subsequent induction of plant defense responses. Crosses between Arabidopsis ecotypes Col-0 (resistant) and Po-1 (susceptible) revealed segregation of more than one gene that controls resistance to P. syringae that express avrRpt2. Many F(2) and F(3) progeny exhibited intermediate resistance phenotypes. In addition to RPS2, at least one additional genetic interval associated with this defense response was identified and mapped using quantitative genetic methods. Further genetic and molecular genetic complementation experiments with cloned RPS2 alleles revealed that the Po-1 allele of RPS2 can function in a Col-0 genetic background, but not in a Po-1 background. The other resistance-determining genes of Po-1 can function, however, as they successfully conferred resistance in combination with the Col-0 allele of RPS2. Domain-swap experiments revealed that in RPS2, a polymorphism at six amino acids in the LRR region is responsible for this allele-specific ability to function with other host factors.     

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana

Background

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana.

Results

Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species.

Conclusion

This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-3) contains supplementary material, which is available to authorized users.     

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily

The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.     

Genome-wide identification of NBS-encoding resistance genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Nucleotide-binding site (NBS)-encoding resistance genes are key plant disease-resistance genes and are abundant in plant genomes, comprising up to 2% of all genes. The availability of genome sequences from several plant models enables the identification and cloning of NBS-encoding genes from closely related species based on a comparative genomics approach. In this study, we used the genome sequence of Brassica rapa to identify NBS-encoding genes in the Brassica genome. We identified 92 non-redundant NBS-encoding genes [30 CC-NBS-LRR (CNL) and 62 TIR-NBS-LRR (TNL) genes] in approximately 100 Mbp of B. rapa euchromatic genome sequence. Despite the fact that B. rapa has a significantly larger genome than Arabidopsis thaliana due to a recent whole genome triplication event after speciation, B. rapa contains relatively small number of NBS-encoding genes compared to A. thaliana, presumably because of deletion of redundant genes related to genome diploidization. Phylogenetic and evolutionary analyses suggest that relatively higher relaxation of selective constraints on the TNL group after the old duplication event resulted in greater accumulation of TNLs than CNLs in both Arabidopsis and Brassica genomes. Recent tandem duplication and ectopic deletion are likely to have played a role in the generation of novel Brassica lineage-specific resistance genes.