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Phylogeny and genomic organization of the TIR and non-TIR NBS-LRR resistance gene family in
Abstract
Sequences homologous to the nucleotide binding site (NBS) domain of NBS-leucine-rich repeat (LRR) resistance genes were retrieved from the model legume M. truncatula through several methods. Phylogenetic analysis classified these sequences into TIR (toll and interleukin-1 receptor) and non-TIR NBS subfamilies and further subclassified them into several well-defined clades within each subfamily. Comparison of M. truncatula NBS sequences with those from several closely related legumes, including members of the tribes Trifoleae, Viceae, and Phaseoleae, reveals that most clades contain sequences from multiple legume species. Moreover, sequences from species within the closely related Trifoleae and Viceae tribes (e.g., Medicago and Pisum spp.) tended to be cophyletic and distinct from sequences of Phaseoleae species (e.g., soybean and bean). These results suggest that the origin of major clades within the NBS-LRR family predate radiation of these Papilionoid legumes, while continued diversification of these sequences mirrors speciation within this legume subfamily. Detailed genetic and physical mapping of both TIR and non-TIR NBS sequences in M. truncatula reveals that most NBS sequences are organized into clusters, and few, if any, clusters contain both TIR and non-TIR sequences. Examples were found, however, of physical clusters that contain sequences from distinct phylogenetic clades within the TIR or non-TIR subfamilies. Comparative mapping reveals several blocks of resistance gene loci that are syntenic between M. truncatula and soybean and between M. truncatula and pea.
... A comparable case has been reported earlier by Pan et al 32 in Solanaceae, where potato and tomato sequences generated tight clusters in a phylogenetic tree while some other tomato derived sequences are more distantly related than potato homologs. In another phylogenetic analysis, Zhu et al 33 have shown that sequences of two species of Medicago, M. sativa and M. truncatula, are placed in close proximity. It was presumed that due to close phylogenetic relationship of resistance gene analogs of these two species, the functional R gene of M. truncatula against Erysiphe pisi (powdery mildew pathogen) could be transferred across species boundaries by transgenic approach 33 . ...
... In another phylogenetic analysis, Zhu et al 33 have shown that sequences of two species of Medicago, M. sativa and M. truncatula, are placed in close proximity. It was presumed that due to close phylogenetic relationship of resistance gene analogs of these two species, the functional R gene of M. truncatula against Erysiphe pisi (powdery mildew pathogen) could be transferred across species boundaries by transgenic approach 33 . If this proposition holds true, then the resistance factor against YMV, present in YMV-resistant V. mungo, could also be transferred to YMV-susceptible cultivars of G. max, V. unguiculata and V. radiata, due to their proximity in the phylogenetic tree (vide TA1 sub-clade of T1 clade, Fig. 1). ...
... It has been contemplated that single genotypes of plant pathogens are able to infect closely related legume species indicating taxonomically restricted R gene diversity 33 . In the present study, we have found that R gene sequences of the genera under the tribe Phaseoleae are grouped into TA1 and NTA1, while, those of tribe Trifolieae represented in TB1, TB2 and NTB1. ...
In higher plants, disease resistance (R) genes are present in abundance and majority of these encode proteins with a nucleotide-binding site (NBS). N-terminal end of the NBS is either endowed with TIR or by Non-TIR sequences. We have cloned and sequenced one yellow mosaic virus-resistance linked R gene homolog (RGH) from Vigna mungo, line VM-1, GenBank accession number AY297425. Later, two other RGHs from YMV resistant lines, V. mungo WBU 108 and V. radiata Pusa 9072 were selectively amplified using R gene targeted degenerate primers and were cloned subsequently (AY301991 and trIQ7XZT9, respectively). Characterization of these three RGHs and analysis of a total of 221 R genes and RGHs lead to the question of the evolution and distribution of the R genes/RGHs in the family Fabaceae, to which the primary hosts of the YMV belong. These 14 species mainly comprised grain legumes and the R gene phylogeny were reconstructed. The phylogenetic analyses indicate that two-third of the sequences are of the TIR-NBS type, while about one third represent the Non-TIR subfamily. Simultaneous presence of the TIR and the Non-TIR domains within the Fabaceae indicates divergent evolution and heterogeneity within the NBS domain. The plausible mechanism of continued diversification of the NBS sequences within the family Fabaceae has been substantiated by the supportive published evidences. It is presumed that such cases of divergent architectures of NB-domains of R genes within a single species or in the cultivars of the species has been influenced considerably through human interference during domestication, as against, evolution and random selection in nature. This finding reflects that the successful introgression of the functional R gene could be possible to the disease susceptible cultivars within the tribes Phaseoleae and Trifoleae.
... Over the past decade researchers have developed the tools and infrastructure for basic research in M. truncatula, including efficient transformation systems (Chabaud et al., 2003;Zhou et al., 2004), collections of induced variation (Penmetsa and Cook, 2000), well-characterized cytogenetics (Kulikova et al., 2001), and a collaborative research network (http://www.medicago.org). Research efforts on M. truncatula encompass a broad range of issues in plant biology, from studies of population biology (Bonnin et al., 1996b;Bonnin et al., 1996a) and resistance gene evolution (Cannon et al., 2002;Zhu et al., 2002) to the molecular biology of symbiotic interactions (e.g., (Endre et al., 2002a;Limpens et al., 2003;Ane et al., 2004;Levy et al., 2004), plant natural products (Dixon and Sumner, 2003), and micronutrient homeostasis (Ellis et al., 2003). Of importance to these hypothesis-driven investigations is the parallel development of tools for genome be translated efficiently and potentially to great advantage for gene discovery in related species. ...
... Over the past decade researchers have developed the tools and infrastructure for basic research in M. truncatula, including efficient transformation systems (Chabaud et al., 2003;Zhou et al., 2004), collections of induced variation (Penmetsa and Cook, 2000), well-characterized cytogenetics (Kulikova et al., 2001), and a collaborative research network (http://www.medicago.org). Research efforts on M. truncatula encompass a broad range of issues in plant biology, from studies of population biology (Bonnin et al., 1996b;Bonnin et al., 1996a) and resistance gene evolution (Cannon et al., 2002;Zhu et al., 2002) to the molecular biology of symbiotic interactions (e.g., (Endre et al., 2002a;Limpens et al., 2003;Ane et al., 2004;Levy et al., 2004), plant natural products (Dixon and Sumner, 2003), and micronutrient homeostasis (Ellis et al., 2003). Of importance to these hypothesis-driven investigations is the parallel development of tools for genome be translated efficiently and potentially to great advantage for gene discovery in related species. ...
... A combination of genetic and physical mapping was used to assign the genetic position of a minimum of 150 distinct NBS-LRR homologs in M. truncatula. In many cases, the mapped RGHs are also organized into clusters, and, few, if any, of these clusters contain both TIR-and CC-NBS-LRR sequences (Zhu et al., 2002). Phylogenetic analysis of these R-like protein sequences indicates a high level of diversity in M. truncatula. ...
Anthracnose, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. Cloning and characterization of the host resistance (R) genes against the pathogen will improve our knowledge of molecular mechanisms underlying host resistance and facilitate the development of resistant alfalfa cultivars. However, the intractable genetic system of cultivated alfalfa, owing to its tetrasomic inheritance and outcrossing nature, limits the ability to carry out genetic analysis in alfalfa. Nonetheless, the model legume Medicago truncatula, a close relative of alfalfa, provides a surrogate for cloning the counterparts of many agronomically important genes in alfalfa. In this study, we used genetic map-based approach to clone RCT1, a host resistance gene against C. trifolii race 1, in M. truncatula. The RCT1 locus was delimited within a physical interval spanning ~200 kilo-bases located on the top of M. truncatula linkage group 4. Complementation tests of three candidate genes on the susceptible alfalfa clones revealed that RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad spectrum anthracnose resistance. Thus, RCT1 offers a novel resource to develop anthracnose-resistant alfalfa cultivars. Furthermore, the cloning of RCT1 also makes a significant contribution to our understanding of host resistance against the fungal genus Colletotrichum.
... Modern biotechnology is based on scientific advances that make it possible to isolate and clone specific pieces of DNA-containing genes, and to sequence the nucleotides in a DNA molecule (the genetic code) so that the precise location and structure of genes can be studied at the molecular level (Goodman and Kiser, 1985). Numerous disease-resistance (R) gene homologues have been identified in legume species by sequence identity of conserved motifs with known R genes (Kanazin et al., 1996;Yu et al., 1996;Zhu et al., 2002). The challenge is to identify the specific genes conferring resistance to a particular pathogen. ...
... Legumes are a rich source of flavonoids, notably isoflavones and isoflavanones, which are not found in Arabidopsis (Arabidopsis thaliana). Legume nodules are also rich sources of cysteine cluster proteins, some of which have been shown to have antimicrobial activity and may play a role in protecting nodules from pathogens (Yu et al., 1996;Zhu et al., 2002). SNF and atmospherically fixed nitrogen are important alternative sources of usable nitrogen for legume (and other) crops for improved yields, especially on degraded soils and under low external input cropping systems (Vincent, 1982;FAO, 1983;Buttery et al., 1992). ...
The book is divided into 6 sections (20 chapters) which deal with history and importance, botany, genetics and plant physiology, production, plant protection, utilization and marketing and trade of soyabeans.
... Within the angiosperms, the CC-receptor domain is present in all clades; thus, no phylogenetic signal is detectable for the presence of that domain. In contrast, TIR-receptor domains have been lost from the cereal clade Poales (138,193). Thus, although it is also of ancient origin, unlike the CC domain, TIR shows a strong phylogenetic signal across the phylogeny of the angiosperms. ...
... amino-terminal domain of NBS-LRR proteins domains have been lost from the cereal clade Poales (138,193), so TIR shows a strong phylogenetic signal when measured across the phylogeny of all angiosperms (as in Figure 2, clade A) but not if grasses are excluded from the study (as in Figure 2, clade B). ...
An explicit phylogenetic perspective provides useful tools for phytopathology and plant disease ecology because the traits of both plants and microbes are shaped by their evolutionary histories. We present brief primers on phylogenetic signal and the analytical tools of phylogenetic ecology. We review the literature and find abundant evidence of phylogenetic signal in pathogens and plants for most traits involved in disease interactions. Plant nonhost resistance mechanisms and pathogen housekeeping functions are conserved at deeper phylogenetic levels, whereas molecular traits associated with rapid coevolutionary dynamics are more labile at branch tips. Horizontal gene transfer disrupts the phylogenetic signal for some microbial traits. Emergent traits, such as host range and disease severity, show clear phylogenetic signals. Therefore pathogen spread and disease impact are influenced by the phylogenetic structure of host assemblages. Phylogenetically rare species escape disease pressure. Phylogenetic tools could be used to develop predictive tools for phytosanitary risk analysis and reduce disease pressure in multispecies cropping systems. Expected final online publication date for the Annual Review of Phytopathology Volume 54 is August 04, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... Modern biotechnology is based on scientific advances that make it possible to isolate and clone specific pieces of DNA-containing genes, and to sequence the nucleotides in a DNA molecule (the genetic code) so that the precise location and structure of genes can be studied at the molecular level (Goodman and Kiser, 1985). Numerous disease-resistance (R) gene homologues have been identified in legume species by sequence identity of conserved motifs with known R genes (Kanazin et al., 1996;Yu et al., 1996;Zhu et al., 2002). The challenge is to identify the specific genes conferring resistance to a particular pathogen. ...
... Legumes are a rich source of flavonoids, notably isoflavones and isoflavanones, which are not found in Arabidopsis (Arabidopsis thaliana). Legume nodules are also rich sources of cysteine cluster proteins, some of which have been shown to have antimicrobial activity and may play a role in protecting nodules from pathogens (Yu et al., 1996;Zhu et al., 2002). SNF and atmospherically fixed nitrogen are important alternative sources of usable nitrogen for legume (and other) crops for improved yields, especially on degraded soils and under low external input cropping systems (Vincent, 1982;FAO, 1983;Buttery et al., 1992). ...
... Modern biotechnology is based on scientific advances that make it possible to isolate and clone specific pieces of DNA-containing genes, and to sequence the nucleotides in a DNA molecule (the genetic code) so that the precise location and structure of genes can be studied at the molecular level (Goodman and Kiser, 1985). Numerous disease-resistance (R) gene homologues have been identified in legume species by sequence identity of conserved motifs with known R genes (Kanazin et al., 1996;Yu et al., 1996;Zhu et al., 2002). The challenge is to identify the specific genes conferring resistance to a particular pathogen. ...
... Legumes are a rich source of flavonoids, notably isoflavones and isoflavanones, which are not found in Arabidopsis (Arabidopsis thaliana). Legume nodules are also rich sources of cysteine cluster proteins, some of which have been shown to have antimicrobial activity and may play a role in protecting nodules from pathogens (Yu et al., 1996;Zhu et al., 2002). SNF and atmospherically fixed nitrogen are important alternative sources of usable nitrogen for legume (and other) crops for improved yields, especially on degraded soils and under low external input cropping systems (Vincent, 1982;FAO, 1983;Buttery et al., 1992). ...
... Sur la base de leurs séquences N-terminale, les protéines NB-LRR sont subdivisées en deux classes. La première catégorie (TNL) est constituée par les TIR-NB-LRR, en raison des homologies trouvées avec le domaine protéique TIR (Toll Interleukine-Receptor) chez la drosophile (Whitham et al. 1994 (Zhu et al. 2002, Ameline-Torregrosa et al. 2008b. Cette homogénéité des clusters d'RGAs a été aussi notée chez le soja (Kanazin et al. 1996;Yu et al. 1996). ...
... ).Les programmes de séquençage des différentes plantes couplés aux analyses bioinformatiques ont permis de révéler que les gènes R les plus représentés se regroupaient au sein de certaines régions des génomes analysés. Ainsi, le séquençage de Medicago truncatula a permis de mettre en évidence l'existence de plusieurs gènes R ayant une homologie avec les NB-LRRs(Zhu et al. 2002). La cartographie génétique et physique a montré que les TNL et les CNL sont groupés en clusters chez cette légumineuse, et que peu de clusters contiennent ces deux types de gènes NB-LRR réunis ensembles ...
... The presence of these conserved domains has enabled rapid isolation of resistance gene candidate (RGC) from different plant species by using a polymerase chain reaction (PCR) with degenerate oligonucleotide primers designed from these domains. RGCs were isolated from several plant species, such as cotton (He et al., 2004), potato (Leister et al., 1996), soybean (Yu et al., 1996), lettuce (Shen et al., 1998), tomato (Pan et al., 2000), rice (Mago et al., 1999), barley (Leister et al., 1998), wheat (Seah et al., 2000), chickpea (Huettel et al., 2002), Medicago truncatula (Zhu et al., 2002) and Sunflower (Radwan et al., 2003Radwan et al., , 2004Radwan et al., , 2008). Peanut or groundnut (Arachis hypogaea L.) is the fourth most important oil seed crop in the world, cultivated mainly in tropical, subtropical and warm temperate climates (FAO, 2004). ...
... This classification is supported by high bootstrap resembling (Fig. 2). The number of subfamilies is similar to that reported in other species such as soybean (Kanazin et al., 1996; Yu et al., 1996), apple (Lee et al., 2003), Medicago truncatula (Zhu et al., 2002), Arabidopsis (Meyers et al., 2003), cotton (He et al., 2004) and sunflower (Radwan et al., 2008 ). The number of subfamilies, which identified in this work, is greater than these identified by Yuksel et al. (2005) and equal the subfamily numbers identified by Bertioli et al. (2003). ...
The nucleotide-binding-site-leucine-rich-repeat (NBS-LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. Here, we describe a collection of peanut NBS-LRR resistance gene candidates (RGCs) isolated from peanut (Arachis) species by mining Gene Bank data base. NBS-LRR sequences assembled into TIR-NBS-LRR (75.4%) and non-TIR-NBS-LRR (24.6%) subfamilies. Total of 20 distinct clades were identified and showed a high level of sequence divergence within TIR-NBS and non-TIR-NBS subfamilies. Thirty-four primer pairs were designed from these RGC sequences and used for screening different genotypes belonging to wild and cultivated peanuts. Therefore, peanut RGC identified in this study will provide useful tools for developing DNA markers and cloning the genes for resistance to different pathogens in peanut.
... Mt3/), the very low number of in silico matches between Mt-6 and multiple T. repens sequence sources is not a surprise for several reasons. Mt-6 is atypical of the other Mt chromosomes as it contains an over-representation of resistance gene analogues and leucine rich repeats [65], the greatest proportion of heterochromatin [66], and a corresponding under-representation of randomly selected and mapped EST markers [65,67]. Furthermore, comparative alignment with other legumes reveals Mt-6 to have reduced marker-based synteny [67]. ...
... Mt3/), the very low number of in silico matches between Mt-6 and multiple T. repens sequence sources is not a surprise for several reasons. Mt-6 is atypical of the other Mt chromosomes as it contains an over-representation of resistance gene analogues and leucine rich repeats [65], the greatest proportion of heterochromatin [66], and a corresponding under-representation of randomly selected and mapped EST markers [65,67]. Furthermore, comparative alignment with other legumes reveals Mt-6 to have reduced marker-based synteny [67]. ...
Background
White clover (Trifolium repens L.) is a temperate forage legume with an allotetraploid genome (2n=4×=32) estimated at 1093 Mb. Several linkage maps of various sizes, marker sources and completeness are available, however, no integrated map and marker set has explored consistency of linkage analysis among unrelated mapping populations. Such integrative analysis requires tools for homoeologue matching among populations. Development of these tools provides for a consistent framework map of the white clover genome, and facilitates in silico alignment with the model forage legume, Medicago truncatula.
Results
This is the first report of integration of independent linkage maps in white clover, and adds to the literature on methyl filtered GeneThresher®-derived microsatellite (simple sequence repeat; SSR) markers for linkage mapping. Gene-targeted SSR markers were discovered in a GeneThresher® (TrGT) methyl-filtered database of 364,539 sequences, which yielded 15,647 SSR arrays. Primers were designed for 4,038 arrays and of these, 465 TrGT-SSR markers were used for parental consensus genetic linkage analysis in an F1 mapping population (MP2). This was merged with an EST-SSR consensus genetic map of an independent population (MP1), using markers to match homoeologues and develop a multi-population integrated map of the white clover genome. This integrated map (IM) includes 1109 loci based on 804 SSRs over 1274 cM, covering 97% of the genome at a moderate density of one locus per 1.2 cM. Eighteen candidate genes and one morphological marker were also placed on the IM. Despite being derived from disparate populations and marker sources, the component maps and the derived IM had consistent representations of the white clover genome for marker order and genetic length. In silico analysis at an E-value threshold of 1e-20 revealed substantial co-linearity with the Medicago truncatula genome, and indicates a translocation between T. repens groups 2 and 6 relative to M. truncatula.
Conclusions
This integrated genetic linkage analysis provides a consistent and comprehensive linkage analysis of the white clover genome, with alignment to a model forage legume. Associated marker locus information, particularly the homoeologue-specific markers, offers a new resource for forage legume research to enable genetic analysis and improvement of this forage and grassland species.
... In the same region of chromosome 3, a supercluster of nucleotide binding site leucine-rich repeat (NBS-LRR) genes was identified based on the Mt1.0 assembly genome, encoding ≈40% of all coiled-coil NBS-LRRs (2). Re- sistance gene analog (RGA) markers were developed especially for NBS-LRR genes on chromosome 3 (46). In our study, we could additionally map one of these RGA markers, namely R-24D15R, between markers E12M47.457 and E12M49.246 of the linkage group built around AER1, at a genetic distance of 8.6 cM from AER1 (Fig. 3). ...
... In our study, we could additionally map one of these RGA markers, namely R-24D15R, between markers E12M47.457 and E12M49.246 of the linkage group built around AER1, at a genetic distance of 8.6 cM from AER1 (Fig. 3). The RGA cluster identified on LG3 contained sequences from different phylogenetic groups of RGAs that were cophyletic with RGAs present in at least one other cluster identified on the M. truncatula genome (46). Such heterogeneous RGA clusters suggest occurrence of genomic evolution events in the region, such as sequence divergence due to diversifying selection after duplication, selective gene loss, or ectopic recombination among physically distinct clusters. ...
... The phylogenetic analysis of NBS sequences revealed no species specific clade, which is consistent with findings in many other plant NBS studies (e.g., Zhu et al. 2002; Yaish et al. 2004; Xu et al. 2007; Samuelian et al. 2008), suggesting functional conservation of the plant/pathogen-interacting components and a signaling cascade of NBS sequences across plant families (Xiao et al. 2003; Takken et al. 2006; Tiffin and Moeller 2006). It is likely that single genotypes of plant pathogens are able to infect different Rhododendron species, indicating guard functions of different R genes are conserved within a given plant family. ...
... The majority of isolated NBS sequences were the CNLtype in Rhododendron. The preferential amplification of CNL-type NBS sequences is probably related to their being the major NBS-LRR genes against pathogens and in general agrees with findings of other studies (e.g., Zhu et al. 2002; Yaish et al. 2004; Xu et al. 2007). Our NBS amino acid sequences in different clades (clades A+B, clades C+D, clade E, and clade F;Fig. ...
To further understand natural variation and local adaptation in the evolution of plant defense, we analyzed polymorphism data of nucleotide-binding site (NBS) sequences of Rhododendron at both the species and population levels. Multiple duplication events were found in NBS sequence evolution in Rhododendron genomes, which resulted in six clades: A–F. Our results of several NBS clade pair comparisons showed significant evolutionary rate changes based on differences in substitution rates between NBS-encoding protein clades (type I functional divergence). Pairwise comparisons of NBS clades further revealed that many amino acids displayed radical biochemical property changes causing a shift in amino acid preferences between NBS-encoding protein clades (type II functional divergence). Such divergent evolution of NBSs is likely a consequence of positive selection related to differentiation of recognition signals in response to different pathogens. Primers specific to clades B and C, which differed in the number of radical amino acid changes causing type II functional divergence and levels of nucleotide diversities, were further used to amplify population clades B and C NBS sequences of Rhododendron formosanum populations. Higher levels of net nucleotide divergences (measured by D
a) between R. formosanum populations were found based on NBS sequences of population clade B compared to population clade C, suggesting local adaptation of population clade B NBS sequences. Local adaptation can be further inferred for R. formosanum population clade B NBS sequences because of significant Φ
ST based on variation in nonsynonymous substitutions. Furthermore, local adaptation was also suggested by no significant correlation of population pairwise F
ST between population clades B and C in R. formosanum.
... Of the pea NBS LRR genes discussed in [15], only one, AF123702, fell within an IBD region, on linkage group VI close to Lst. There were 225 positions on the "Caméor" v1a assembly that corresponded to NBS LRR gene sequences, and an additional 22 mapped to scaffolds (Table S1). ...
Biparental recombinant inbred line (RIL) populations are sets of genetically stable lines and have a simple population structure that facilitates the dissection of the genetics of interesting traits. On the other hand, populations derived from multiparent intercrosses combine both greater diversity and higher numbers of recombination events than RILs. Here, we describe a simple population structure: a three-way recombinant inbred population combination. This structure was easy to produce and was a compromise between biparental and multiparent populations. We show that this structure had advantages when analyzing cultivar crosses, and could achieve a mapping resolution of a few genes.
... The sequence similarity study using known R genes with the conserved motifs resulted in the identification of many R genes in legumes. (Zhu et al. 2002;Yu et al. 1996) but the biggest constraint is mapping of disease resistance gene to pathogen. For, example the Rpg1-b gene in soybean confers resistance to pathogen Pseudomonas syringae pv glycinea which carries virulent gene (avrB gene) and causes blight disease (Ashfield et al. 2004). ...
Legumes are the major sources of protein, dietary fibre, high-quality food & feed. In the world crop production, legumes occupy third place only after cereals and oilseeds. Leguminous crops are known to fix the atmospheric nitrogen for their growth through bacterial association and can be grown in low fertile soils. The requirement of synthetic nitrogenous fertilizers for leguminous crops is far lesser than the other crops, there by reduces the emission of greenhouse gases. Because of these qualities, legumes are considered as potential crops for sustainable agriculture. Due to continuous change in climatic conditions and modern agriculture systems, leguminous crops are frequently exposed to biotic and abiotic stresses. These stresses are considered as major constraints which mainly affects the physiology, metabolism and finally the cultivation and yield of leguminous crops. The yield losses in leguminous crops were accounted for 30–100% based on the magnitude and severity of biological and physical stresses. Hence, it’s required to improve productivities of leguminous plants to harness the potential nutritional values. Developing stress tolerant varieties is one of the strategies to overcome this. So far, traditional breeding approaches have been exploited to develop stress tolerant varieties, however, these are laborious and time consuming. The recent establishments of biotechnological tools in model leguminous crops such as Medicago truncatula and Lotus japonicas, have helped in understanding the regulation and mechanism of action of stress related genes. In the current book chapter, we have discussed about the major biotic and abiotic stresses and the application of various biotechnological tools in developing stress tolerant leguminous plants for improved productivity.
... Upon pathogen attack, resistance proteins coded by R genes are often involved in direct or indirect activation of the plant effectortriggered immune (ETI) system. Several R genes have been identified in different plant species including Arabidopsis, Wheat, Maize, Medicago and Sorghum (Collins et al., 1999;Seah et al., 1998;Meyers et al., 2003;Zhu et al., 2002;Totad et al., 2005;Yang and Wang, 2016;Xu et al., 2018). However, there is no genome-wide study of R genes in any plant species that may provide a complete list of all resistance genes with comprehensive classification (Gururani et al., 2012;. ...
Resistance (R) genes are directly or indirectly involved in the activation of the plant immune systems. Despite their essential roles in defense against a variety of pathogens, very little is known about their classification as well as associated decoy domains, particularly in crop plants. Using advanced bioinformatics strategies, this study provided a complete classification of all R genes in three cotton species, G. arboreum (Ga), G. raimondii (Gr) and G. hirsutum (Gh)). A total of 3085, 3024 and 5355 R genes were identified in Ga, Gr and Gh, respectively and these were classified into fifteen major classes based on R domains. Moreover, we identified several decoy domains associated with the R domains mainly comprising DUF, Lectin_legB, WRKY and Calmodulin_binding domains. Interestingly, most of the identified decoy domains belong to stress-responsive protein families. Furthermore, the comparison among the cotton genomes suggested the gain and loss of R gene classes displaying the evolutionary divergence of these three genomes. Finally, promoter analysis also predicted different cis-acting sites in different NBS-containing R gene classes. Overall this study provides in-depth genome-wide insights into R genes and their associated decoy domains in cotton, and potentially also appears as a reference study on R genes for other agronomically important crops.
... No mixed clusters of TNLs and CNLs were found in the B. napus pangenome. The separate clustering of TIR and non-TIR-NBS-LRR sequences may be contributed to the ancient divergence of these two subfamilies, for example, by restricting locally acting mechanisms for sequence homogenization such as unequal crossing over (Zhu et al., 2002). The current results revealed that the NBS-LRR and TM-LRR classes are abundant and widely distributed throughout the genome and NBS-LRR variable genes were more likely to be found in clusters. ...
Methods based on single nucleotide polymorphism (SNP), copy number variation (CNV) and presence/absence variation (PAV) discovery provide a valuable resource to study gene structure and evolution. However, as a result of these structural variations, a single reference genome is unable to cover the entire gene content of a species. Therefore pangenomics analysis is needed to ensure that the genomic diversity within a species is fully represented. Brassica napus is one of the most important oilseed crops in the world and exhibits variability in its resistance genes across different cultivars. Here, we characterised resistance gene distribution across 50 B. napus lines. We identified a total of 1,749 resistance gene analogs (RGAs), of which 996 are core and 753 are variable, 368 of which are not present in the reference genome (cv. Darmor‐bzh) . In addition, a total of 15,318 SNPs were predicted within 1,030 of the RGAs. The results showed that core R‐genes harbour more SNPs than variable genes. More nucleotide binding site leucine‐rich repeat (NBS‐LRR) genes were located in clusters than as singletons, with variable genes more likely to be found in clusters. We identified 106 RGA candidates linked to blackleg resistance quantitative trait locus (QTL). This study provides a better understanding of resistance genes to target for genomics‐based improvement and improved disease resistance.
... The TNL and non-TNL genes located separately in phylogenetic tree constructed by the NBS domains in the Fragaria genomes (Additional file 3: Figure S1), legume family and other plants [3,14,32,42]. In addition, TNL and non-TNL genes differ in terms of the topologies of phylogenetic analysis, especially the distinct branch lengths between the two type genes. ...
Background:
Plant disease resistance (R) genes are evolving rapidly and play a critical role in the innate immune system of plants. The nucleotide binding sites-leucine rich repeat (NBS-LRR) genes are one of the largest classes in plant R genes. Previous studies have focused on the NBS-LRR genes from one or several species of different genera, and the sequenced genomes of the genus Fragaria offer the opportunity to study the evolutionary processes of these R genes among the closely related species.
Results:
In this study, 325, 155, 190, 187, and 133 NBS-LRRs were discovered from F. x ananassa, F. iinumae, F. nipponica, F. nubicola, and F. orientalis, respectively. Together with the 144 NBS-LRR genes from F. vesca, a total of 1134 NBS-LRRs containing 866 multi-genes comprised 184 gene families across the six Fragaria genomes. Extremely short branch lengths and shallow nodes were widely present in the phylogenetic tree constructed with all of the NBS-LRR genes of the six strawberry species. The identities of the orthologous genes were highly significantly greater than those of the paralogous genes, while the Ks ratios of the former were very significantly lower than those of the latter in all of the NBS-LRR gene families. In addition, the Ks and Ka/Ks values of the TIR-NBS-LRR genes (TNLs) were significantly greater than those of the non-TIR-NBS-LRR genes (non-TNLs). Furthermore, the expression patterns of the NBS-LRR genes revealed that the same gene expressed differently under different genetic backgrounds in response to pathogens.
Conclusions:
These results, combined with the shared hotspot regions of the duplicated NBS-LRRs on the chromosomes, indicated that the lineage-specific duplication of the NBS-LRR genes occurred before the divergence of the six Fragaria species. The Ks and Ka/Ks ratios suggested that the TNLs are more rapidly evolving and driven by stronger diversifying selective pressures than the non-TNLs.
... The other family, where the TIR is absent or in its place a coiledcoil (CC) N-terminal domain involved in protein-protein interactions and signaling present, is known as non-TIR-NBS-LRR (non-TNL) or sometimes as CC-NBS-LRR (CNL) [7,8]. Moreover, the sequences of conserved motifs, especially those within the NBS domain, have been used extensively to identify novel disease resistance genes in the model and crop plants [9][10][11]. ...
The nucleotide binding site-leucine rich repeat (NBS-LRR) proteins play an important role in the defense mechanisms against pathogens. Using bioinformatics approach, we identified and annotated 104 NBS-LRR genes in chickpea. Phylogenetic analysis points to their diversification into two families namely TIR-NBS-LRR and non-TIR-NBS-LRR. Gene architecture revealed intron gain/loss events in this resistance gene family during their independent evolution into two families. Comparative genomics analysis elucidated its evolutionary relationship with other fabaceae species. Around 50% NBS-LRRs reside in macro-syntenic blocks underlining positional conservation along with sequence conservation of NBS-LRR genes in chickpea. Transcriptome sequencing data provided evidence for their transcription and tissue-specific expression. Four cis-regulatory elements namely WBOX, DRE, CBF, and GCC boxes, that commonly occur in resistance genes, were present in the promoter regions of these genes. Further, the findings will provide a strong background to use candidate disease resistance NBS-encoding genes and identify their specific roles in chickpea.
... This result is consistent with previous observation that MtChr-6 is relatively rich in heterochromatic DNA regions and lacks in transcribed genes (Choi et al. 2004a, b;Kulikova et al. 2001). Instead, it was found that MtChr-6 was enriched largely with resistance gene analogs (Young et al. 2011;Zhu et al. 2002). Among others, two legumes belonging to the same genus, M. truncatula and M. sativa, showed the most extensive synteny to each other, which would be easily predictable. ...
The Fabaceae (legume family) is the third largest and the second of agricultural importance among flowering plant groups. In this study, we report the reconstruction of a composite comparative map composed of ten legume genomes, including seven species from the galegoid clade (Medicago truncatula, Medicago sativa, Lens culinaris, Pisum sativum, Lotus japonicus, Cicer arietinum, Vicia faba) and three species from the phaseoloid clade (Vigna radiata, Phaseolus vulgaris, Glycine max). To accomplish this comparison, a total of 209 cross-species gene-derived markers were employed. The comparative analysis resulted in a single extensive genetic/genomic network composed of 93 chromosomes or linkage groups, from which 110 synteny blocks and other evolutionary events (e.g., 13 inversions) were identified. This comparative map also allowed us to deduce several large scale evolutionary events, such as chromosome fusion/fission, with which might explain differences in chromosome numbers among compared species or between the two clades. As a result, useful properties of cross-species genic markers were re-verified as an efficient tool for cross-species translation of genomic information, and similar approaches, combined with a high throughput bioinformatic marker design program, should be effective for applying the knowledge of trait-associated genes to other important crop species for breeding purposes. Here, we provide a basic comparative framework for the ten legume species, and expect to be usefully applied towards the crop improvement in legume breeding.
Electronic supplementary material
The online version of this article (doi:10.1007/s13258-016-0481-8) contains supplementary material, which is available to authorized users.
... Zinc fingers proteins are members of a super family involved in resistance and regulatory mechanisms for various biotic stresses [71,72]. The presence of zinc finger DNA binding domains in nucleotide binding site-leucine rich repeats (NBS-LRR) determines the regulatory function of this protein in stress conditions [73]. Most plant disease resistance R proteins contain a series of NBS-LRRs. ...
The common bean (Phaseolus vulgaris L.) is the world's most important legume for human consumption. Anthracnose (ANT; Colletotrichum lindemuthianum) and angular leaf spot (ALS; Pseudocercospora griseola) are complex diseases that cause major yield losses in common bean. Depending on the cultivar and environmental conditions, anthracnose and angular leaf spot infections can reduce crop yield drastically. This study aimed to estimate linkage disequilibrium levels and identify quantitative resistance loci (QRL) controlling resistance to both ANT and ALS diseases of 180 accessions of common bean using genome-wide association analysis. A randomized complete block design with four replicates was performed for the ANT and ALS experiments, with four plants per genotype in each replicate. Association mapping analyses were performed for ANT and ALS using a mixed linear model approach implemented in TASSEL. A total of 17 and 11 significant statistically associations involving SSRs were detected for ANT and ALS resistance loci, respectively. Using SNPs, 21 and 17 significant statistically associations were obtained for ANT and angular ALS, respectively, providing more associations with this marker. The SSR-IAC167 and PvM95 markers, both located on chromosome Pv03, and the SNP scaffold00021_89379, were associated with both diseases. The other markers were distributed across the entire common bean genome, with chromosomes Pv03 and Pv08 showing the greatest number of loci associated with ANT resistance. The chromosome Pv04 was the most saturated one, with six markers associated with ALS resistance. The telomeric region of this chromosome showed four markers located between approximately 2.5 Mb and 4.4 Mb. Our results demonstrate the great potential of genome-wide association studies to identify QRLs related to ANT and ALS in common bean. The results indicate a quantitative and complex inheritance pattern for both diseases in common bean. Our findings will contribute to more effective screening of elite germplasm to find resistance alleles for marker-assisted selection in breeding programs.
... The highly conserved nature of the major motifs of the NBS domain has allowed the use of a degenerate oligonucleotide-primed polymerase chain reaction (PCR) strategy to identify homologous sequences called resistance gene analogues (RGAs) (Kanazin et al., 1996) or resistance gene candidates (Shen et al., 1998). Using this approach, several RGAs have been isolated from various species, including wheat (Zhang et al., 2011;Bouktila et al., 2014), A. thaliana (Meyers et al., 2003), faba bean and chickpea (Palomino et al., 2006), soybean (Graham et al., 2000), pea (Timmerman-Vaughan et al., 2000), and Medicago truncatula (Zhu et al., 2002). The identified RGAs correspond to different disease specificities, and, in some cases, their mapping has provided evidence that they co-segregate with resistance markers related to disease (Meyers et al., 1999). ...
Pea (Pisum sativum) is one of the most cultivated le-gumes in the world, and its yield and seed quality are affected by a variety of pathogens. In plants, NBS-LRR (nucleotide binding site-leucine-rich repeat) is the main class of disease resistance genes. Using degenerate primers deduced from conserved motifs in the NBS domain of known resistance genes, we identified 10 NBS sequences in three varieties of P. sativum. The deduced amino acid sequences of the iden-tified resistance gene analogues (RGAs) exhibited the typical motifs of the NBS domain (P-loop, kinase-2, kinase-3a, and the hydrophobic domain, GLPL) present in the majority of plant proteins belonging to the NBS-LRR class. Phylogenetic analysis showed that seven RGAs belonged to the non-TIR-NBS-LRR subclass and three to the TIR-NBS-LRR subclass. The results of this study provide insights into the structure of this class of resistance genes in the pea, and their evolution-ary relationships with those of other plant species.
... As in other plants, the RGA genes in the G. raimondii genome reside in clusters ( Fig. 3; Additional file 2: Figure S3; Additional file 1: Table S6). Previous studies have shown that the clustering of RGA genes is usually caused by tandem duplications [7,[62][63][64] or sequence exchanges [9], which have been detected in many RGA gene clusters [17,19,26,[65][66][67]. Similar results were found in the G. raimondii genome, where most of the RGA Figure S2; Additional file 2: Figure S4; Additional file 1: Table S4), indicating that tandem duplication or sequence exchanges could have occurred frequently in the evolution of RGA genes or Rgrcs. ...
Gossypium raimondii is a Verticillium wilt-resistant cotton species whose genome encodes numerous disease resistance genes that play important roles in the defence against pathogens. However, the characteristics of resistance gene analogues (RGAs) and Verticillium dahliae response loci (VdRLs) have not been investigated on a global scale. In this study, the characteristics of RGA genes were systematically analysed using bioinformatics-driven methods. Moreover, the potential VdRLs involved in the defence response to Verticillium wilt were identified by RNA-seq and correlations with known resistance QTLs.
The G. raimondii genome encodes 1004 RGA genes, and most of these genes cluster in homology groups based on high levels of similarity. Interestingly, nearly half of the RGA genes occurred in 26 RGA-gene-rich clusters (Rgrcs). The homology analysis showed that sequence exchanges and tandem duplications frequently occurred within Rgrcs, and segmental duplications took place among the different Rgrcs. An RNA-seq analysis showed that the RGA genes play roles in cotton defence responses, forming 26 VdRLs inside in the Rgrcs after being inoculated with V. dahliae. A correlation analysis found that 12 VdRLs were adjacent to the known Verticillium wilt resistance QTLs, and that 5 were rich in NB-ARC domain-containing disease resistance genes.
The cotton genome contains numerous RGA genes, and nearly half of them are located in clusters, which evolved by sequence exchanges, tandem duplications and segmental duplications. In the Rgrcs, 26 loci were induced by the V. dahliae inoculation, and 12 are in the vicinity of known Verticillium wilt resistance QTLs.
... Most of these R-genes encode nucleotide binding site (NBS)-leucine-rich repeat (LRR) class R proteins (Yue et al., 2012). Additionally, many resistance gene analogues and candidates RGAs/RGCs, which are potential sources of active R-genes (Zhu et al., 2002), have been cloned from economical plants by homology cloning using degenerate primers (Kanazin et al., 1996;Quirin et al., 2012). These R-genes were identified based on the four conserved motifs of the NBS-LRR R protein: (1) kinase-1a or P-loop, (2) kinase-2, (3) kinase-3a, and (4) unknown function motifs such as GLPL motifs (DeYoung and Innes, 2006;Yue et al., 2012). ...
... This observation seems consistent with previous reports on comparative analysis done with limited number of genetic markers [29] and recent M. truncatula whole genome analysis [13] that MtChr6 is rich in heterochromatic DNA and relatively poor in expressed genes [12]. Furthermore, it has been known that the majority of NBS-LRR resistance analogous genes were localized as clusters in the MtChr6 [30], indicating that the MtChr6 is functionally specialized for the resistance mechanism against pathogen infections. Likewise, functionally specialized genomic regions for the ABS responses might exist and remain to be further elucidated with more extended data in the future. ...
Cross-species translation of genomic information may play a pivotal role in applying biological knowledge gained from relatively simple model system to other less studied, but related, genomes. The information of abiotic stress (ABS)-responsive genes in Arabidopsis was identified and translated into the legume model system, Medicago truncatula. Various data resources, such as TAIR/AtGI DB, expression profiles and literatures, were used to build a genome-wide list of ABS genes. tBlastX/BlastP similarity search tools and manual inspection of alignments were used to identify orthologous genes between the two genomes. A total of 1,377 genes were finally collected and classified into 18 functional criteria of gene ontology (GO). The data analysis according to the expression cues showed that there was substantial level of interaction among three major types (i.e., drought, salinity and cold stress) of abiotic stresses. In an attempt to translate the ABS genes between these two species, genomic locations for each gene were mapped using an in-house-developed comparative analysis platform. The comparative analysis revealed that fragmental colinearity, represented by only 37 synteny blocks, existed between Arabidopsis and M. truncatula. Based on the combination of E-value and alignment remarks, estimated translation rate was 60.2% for this cross-family translation. As a prelude of the functional comparative genomic approaches, in-silico gene network/interactome analyses were conducted to predict key components in the ABS responses, and one of the sub-networks was integrated with corresponding comparative map. The results demonstrated that core members of the sub-network were well aligned with previously reported ABS regulatory networks. Taken together, the results indicate that network-based integrative approaches of comparative and functional genomics are important to interpret and translate genomic information for complex traits such as abiotic stresses.
... When comparing M. truncatula genome with Lotus japonica [40], similar results were obtained. These findings demonstrate the lack of marker-based synteny with pea [41] and the abundance of nucleotide-binding site-Leu-rich repeat genes [42]. The unusual high proportion of heterochromatin in this chromosome as it was previously reported [43] may explain why we found less homologous mRNA sequences in chromosome 6 of M. truncatula. ...
Prosopis alba (Fabaceae) is an important native tree adapted to arid and semiarid regions of north-western Argentina which is of great value as multipurpose species. Despite its importance, the genomic resources currently available for the entire Prosopis genus are still limited. Here we describe the development of a leaf transcriptome and the identification of new molecular markers that could support functional genetic studies in natural and domesticated populations of this genus.
Next generation DNA pyrosequencing technology applied to P. alba transcripts produced a total of 1,103,231 raw reads with an average length of 421 bp. De novo assembling generated a set of 15,814 isotigs and 71,101 non-assembled sequences (singletons) with an average of 991 bp and 288 bp respectively. A total of 39,000 unique singletons were identified after clustering natural and artificial duplicates from pyrosequencing reads.Regarding the non-redundant sequences or unigenes, 22,095 out of 54,814 were successfully annotated with Gene Ontology terms. Moreover, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 5,992 and 6,236 markers, respectively, throughout the genome. For the validation of the the predicted SSR markers, a subset of 87 SSRs selected through functional annotation evidence was successfully amplified from six DNA samples of seedlings. From this analysis, 11 of these 87 SSRs were identified as polymorphic. Additionally, another set of 123 nuclear polymorphic SSRs were determined in silico, of which 50% have the probability of being effectively polymorphic.
This study generated a successful global analysis of the P. alba leaf transcriptome after bioinformatic and wet laboratory validations of RNA-Seq data.The limited set of molecular markers currently available will be significantly increased with the thousands of new markers that were identified in this study. This information will strongly contribute to genomics resources for P. alba functional analysis and genetics. Finally, it will also potentially contribute to the development of population-based genome studies in the genera.
... Similarly, analysis of the Arabidopsis genome also indicate that numerous small-scale genomic duplications have copied or translocated one or several NBS-LRR genes from these clusters to distal and probably random locations in the genome. Molecular studies have demonstrated that this clustering usually results from tandem duplications of paralogues sequences (Meyers et al. 2003;Michelmore and Meyers 1998;Zhu et al. 2002). Duplication and insertion of repetitive sequences such as transposons may result in genic and intergenic sequence repeats within NBS-LRR R genes, which can cause mispairing during recombination events. ...
Plants are under strong evolutionary pressure in developing new and noble R genes to recognize pathogen avirulence (avr) determinants and bring about stable defense for generation after generations. Duplication, sequence variation by mutation, disparity in the length and structure of leucine rich repeats etc., causes tremendous variations within and among R genes in a plant thereby developing diverse recognitional specificity suitable enough for defense against new pathogens. Recent studies on genome sequencing, diversity and population genetics in different plants have thrown new insights on the molecular evolution of these genes. Tandem and segmental duplication are important factors in R gene abundance as inferred from the distribution of major nucleotide binding site-leucine rich repeats (NBS-LRRs) type R-genes in plant genomes. Likewise, R-gene evolution is also thought to be facilitated by cluster formation thereby causing recombination and sequence exchange and resulting in haplotypic diversity. Population studies have further proven that balancing selection is responsible for the maintenance of allelic diversity in R genes. In this review, we emphasize and discuss on improved perspectives towards the molecular mechanisms and selection pressure responsible for the evolution of NBS-LRR class resistance genes in plants.
... High conservation of gene content and order between M. truncatula and G. max was observed based on the number of Mt-FTQTL6-linked markers that could be obtained and their order on the genetic linkage maps constructed using either the LR3 or the RHL-derived population. These results extend previous observations on synteny conservation between M. truncatula and G. max and are particularly important since they concern M. truncatula chromosome 6, reported to be the shortest among all eight M. truncatula chromosomes, the most heterochromatic (Kulikova et al. 2001) and the richest in nucleotide-binding siteleucine-rich repeat genes (Zhu et al. 2002). Taking into account the effectiveness of this strategy, it can be argued that similar efforts may be helpful to provide markers linked to other residual gaps in the M. ...
Freezing is one of the most serious abiotic stress factors that affect cool-season legumes. It limits species geographic distribution and causes severe yield losses. Improving tolerance to freezing has long been a main concern for legume breeders. Medicago truncatula Gaertn. has been selected as a model species for legume biology. Various studies have shown significant macrosynteny between M. truncatula and agronomically important crop legumes. A major freezing tolerance quantitative trait locus (QTL), herein referred to as Mt-FTQTL6, was previously identified on M. truncatula chromosome 6. The physical location of this QTL was determined in this study and its corresponding chromosomal interval was enriched with additional markers. Markers were first developed using the draft sequence of M. truncatula euchromatin (release versions Mt3.0 and Mt3.5). Because Mt-FTQTL6 was found to coincide with an assembly gap, the Glycine max (L.) Merr. genome sequence was also used to generate markers. Five Mt-FTQTL6-linked markers were found to be common to a region on Pisum sativum L. linkage group VI harboring a QTL for freezing damage. A subset of markers was tested for transferability across 11 additional legume species. This study lays the groundwork for identifying the molecular basis of Mt-FTQTL6. Cross-legume markers will be useful in future efforts aiming to investigate the conservation of Mt-FTQTL6 in cool-season legumes and subsequently the existence of common mechanisms for response to freezing between M. truncatula and crop legumes.
... The advent of next generation sequencing technologies has dramatically accelerated research in genome analysis. Data-mining approaches have been developed and utilized for identification of RGAs in ESTs of sugarcane ( Rossi et al., 2003), wheat ( Dilbirligi and Gill, 2003), and Medicago ( Zhu et al., 2002). With the whole-genome sequences of many plants becoming available, genome-wide R gene detection has been undertaken in rice ( Monsi et al., 2004), Arabidopsis ( Meyers et al., 2003), Medicago (Ameline Torregrosa et al., 2008), and tomato ( Andolfo et al., 2014). ...
... In all chromosomes associated with Vc, at least one cluster of the genes mapping to clade C is present ( Figure 2 and Table S6). In general, genes of the same cluster have almost contiguous tree topologies, as expected if local gene tandem duplication was the mechanism generating clusters [19,[25][26][27]. ...
... The progress achieved previously in the transformation of the model legume, Medicago truncatula, has allowed testing T-DNA-mediated insertion mutagenesis as an approach to investigate legume-specific biological processes such as symbiosis with bacteria or fungi, production of specific secondary metabolites as well as the biology of resistance genes [7]. These processes are either absent or different in Arabidopsis thaliana and, therefore, cannot be studied in this model plant. ...
The development of efficient transformation protocols in several legume species has opened the possibility to use T-DNA insertional mutagenesis to investigate legume-specific processes such as symbiosis. A T-DNA mutagenesis approach was initiated in the model legume Medicago truncatula by constructing a small collection of 1000 M. truncatula T-DNA lines, using in vitro-mediated transformation-regeneration protocols. In this T-DNA collection, mutant phenotypes were observed but generally did not result from the disruption of genes by the T-DNA but most probably from somaclonal mutations induced by the in vitro regeneration process or by unsuccessful T-DNA integration events resulting in small (undetectable) insertions or deletions in the genome of the regenerated plants. In addition, a promoter trap approach was also not very successful for gene discovery because aberrant gene fusions were observed in this collection. Therefore, the T-DNA mutagenesis approach in M. truncatula seems to be limited by the lack of an efficient in planta transformation protocol that would allow the generation of a large mutant collection suitable for reverse genetic studies. Thus, there is a need for the development of alternative tools for efficient gene disruption in legumes.
... Since NBS sequences are abundant in plant genomes (Meyers et al. 1999), NBS-derived primers amplify multiple DNA fragments in each PCR (Shi et al. 2001;Wen et al. 2008). Although NBS-primer amplified DNA polymorphisms may, or may not, be related to plant disease resistance genes (Zhu et al. 2002), RGAP provides a fast, easy and reliable DNA fingerprinting approach, which can be used to compare the genetic diversity at the DNA level between individual plants (Diaz & Ferrer 2003). The RGAP may also be applicable in the DAFWA waxflower breeding programme for the identification of interspecific waxflower hybrids according to the following hypothesis (Fig. 2). ...
Waxflower is one of Australia's major native cut flowers for the export market. A number of interspecific hybrid cultivars such as the ‘Pearl’ series bred by the Department of Agriculture and Food, Western Australia have increased the competitiveness of waxflower on world markets. To improve the breeding efficiency, resistance gene analog polymorphisms (RGAP) are investigated as molecular markers for the early identification of interspecific hybrids between Chamelaucium uncinatum and C. megalopetalum. The results show that RGAP can be effectively applied to generate DNA markers to identify true waxflower hybrids. The RGAP marker system provides a reliable, simple, fast and inexpensive approach for hybrid identification in waxflower breeding.
... These genes can be classified by the presence or absence of a Toll/interleukin receptor domain (TIR) region. Retrieval of M. truncatula sequences homologous to the NBS domain of resistance genes identified at least 150 resistance gene analogs, of which more than 100 have been mapped Zhu et al., 2002). Phylogenetic analysis classified these sequences into several clades within the TIR and non-TIR subfamilies. ...
... The primers NBS F1 and NBS R1 were designed by Yu et al. (1996) based on the amino acid sequences of two highly conserved motifs of the nucleotide-binding site in tobacco N and Arabidopsis RPS2 genes. The second set (four primer pairs) of degenerate primers was designed from the known RGH genes of the model legume Medicago truncatula (Zhu et al. 2002) in order to obtain a broad spectrum of RGH genes. Polymerase chain reaction (PCR) amplification was carried out in a total reaction volume of 20 ll, containing 100 ng of template DNA, 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 0.25 lM each primer and 1 U of Taq DNA polymerase (Applied Biosystems, Foster City, CA, USA). ...
With 2 figures and 2 tables
The majority of functionally characterized plant disease resistance genes are of the nucleotide-binding site (NBS)-LRR gene family, encoding proteins with a central NBS domain, a carboxy-terminal leucine-rich repeat domain, and a variable N-terminal region with or without homology to the Toll interleukin 1-like receptor (TIR) domain, referred to as TIR and non-TIR resistance gene homologues (RGH), respectively. Degenerate primers designed from conserved motifs within the NBS sequence were used to amplify, clone and sequence NBS-RGH from the sweetpotato genome. Two hundred and twenty-five distinct sweetpotato NBS sequences with similarity to known RGH genes were identified. Additional 50 sweetpotato RGHs were mined from the public genomic sequence database. Thus, a total of 275 RGH sequences were obtained using both PCR-based method and data-mining approach, from which 237 were non-TIR sequences organized into 35 singletons and 35 groups after reduced to 90% nucleotide identity, and 38 were TIR sequences divided into three primary phylogenetic clades. A bias of non-TIR vs. TIR was observed not only in genomic RGH sequences, but also in expressed sequence tags-RGH sequences. A subset of sweetpotato non-TIR RGH genes contained a conserved intron within the NBS sequences. The exploration of RGH diversity enables resistance gene evolutionary study and may facilitate the isolation of new and functional alleles. These new RGH sequences provided a resource of candidate genes and molecular markers for disease resistance research in sweetpotato.
... RGAs are the small parts of the R gene that are amplifiable by PCR and provide a short cut to full-length R genes. RGAs can be cloned easily because PCR-based isolation procedures are used, and this approach has been applied efficiently in a variety of plants, including potato (Leister et al. 1996), A. thaliana (Aarts et al. 1998), sugar beet , apricot (Soriano et al. 2005), chickpea (Huettel et al. 2002), Medicago truncatula (Zhu et al. 2002) and maize (Xiao et al. 2006) for the isolation of NBS-LRR domains encoding RGAs through the design of degenerative as well as specific primers. The results of genetic mapping studies have shown that many of the RGAs either co-segregate or are closely associated with disease resistance loci (Shen et al. 1998;Pan et al. 2000). ...
Four cotton species (genus Gossypium) produce spinable fiber. The two diploid species of Asiatic origin, Gossypium
arboreum and G. herbaceum, have been largely replaced by G. hirsutum. However, these diploid species are potentially a rich source of genes for the improvement of G. hrisutum, particularly in terms of providing resistance against biotic and abiotic stresses. As a first step towards understanding
the mechanisms of resistance in cotton, we designed 24 non-degenerate primers based on resistance gene analogs (RGAs) cloned
from G. hirsutum for screening a number of cotton species with the A and D genomes. Most of these RGAs are conserved on the A genome (G. arboreum), suggesting a bias towards this genome. The amplified RGAs from G. arboreum were cloned and their nucleotide and amino acid sequences compared with RGA sequences available in public databases. The
majority of the RGAs identified were homologous to those isolated from G. hirsutum and G. barbadense, but their diversity was greater than expected at both the nucleotide and amino acid levels. These RGAs provide useful tools
for the identification of full-length resistance genes from bacterial artificial chromosome and cDNA libraries.
Keywords
Gossypium arboreum
–RGAs–NBS-LRR
... A large number of resistance gene analogs (RGAs) have been isolated from many plant species by PCR amplification using degenerate primers (Friederike and Bodo 2005;Noir et al. 2001;Zhu et al. 2002), as well as by genomic and EST sequencing projects (Xiao et al. 2006;Rossi et al. 2003). Genetic studies of these R genes and RGAs have provided insight into the evolution of R genes and the mechanisms that generate variation in these genes. ...
The well-conserved NBS domain of resistance (R) genes cloned from many plants allows the use of a PCR-based approach to isolate resistance gene analogs (RGAs). In this
study, we isolated an RGA (CapRGC) from Capsicum annuum “CM334” using a PCR-based approach. This sequence encodes a protein with very high similarity to Rx genes, the Potato Virus X (PVX) R genes from potato. An evolutionary analysis of the CapRGC gene and its homologs retrieved by an extensive search of a Solanaceae database provided evidence that Rx-like genes (eight ESTs or genes that show very high similarity to Rx) appear to have diverged from R1 [an NBS-LRR R gene against late blight (Phytophthora infestans) from potato]-like genes. Structural comparison of the NBS domains of all the homologs in Solanaceae revealed that one novel
motif, 14, is specific to the Rx-like genes, and also indicated that several other novel motifs are characteristic of the R1-like genes. Our results suggest that Rx-like genes are ancient but conserved. Furthermore, the novel conserved motifs can provide a basis for biochemical structural–function
analysis and be used for degenerate primer design for the isolation of Rx-like sequences in other plant species. Comparative mapping study revealed that the position of CapRGC is syntenic to the locations of Rx and its homolog genes in the potato and tomato, but cosegregation analysis showed that CapRGC may not be the R gene against PVX in pepper. Our results confirm previous observations that the specificity of R genes is not conserved, while the structure and function of R genes are conserved. It appears that CapRGC may function as a resistance gene to another pathogen, such as the nematode to which the structure of CapRGC is most similar.
Keywords
Rx
–
CapRGC
–Solanaceae–Resistance gene analogs (RGAs)–Evolution
The transformation of conventional approaches towards modernization
in agriculture has escorted by the influence of technology. Few notable
technologies include drone imaging for pest detection, genome editing to
augment disease resistance in crops, Data mining for irrigation control and
optimization of natural resources and electrically powered tractors for ebbing
pollution. Implementing these technologies alleviate water wastage,
pollutions, erratic climatic conditions and use of pesticides for improving
sustainability in agriculture. In this review, a glimpse of several agricultural
technologies was discussed in the aspects of its relevance, advantages, and
setbacks. The impediments on the data-driven technology are gradually
getting replaced towards whole automated systems in the way of providing a
better yield of crops. Moreover, we endeavor to provide the utmost
information to deliver all the recent technologies from various fields of
science in this book. We conclude that future technology should involve in
development of farmer friendly software and easier operation of technology
to enhance agricultural production.
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. They are classified into two major subfamilies, toll/interleukin-1 receptor (TIR)-NBS-LRR (TNL) and coiled-coil (CC)-NBS-LRR (CNL) proteins. We have identified and characterised 641 NBS-LRR genes in Brassica napus, 249 in B. rapa and 443 in B. oleracea. A ratio of 1 : 2 of CNL : TNL genes was found in the three species. Domain structure analysis revealed that 57% of the NBS-LRR genes are typical resistance genes and contain all three domains (TIR/CC, NBS, LRR), whereas the remaining genes are partially deleted or truncated. Of the NBS-LRR genes, 59% were found to be physically clustered, and individual genes involved in clusters were more polymorphic than those not clustered. Of the NBS-LRR genes in B. napus, 50% were identified as duplicates, reflecting a high level of genomic duplication and rearrangement. Comparative analysis between B. napus and its progenitor species indicated that >60% of NBS-LRR genes are conserved in B. napus. This study provides a valuable resource for the identification and characterisation of candidate NBS-LRR genes.
The nucleotide binding site and leucine rich repeat (NBS–LRR) class of R genes is the most comprehensively studied in terms of sequence evolution; however, in coconut palm and, more generally, in the family of Arecaceae, our understanding of the evolution of these genes is rather limited. In this study, disease resistance gene candidates (RGCs) of the nucleotide binding site (NBS) type of coconut palm were used to investigate evolutionary relationships in Arecaceae, Poaceae and Brassicaceae species. The results indicate a species-specific evolution of RGCs in coconut palm. However, strikingly similar RGCs between species of Arecales indicate a high conservation of specific RGCs of this family, suggesting a monophyletic origin of three genera. The phylogenetic relationship between RGCs of Arecales and Brassicales suggests that these sequences possibly emerged before being divided between monocots and dicots. Finally the comparative analysis of the expression of four RGCs in healthy coconut palm and those affected with lethal yellowing disease revealed differences in their expression profiles. This study provides new insights for future efforts towards the improvement of disease resistance in coconut palm and other species of Arecaceae.
A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F2 population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.
Late blight caused by the oomycete Phytophthora infestans is one of the most severe threats to potato production worldwide. Numerous studies suggest that the most effective protective strategy against the disease would be to provide potato cultivars with durable resistance (R) genes. However, little is known about the origin and evolutional history of these durable R-genes in potato. Addressing this might foster better understanding of the dynamics of these genes in nature and provide clues for identifying potential candidate R-genes. Here, a systematic survey was executed at RB/Rpi-blb1 locus, an exclusive broad-spectrum R-gene locus in potato. As indicated by synteny analysis, RB/Rpi-blb1 homologs were identified in all tested genomes, including potato, tomato, pepper, and Nicotiana, suggesting that the RB/Rpi-blb1 locus has an ancient origin. Two evolutionary patterns, similar to those reported on RGC2 in Lactuca, and Pi2/9 in rice, were detected at this locus. Type I RB/Rpi-blb1 homologs have frequent copy number variations and sequence exchanges, obscured orthologous relationships, considerable nucleotide divergence, and high non-synonymous to synonymous substitutions (Ka/Ks) between or within species, suggesting rapid diversification and balancing selection in response to rapid changes in the oomycete pathogen genomes. These characteristics may serve as signatures for cloning of late blight resistance genes.
Cao, K., Wang, L. R., Zhu, G. R., Fang, W. CH. and Chen, CH. W. 2011. Isolation, characterisation and phylogenetic analysis of resistance gene analogues in a wild species of peach (Prunus kansuensis). Can. J. Plant Sci. 91: 961-970. Conserved motifs, such as nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains, have been found in resistance (R) genes cloned from plant species. These allow the study of plant defence mechanisms and isolating candidate genes in several species including peaches. Seventy-five resistance gene analogues (RGA) were identified using two different degenerative primer pairs in the Honggengansutao (Prunus kansuensis), a wild species of peach resistant to drought and nematodes. Through aligning their amino-acid sequences, P-loop and GLPL motifs were found in 48 RGAs with open-reading frames (ORF). These RGAs and 17 RGAs from Arabidopsis thallium, Capsicum annuum and Solanum lycopersicum were grouped into two classes by phylogenetic analysis: toll and interleukin-1 receptor (TIR)- and non-TIR-NBS. Most Honggengansutao RGAs were TIR-NBS. A semiquantitative RT-PCR analysis revealed transcript-level variations of 22 RGAs in the young leaves, flowers, fruits and roots of the Honggengansutao, demonstrating their probable role in resistance against diseases attacking the organs. This is the first large-scale analysis of NBS-LRR RGAs in P. kansuensis, this technique has the potential for involvement in rootstock breeding. It will foster further R gene isolation and exploitation.
Coconut, an important crop of the tropics and subtropics, is susceptible to a variety of diseases and enhancing disease resistance has been the major goal of coconut breeding programs all over the world. Information on the presence and distribution of disease resistance (R) genes, which play a primary role in the detection of pathogens and the initiation of specific plant defenses, is scarce in coconut. In this study, RNA-Seq was used to generate the transcriptome of leaf samples of coconut root (wilt) disease-resistant cultivar Chowghat Green Dwarf. Comprehensive bioinformatics analysis identified 243 resistance gene analog (RGA) sequences, comprising 6 classes of RGAs. Domain and conserved motif predictions of clusters were performed to analyze the architectural diversity. Phylogenetic analysis of deduced amino acid sequences revealed that coconut NBS-LRR type RGAs were classified into distinct groups based on the presence of TIR or CC motifs in the N-terminal regions. Furthermore, qRT-PCR analysis validated the expression of randomly selected NBS-LRR type RGAs. The results of this study provide a sequence resource for development of RGA-tagged markers in coconut, which would aid mapping of disease-resistant candidate genes. In addition, we hope that this study will provide a genomic framework for isolation of additional RGAs in coconut via comparative genomics and also contribute to the deciphering of mode of evolution of RGAs in Arecaceae.
The agronomic effects of drought on forage species and grassland are related with productivity and pasture persistence. The water shortage produce a growth reduction caused by decreased turgor at cellular level, changes in membrane fluidity and composition, decrease of photosynthetic electron transport, changes in solute concentration and metabolism in general. Plants have developed several mechanisms for tolerate drought stress in additive action and synergy. They are the origin for morphological adaptations, which will give place to a stable acclimation for drought tolerance. In grasses, water stress produce decrease in leaf growth and the number of tillers, in white clover result in a severe reduction in stolon elongation. The roots architecture, mass and depth, for specie, in particular, can be determinant in its response to water stress. Reduction of soil water availability in pastures also produces changes in fertilizer use efficiency, nutritive value, incidence of pests and diseases, and grazing animals performance. Morphological modifications reduce water loss which are associated to changes in leaf anatomy as presence of trichomes, decrease of leaf area or loss of any leaves, change in position respect to radiation, increase of cuticle thickness and decrease of stomatal density. Leaves can modify cuticle thickness or compositions thanks to a phenotypic plasticity given for regulation of cutin pathway biosynthesis and transport of lipidic component to a leaf surface. In this section the agronomical aspects of drought stress in forage plants are presented. The cuticle formation in plant is a matter revised specifically centered in the effect over drought tolerance and considered as a character for plant breeding selection. Additionally, advances in forage perennial legumes for increasing drought tolerance will be discussed and some results of the Forage Breeding Program of INIA-Chile will be presented as example.
Following a brief introduction on the phylogeny, taxonomy, production, uses, diseases, pests, environmental stress and variation in genomes of grain legumes, this review focuses on the available genetic resources of key legume crops (tropical and temperate legumes, and model species related to grain legumes); management and utilization of legume genetic resources; impact of genetic resources in conventional legume breeding (germplasm distribution, domesticated germplasm and breeding gains, wild germplasm, conventional manipulation of genetic resources); enhanced molecular strategies for manipulating novel genetic variation for legume breeding (interspecific hybridization, linkage mapping and QTL detection, linkage disequilibrium and association mapping, dissection and manipulation of legume physiology); advanced applications in legume molecular breeding (comparative genomics and allele mining, functional genomics and gene discovery, new technologies for marker-assisted selection, and molecular breeding in legumes
Subterranean clover mottle virus (SCMoV), which causes an important disease of annual clover pastures, was inoculated to the annual pasture legume Medicago truncatula, a model legume species used to help understand legume genome structure and function. Two hundred and nine accessions representing the core collection of M. truncatula were inoculated with infective sap containing SCMoV to determine their disease phenotypes. Forty-two of these accessions remained uninfected systemically and so were potentially resistant to the virus. Accession DZA315.16 developed a localised hypersensitive resistance reaction. In an F8 mapping population from a cross between the susceptible parent Jemalong 6/A17 and resistant accession DZA315.16, a total of 166F8 recombinant inbred lines (RILs) were phenotyped for resistance and susceptibility to SCMoV. Resistant and susceptible lines showed parental phenotypic responses: 84 were susceptible and 82 were resistant, suggesting presence of a single resistance (R) gene. The phenotypic data were combined with genotypic data (76 polymorphic molecular markers) for this RIL population to provide a framework map. Genetic analysis located a single resistance locus termed RSCMoV1 on the long arm of chromosome 6. These results provide a basis for fine mapping the RSCMoV1 gene.
A rapid resistance/susceptibility test for Peronospora parasitica (downy mildew) was established by inoculating leaf-disks of four Brassica oleracea accessions. Several conditions were tested: disk disinfection or not, agar medium with or without nutrients and with 50 or 100 ppm of benzimidazole. Using disinfected disks placed on agar (no nutrient and benzimidazole at 50 or 100 ppm), the responses of leaf-disks to four isolates were similar to those obtained using the classical cotyledon test, whereas undesired contaminations occurred in all other conditions. The possible effect of the particular leaf used for obtaining the disks was also studied. In each incompatible interaction tested, disks were resistant whatever the leaf used. In compatible interactions, susceptible phenotypes were observed on disks derived from the six lowest leaves, but disks from upper leaves were resistant. The genetic basis of resistance in a F1 hybrid broccoli was assessed, by testing six isolates on an F2 population derived from this hybrid. The cotyledon test only allows inoculation of two isolates per seedling, whereas many isolates can be tested on each plant by using leaf-disks. The segregation of the resistance to each of the six isolates was analysed: two dominant genes (tightly linked) control resistance to all isolates (one to five isolates; the other to only one isolate).
Medicagotruncatula, barrel medic, is an important forage crop that also is considered a model legume for laboratory studies. It is genetically
tractable with a relative small genome of ~470 million base pairs, has simple Mendelian genetics, a short seed-to-seed generation
time, a relatively high transformation efficiency, an excellent collection of phenotypic mutants, and several large collections
of diverse, naturally occurring ecotypes. The recent work of D. Cook and D.J. Kim, University of California at Davis has resulted
in constructing an ~20-fold coverage BAC library and fingerprinting it to a depth of ~12- fold, and the Noble Foundation and
The Institute for Genome Research have generated over 180,000 expressed sequence tags (ESTs) representing genes expressed
in almost every M.truncatula tissue, developmental stage and growth condition. To complement these efforts, we recently began to sequence the M. truncatula genome. By collecting sample sequence data through an initial whole genome shotgun approach, we confirmed earlier cytogenetic
data that indicates the eight chromosomes of M.truncatula are organized into distinct gene-rich euchromatic and separate pericentromeric repeat-rich regions. We now have sequenced
almost 1000 gene-rich bacterial artificial chromosome (BAC) clones. The results of these studies indicate that the gene density
in M. truncatula is of the order of one gene in every 6–7 kilobase pairs (kbp). The ~200 Mbp of euchromatic regions therefore encodes ~30,000
to 33,000 genes, of which ~66% are represented by ESTs. Following in the tradition of other genome projects, all our sequence
data is freely available through the international databases.
The importance of high-quality fruit, and the intrinsic difficulties of breeding in a perennial species, requires the development
and application of structural and functional genomics databases for the sustained improvement of fruit tree crops. Identification
and characterization of genes controlling the genetic basis of the traits, and their tagging with molecular markers, permits
a more realistic estimate of the effort needed to complete the introgression and to produce a new variety combining the best
of traits formerly isolated in separate varieties. It also reduces effort and time, and improves the accuracy of marker-assisted
selections. Thus, field evaluation is limited to trees containing the genes of interest, significantly reducing the costs
associated with maintaining undesirable trees to maturity. The ability to pre-select seedlings, using DNA based markers, for
traits such as sugar and acid levels, color, firmness, and fruit size while introgressing traits, such as biotic and abiotic
stress resistance from exotic germplasm, speeds the development of commercially acceptable cultivars. Having the cloned gene
sequences controlling the traits of interest also provides a means to directly move the character through the use of transgenic
technologies significantly reducing the breeding time required to obtain cultivars with commercially desirable qualities.
The tomato Cf-4 and Cf-9 genes are the founder members of a large gene family of homologues of Cladosporium fulvum resistance gene Cf-9 (Hcr9 genes), several of which confer resistance against C. fulvum through recognition of different pathogen-encoded avirulence determinants. Three loci of tandemly repeated Hcr9 genes--Southern Cross (SC), Milky Way (MW), and Northern Lights (NL)--are located on the short arm of tomato chromosome 1. Comparisons between 2 SC-Hcr9s, 11 from MW, and 5 from NL implicated sequence exchange between gene family members in their evolution. The extent to which novel variants can be generated by recombination depends on the degree of sequence polymorphism available within the gene family. Here we show that physical separation of Hcr9 genes can be associated with elevated sequence divergence. Two diverged subclasses of Hcr9s could be defined. These are physically separated from each other, with members of one class exclusively residing at Northern Lights. One exceptional Hcr9 at Northern Lights carried sequence features specific for Hcr9s at other loci, suggesting a recent transfer of this gene by an interlocus recombination event. As members of diverged subclasses are brought into physical vicinity within a tandem repeat, a larger spectrum of sequence variants can potentially be generated by subsequent interhomologue sequence exchange.
Plant disease resistance (R) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional
polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most R loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast,
the resistance to Pseudomonas syringae pathovar maculicola (RPM1) bacterial resistance gene is completely absent (rpm1-null) in 5/5 Arabidopsis thaliana accessions that lack RPM1 function. The rpm1-null locus contains a 98-bp segment of unknown origin in place of the RPM1 gene. We undertook comparative mapping of RPM1 and flanking genes in Brassica napus to determine the ancestral state of the RPM1 locus. We cloned two B. napus RPM1 homologs encoding hypothetical proteins with ≈81% amino acid identity to Arabidopsis RPM1. Collinearity of genes flanking RPM1 is conserved between B. napus and Arabidopsis. Surprisingly, we found four additional B. napus loci in which the flanking marker synteny is maintained but RPM1 is absent. These B. napus rpm1-null loci have no detectable nucleotide similarity to the Arabidopsis rpm1-null allele. We conclude that RPM1 evolved before the divergence of the Brassicaceae and has been deleted independently in the Brassica and Arabidopsis lineages. These results suggest that functional polymorphism at R gene loci can arise from gene deletions.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic, and statistical refinements permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is described for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position Specific Iterated BLAST (PSLBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities.
The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans--the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.
The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88øamino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.
The tobacco N and Arabidopsis RPS2 genes, among several recently cloned disease-resistance genes, share highly conserved structure, a nucleotide-binding site (NBS). Using degenerate oligonucleotide primers for the NBS region of N and RPS2, we have amplified and cloned the NBS sequences from soybean. Each of these PCR-derived NBS clones detected low-or moderate-copy soybean DNA sequences and belongs to 1 of 11 different classes. Sequence analysis showed that all PCR clones encode three motifs (P-loop, kinase-2, and kinase-3a) of NBS nearly identical to those in N and RPS2. The intervening region between P-loop and kinase-3a of the 11 classes has high (26% average) amino acid sequence similarity to the N gene although not as high (19% average) to RPS2. These 11 classes represent a superfamily of NBS-containing soybean genes that are homologous to N and RPS2. Each class or subfamily was assessed for its positional association with known soybean disease-resistance genes through near-isogenic line assays, followed by linkage analysis in F2 populations using restriction fragment length polymorphisms. Five of the 11 subfamilies have thus far been mapped to the vicinity of known soybean genes for resistance to potyviruses (Rsv1 and Rpv), Phytophthora root rot (Rps1, Rps2, and Rps3), and powdery mildew (rmd). The conserved N- or RPS2-homologous NBS sequences and their positional associations with mapped soybean-resistance genes suggest that a number of the soybean disease-resistance genes may belong to this superfamily. The candidate subfamilies of NBS-containing genes identified by genetic mapping should greatly facilitate the molecular cloning of disease-resistance genes.
Sequences of cloned resistance genes from a wide range of plant taxa reveal significant similarities in sequence homology and structural motifs. This is observed among genes conferring resistance to viral, bacterial, and fungal pathogens. In this study, oligonucleotide primers designed for conserved sequences from coding regions of disease resistance genes N (tobacco), RPS2 (Arabidopsis) and L6 (flax) were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing of amplification products indicated that at least nine classes of resistance gene analogs (RGAs) were detected. Genetic mapping of members of these classes located them to eight different linkage groups. Several RGA loci mapped near known resistance genes. A bacterial artificial chromosome library of soybean DNA was screened using primers and probes specific for eight RGA classes and clones were identified containing sequences unique to seven classes. Individual bacterial artificial chromosomes contained 2-10 members of single RGA classes. Clustering and sequence similarity of members of RGA classes suggests a common process in their evolution. Our data indicate that it may be possible to use sequence homologies from conserved motifs of cloned resistance genes to identify candidate resistance loci from widely diverse plant taxa.
The interaction between Arabidopsis and the biotrophic oomycete Peronospora parasitica (downy mildew) provides an attractive model pathosystem to identify molecular components of the host that are required for genotype-specific recognition of the parasite. These components are the so-called RPP genes (for resistance to P. parasitica). Mutational analysis of the ecotype Wassilewskija (Ws-0) revealed an RPP-nonspecific locus called EDS1 (for enhanced disease susceptibility) that is required for the function of RPP genes on chromosomes 3 (RPP1/RPP14 and RPP10) and 4 (RPP12). Genetic analyses demonstrated that the eds1 mutation is recessive and is not a defective allele of any known RPP gene, mapping to the bottom arm of chromosome 3 (approximately 13 centimorgans below RPP1/RPP14). Phenotypically, the Ws-eds1 mutant seedlings supported heavy sporulation by P. parasitica isolates that are each diagnostic for one of the RPP genes in wild-type Ws-0; none of the isolates is capable of sporulating on wild-type Ws-0. Ws-eds1 seedlings exhibited enhanced susceptibility to some P. parasitica isolates when compared with a compatible wild-type ecotype, Columbia, and the eds1 parental ecotype, Ws-0. This was observed as earlier initiation of sporulation and elevated production of conidiosporangia. Surprisingly, cotyledons of Ws-eds1 also supported low sporulation by five isolates of P. parasitica from Brassica oleracea. These isolates were unable to sporulate on > 100 ecotypes of Arabidopsis, including wild-type Ws-0. An isolate of Albugo candida (white blister) from B. oleracea also sporulated on Ws-eds1, but the mutant exhibited no alteration in phenotype when inoculated with several oomycete isolates from other host species. The bacterial resistance gene RPM1, conferring specific recognition of the avirulence gene avrB from Pseudomonas syringae pv glycinea, was not compromised in Ws-eds1 plants. The mutant also retained full responsiveness to the chemical inducer of systemic acquired resistance, 2,6-dichloroisonicotinic acid; Ws-eds1 seedlings treated with 2,6-dichloroisonicotinic acid became resistant to the Ws-0-compatible and Ws-0-incompatible P. parasitica isolates Emwa1 and Noco2, respectively. In summary, the EDS1 gene appears to be a necessary component of the resistance response specified by several RPP genes and is likely to function upstream from the convergence of disease resistance pathways in Arabidopsis.
Analysis of viral and bacterial pathogenesis has revealed common themes in the ways in which plants and animals respond to pathogenic agents. Pathogenic bacteria use macromolecule delivery systems (types III and IV) to deliver microbial avirulence proteins and transfer DNA-protein complexes directly into plant cells. The molecular events that constitute critical steps of plant-pathogen interactions seem to involve ligand-receptor mechanisms for pathogen recognition and the induction of signal transduction pathways in the plant that lead to defense responses. Unraveling the molecular basis of disease resistance pathways has laid a foundation for the rational design of crop protection strategies.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
As part of the Human Genome Project, the Washington University Genome Sequencing Center has commenced systematic sequencing of human chromsome 7. To organize and supply the effort, we have undertaken the construction of sequence-ready physical maps for defined chromosomal intervals. Map construction is a serial process composed of three main activities. First, candidate STS-positive large-insert PAC and BAC clones are identified. Next, these candidate clones are subjected to fingerprint analysis. Finally, the fingerprint data are used to assemble sequence-ready maps. The fingerprinting method we have devised is key to the success of the overall approach. We present here the details of the method and show that the fingerprints are of sufficient quality to permit the construction of megabase-size contigs in defined regions of the human genome. We anticipate that the high throughput and precision characteristic of our fingerprinting method will make it of general utility.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new
system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing
the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows
the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional
multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order
of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to
be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments
or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with
a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled
on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for
x86 PCs, and Macintosh PowerMac.
The Arabidopsis genes EDS1 and NDR1 were shown previously by mutational analysis to encode essential components of race-specific disease resistance. Here, we examined the relative requirements for EDS1 and NDR1 by a broad spectrum of Resistance (R) genes present in three Arabidopsis accessions (Columbia, Landsberg-erecta, and Wassilewskija). We show that there is a strong requirement for EDS1 by a subset of R loci (RPP2, RPP4, RPP5, RPP21, and RPS4), conferring resistance to the biotrophic oomycete Peronospora parasitica, and to Pseudomonas bacteria expressing the avirulence gene avrRps4. The requirement for NDR1 by these EDS1-dependent R loci is either weak or not measurable. Conversely, three NDR1-dependent R loci, RPS2, RPM1, and RPS5, operate independently of EDS1. Another RPP locus, RPP8, exhibits no strong exclusive requirement for EDS1 or NDR1 in isolate-specific resistance to P. parasitica, although resistance is compromised weakly by eds1. Similarly, resistance conditioned by two EDS1-dependent RPP genes, RPP4 and RPP5, is impaired partially by ndr1, implicating a degree of pathway cross-talk. Our results provide compelling evidence for the preferential utilization of either signaling component by particular R genes and thus define at least two disease resistance pathways. The data also suggest that strong dependence on EDS1 or NDR1 is governed by R protein structural type rather than pathogen class.
At least 10 Dm genes conferring resistance to the oomycete downy mildew fungus Bremia lactucae map to the major resistance cluster in lettuce. We investigated the structure of this cluster in the lettuce cultivar Diana, which contains Dm3. A deletion breakpoint map of the chromosomal region flanking Dm3 was saturated with a variety of molecular markers. Several of these markers are components of a family of resistance gene candidates (RGC2) that encode a nucleotide binding site and a leucine-rich repeat region. These motifs are characteristic of plant disease resistance genes. Bacterial artificial chromosome clones were identified by using duplicated restriction fragment length polymorphism markers from the region, including the nucleotide binding site-encoding region of RGC2. Twenty-two distinct members of the RGC2 family were characterized from the bacterial artificial chromosomes; at least two additional family members exist. The RGC2 family is highly divergent; the nucleotide identity was as low as 53% between the most distantly related copies. These RGC2 genes span at least 3.5 Mb. Eighteen members were mapped on the deletion breakpoint map. A comparison between the phylogenetic and physical relationships of these sequences demonstrated that closely related copies are physically separated from one another and indicated that complex rearrangements have shaped this region. Analysis of low-copy genomic sequences detected no genes, including RGC2, in the Dm3 region, other than sequences related to retrotransposons and transposable elements. The related but divergent family of RGC2 genes may act as a resource for the generation of new resistance phenotypes through infrequent recombination or unequal crossing over.
Primers based on a conserved nucleotide binding site (NBS) found in several cloned plant disease resistance genes were used to amplify DNA fragments from the genome of common bean (Phaseolus vulgaris). Cloning and sequence analysis of these fragments uncovered eight unique classes of disease-resistance related sequences. All eight classes contained the conserved kinase 2 motif, and five classes contained the kinase 3a motif. Gene expression was noted for five of the eight classes of sequences. A clone from the SB3 class mapped 17.8 cM from the Ur-6 gene that confers resistance to several races of the bean rust pathogen Uromyces appendiculatus. Linkage mapping identified microclusters of disease-resistance related sequence in common bean, and sequences mapped to four linkage groups in one population. Comparison with similar sequences from soybean (Glycine max) revealed that any one class of common bean disease-resistance related sequences was more identical to a soybean NBS-containing sequence than to the sequence of another common bean class.
Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.
Genomic positions of phenotypically defined disease resistance genes (R genes) and R gene homologues were analyzed in three solanaceous crop genera, Lycopersicon (tomato), Solanum (potato), and Capsicum (pepper). R genes occurred at corresponding positions in two or more genomes more frequently than expected by chance; however, in only two cases, both involving Phytophthora spp., did genes at corresponding positions have specificity for closely related pathogen taxa. In contrast, resistances to Globodera spp., potato virus Y, tobacco mosaic virus, and tomato spotted wilt virus were mapped in two or more genera and did not occur in corresponding positions. Without exception, pepper homologues of the cloned R genes Sw-5, N, Pto, Prf, and I2 were found in syntenous positions in other solanaceous genomes and in some cases also mapped to additional positions near phenotypically defined solanaceous R genes. This detailed analysis and synthesis of all available data for solanaceous R genes suggests a working hypothesis regarding the evolution of R genes. Specifically, while the taxonomic specificity of host R genes may be evolving rapidly, general functions of R alleles (e.g., initiation of resistance response) may be conserved at homologous loci in related plant genera.
Plant biology is moving rapidly. With the recent completion of the sequence of the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000), researchers have turned their attention to the genomes of other crop plants (Adam, 2000). Many agronomically important crop plants are legumes, such as soybean, pea, and alfalfa. However, the size and complexity of these genomes makes them unwieldy and has slowed progress on the genetic characterization of these crops. Medicago truncatula recently emerged as a model plant for legume genetics and genomics (Cook, 1999). The small diploid genome, autogamous genetics, and ease of transformation make this close relative of alfalfa (Medicago sativa) a good model system. The recent development of genetic and genomic tools for M. truncatula research has propelled M. truncatula into the forefront of legume research as an ideal legume model. Advances in M. truncatula genomics were the subject of a recent workshop preceding the 9th Annual Plant and Animal Genome Meeting in San Diego, California, on January 11 and 12.
The international scope of this workshop underscored the interest in M. trun-catula as a model system for studying plant–microbe interactions, in particular rhizobial and mycorrhizal symbioses. More recently, interest has turned to M. truncatula as a system for examining the very rich production of secondary metabolites by legumes and legume-specific disease resistance (Cook et al., 2000; Harrison, 2000). This workshop, which started out three years ago as a small band of National Science Foundation (NSF) genome grant collaborators sponsored by the NSF Plant Genome Project (award number 9,872,664), has grown into an international effort spanning national boundaries and legume species. This year's participants represented not only the original NSF genome grant collaborators but members of the Institut National de la Recherche Agronomique–Centre National de la Recherche Scientifique (INRA-CNRS), German mycorrhizal and European Union (EU) M. truncatula genome projects (funded by the Deutsche Forschungsgemeinshaft and the European Union 5th Framework Program, respectively), the Noble Foundation M. truncatula project, and legume scientists working with other species such as pea, soybean, and alfalfa. What emerged from the sharing of data, questions, ideas, and future plans was a coordinated effort using worldwide resources to develop M. truncatula as a model plant as easy to use as Arabidopsis with broad applicability to other legume systems.
A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
Phylogenetic studies are contributing greatly to our knowledge of relationships on both sides of the plant-bacteria nodulation symbiosis. Multiple origins of nodulation (perhaps even within the legume family) appear likely. However, all nodulating flowering plants are more closely related than previously suspected, suggesting that the predisposition to nodulate might have arisen only once. Phylogenies of 16S rRNA genes highlight the evolutionary diversity of symbiotic bacteria and appear to rule out any broad coevolution with their plant hosts, but high levels of gene transfer might obscure the relevant pattern. The origins of nodulation, and the extent to which developmental programs are conserved in nodules remain unclear, but an improved understanding of the relationships between nodulin genes is providing some clues.
Alleles, or tightly linked genes, at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to races of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. In this study we demonstrate that Rpg1 maps to a cluster of previously identified resistance genes, including those effective against fungal, viral and nematode
pathogens. Rpg1 is in molecular linkage group (MLG) F, flanked by the markers K644 and B212. The RFLP markers R45, php2265 and php2385 cosegregated
with Rpg1, as did the marker nbs61, which encodes a protein related to previously isolated resistance genes.
Pea (Pisum sativum L.) sequences that are analogous to the conserved nucleotide binding site (NBS) domain found in a number of plant disease
resistance genes (R-genes) were cloned. Using redundant oligonucleotide primers and the polymerase chain reaction (PCR), we
amplified nine pea sequences and characterised their sequences. The pea R-gene analog (RGA)- deduced amino acid sequences
demonstrated significant sequence similarity with known R-gene sequences lodged in public databases. The genomic locations
of eight of the pea RGAs were determined by linkage mapping. The eight RGAs identified ten loci that mapped to six linkage
groups. In addition, the genomic organization of the RGAs was inferred. Both single-copy and multicopy sequence families were
present among the RGAs, and the multicopy families occurred most often as tightly linked clusters of related sequences. Intraspecific
copy number variability was observed in three of the RGA sequence families, suggesting that these sequence families are evolving
rapidly. The genomic locations of the pea RGAs were compared with the locations of known pea R-genes and sym genes involved in the pea-rhizobia symbiosis. Two pea RGAs mapped in the genomic region containing a pea R-gene, Fw, and four pea RGAs mapped in regions of the genome containing sym genes.
To facilitate genome analysis and map-based cloning of symbiotic genes in the model legume Medicago truncatula, a bacterial artificial chromosome (BAC) library was constructed. The library consists of 30 720 clones with an average insert
size of approximately 100 kb, representing approximately five haploid-genome equivalents. The frequency of BAC clones carrying
inserts of chloroplast DNA was estimated to be 1.4%. Screening of the library with single- or low-copy genes as hybridization
probes resulted in the detection of 1–12 clones per gene. Hybridization of the library with repeated sequences such as rDNA
genes and transposon-like elements of M. truncatula revealed the presence of 60 and 374 BAC clones containing the two sequences, respectively. The BAC library was pooled for
screening by polymerase chain reaction (PCR)-amplification. To demonstrate the utility of this system, we used primers designed
from a conserved region of the ein3-like loci of Arabidopsis thaliana and isolated six unique BAC clones from the library. DNA gel-blot and sequence analyses showed that these ein3-like clones could be grouped into three classes, an observation consistent with the presence of multiple ein3-like loci in M. truncatula. These results indicate that the BAC library represents a central resource for the map-based cloning and physical mapping
in M. truncatula and other legumes.
The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans— the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement.
Gene-for-gene plant disease resistance involves two basic processes: perception of pathogen attack, followed by responses to limit disease. Perception involves receptors with high degrees of specificity for pathogen strains, which are encoded by disease resistance genes. Large repertoires of distantly related resistance (R) genes with diverse recognitional specificities are found within a single plant species. The generation of R-gene polymorphism involves gene duplication, followed by DNA-sequence divergence by point mutation, and by deletion and duplication of intragenic DNA repeats encoding blocks of leucine-rich elements. Recombination between related genes reassorts this variation to further diversify gene sequences. Pathogen pressure selects functional resistance specificities and results in the maintenance of R-gene diversity. Recent genome-sequence data reveal that the NBS-LRR (i.e. nucleotide-binding site-leucine-rich repeat) class of R genes represents as much as 1% of the Arabidopsis genome. Experimental data have shown that the LRR has a role in determination of specificity. Mutation experiments, in which R-gene signaling has been dissociated from specificity in constitutive signal mutants, have provided the potential for non-specific resistance to be expressed from pathogen-infection-induced promoters in transgenic plants.
To correlate the classical soybean linkage map with the USDA-ARS molecular map, genes from the classical map must be located relative to molecular markers. The purpose of this study was to integrate the genes for ineffective bradyrhizobia nodulation ( Rj 2), powdery mildew resistance ( Rmd ), and phytophthora root and stem rot resistance ( Rps 2) from classical linkage group 19 with the molecular map. A population of 202 F 2 lines was derived from a cross of two Williams isohnes containing the genes Rj2 Rmd-c Rps2 Ti-a and rj2 rmd rps2 Ti-b . The lines were classified for their reactions to bradyrhizobia, powdery mildew, and phytophthora, and for the Kunitz trypsin mobility variant. Restriction fragment length polymorphism analysis identified three loci from linkage group J (A233, A724, and K375) that were polymorphic between the parent lines. Segregation analysis revealed close linkage between the genes and group J markers, thus correlating linkage group J to classical linkage group 19 The most likely order of the loci was Rps2 Rmd Rj2 A233 A724 K375 (log-likelihood −193 73), with the three genes contained within a 3.8 cM region. This region of the soybean molecular map harbors the densest collection of agronomically important genes identified to date, making it a suitable target for map-based cloning in soybean.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
Plant genes for pathogen resistance can be used to engineer disease resistant crops. Oligonucleotides were designed from sequence motifs conserved between resistance genes of tobacco and Arabidopsis thaliana and used as PCR primers in potato DNA. Amplification products were obtained that were homologous to known resistance genes and linked without recombination with the nematode resistance locus Gro1 and the Phytophthora infestans resistance locus R7 of potato. Map positions of PCR-derived potato gene fragments were also correlated with resistance loci of the related tomato and tobacco genomes. Our results indicate that plant resistance genes that are effective against nematodes, fungi, viruses and bacteria may be isolated based on common sequence motifs and PCR methodology.
Plant disease resistance (R) genes confer an ability to resist infection by pathogens expressing specific corresponding avirulence genes. In Arabidopsis thaliana, resistance to both bacterial and fungal pathogens, mediated by several R gene products, requires the NDR1 gene. Positional cloning was used to isolate NDR1, which encodes a 660-base pair open reading frame. The predicted 219-amino acid sequence suggests that NDR1 may be associated with a membrane. NDR1 expression is induced in response to pathogen challenge and may function to integrate various pathogen recognition signals.
We used conserved domains in the major class (nucleotide binding site plus leucine-rich repeat) of dicot resistance (R) genes to isolate related gene fragments via PCR from the monocot species rice and barley. Peptide sequence comparison of dicot R genes and monocot R-like genes revealed shared motifs but provided no evidence for a monocot-specific signature. Mapping of these genes in rice and barley showed linkage to genetically characterized R genes and revealed the existence of mixed clusters, each harboring at least two highly dissimilar R-like genes. Diversity was detected intraspecifically with wide variation in copy number between varieties of a particular species. Interspecific analyses of R-like genes frequently revealed nonsyntenic map locations between the cereal species rice, barley, and foxtail millet although tight collinear gene order is a hallmark of monocot genomes. Our data suggest a dramatic rearrangement of R gene loci between related species and implies a different mechanism for nucleotide binding site plus leucine-rich repeat gene evolution compared with the rest of the monocot genome.
Classical genetic and molecular data show that genes determining disease resistance in plants are frequently clustered in the genome. Genes for resistance (R genes) to diverse pathogens cloned from several species encode proteins that have motifs in common. These motifs indicate that R genes are part of signal-transduction systems. Most of these R genes encode a leucine-rich repeat (LRR) region. Sequences encoding putative solvent-exposed residues in this region are hypervariable and have elevated ratios of nonsynonymous to synonymous substitutions; this suggests that they have evolved to detect variation in pathogen-derived ligands. Generation of new resistance specificities previously had been thought to involve frequent unequal crossing-over and gene conversions. However, comparisons between resistance haplotypes reveal that orthologs are more similar than paralogs implying a low rate of sequence homogenization from unequal crossing-over and gene conversion. We propose a new model adapted and expanded from one proposed for the evolution of vertebrate major histocompatibility complex and immunoglobulin gene families. Our model emphasizes divergent selection acting on arrays of solvent-exposed residues in the LRR resulting in evolution of individual R genes within a haplotype. Intergenic unequal crossing-over and gene conversions are important but are not the primary mechanisms generating variation.
The co-evolutionary 'arms race' is a widely accepted model for the evolution of host-pathogen interactions. This model predicts that variation for disease resistance will be transient, and that host populations generally will be monomorphic at disease-resistance (R-gene) loci. However, plant populations show considerable polymorphism at R-gene loci involved in pathogen recognition. Here we have tested the arms-race model in Arabidopsis thaliana by analysing sequences flanking Rpm1, a gene conferring the ability to recognize Pseudomonas pathogens carrying AvrRpm1 or AvrB. We reject the arms-race hypothesis: resistance and susceptibility alleles at this locus have co-existed for millions of years. To account for the age of alleles and the relative levels of polymorphism within allelic classes, we use coalescence theory to model the long-term accumulation of nucleotide polymorphism in the context of the short-term ecological dynamics of disease resistance. This analysis supports a 'trench warfare' hypothesis, in which advances and retreats of resistance-allele frequency maintain variation for disease resistance as a dynamic polymorphism.
The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.
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.
Several common themes have shaped the evolution of plant disease resistance genes. These include duplication events of progenitor resistance genes and further expansion to create clustered gene families. Variation can arise from both intragenic and intergenic recombination and gene conversion. Recombination has also been implicated in the generation of novel resistance specificities. Resistance gene clusters appear to evolve more rapidly than other regions of the genome. In addition, domains believed to be involved in recognitional specificity, such as the leucine-rich repeat (LRR), are subject to adaptive selection. Transposable elements have been associated with some resistance gene clusters, and may generate further variation at these complexes.
Genomic approaches are beginning to revolutionize our understanding of plant disease resistance. Large-scale sequencing will reveal the detailed organization of resistance-gene clusters and the genetic mechanisms involved in generating new resistance specificities. Global functional analyses will elucidate the complex regulatory networks and the diversity of proteins involved in resistance and susceptibility.
The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well.
The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.
Plant resistance genes (R genes), especially the nucleotide binding site leucine-rich repeat (NBS-LRR) family of sequences, have been extensively studied in terms of structural organization, sequence evolution and genome distribution. These studies indicate that NBS-LRR sequences can be split into two related groups that have distinct amino-acid motif organizations, evolutionary histories and signal transduction pathways. One NBS-LRR group, characterized by the presence of a Toll/interleukin receptor domain at the amino-terminal end, seems to be absent from the Poaceae. Phylogenetic analysis suggests that a small number of NBS-LRR sequences existed among ancient Angiosperms and that these ancestral sequences diversified after the separation into distinct taxonomic families. There are probably hundreds, perhaps thousands, of NBS-LRR sequences and other types of R gene-like sequences within a typical plant genome. These sequences frequently reside in 'mega-clusters' consisting of smaller clusters with several members each, all localized within a few million base pairs of one another. The organization of R-gene clusters highlights a tension between diversifying and conservative selection that may be relevant to gene families that are unrelated to disease resistance.
The diploid annual legume Medicago truncatula has been developed as a tractable genetic system for studying biological questions that are unique to, or well suited for study in legume species. An efficient mutagenesis protocol using ethyl-methyl sulfonate and a polymorphic ecotype with properties appropriate for use as a mapping parent are described. Isolation and characterization of three developmental mutants are described. The mtapetala mutation results in homeotic conversions of floral organ whorls 2 and 3 into sepals and carpelloid structures, respectively, similar to mutations in the apetala3/pistillata genes of Arabidopsis. The palmyra mutation primarily affects seedling shoot meristem initiation, and thus phenocopies meristem function mutations identified in Arabidopsis such as the zwille locus. The phenotype of the palmyra and mtapetala double mutant is additive, with seedling shoot meristems and floral organs indistinguishable from those of the single palmyra and mtapetala mutants, respectively. These results are consistent with a lack of genetic interaction between these loci. A third mutant, speckle, is characterized by spontaneous necrotic lesion formation on leaves, root, and stems, similar to necrosis mutants identified in other plant species. In addition to documenting the efficient mutagenesis of M. truncatula, the availability of developmental mutants that phenocopy characterized Arabidopsis mutants will provide a basis for establishing orthologous gene function between M. truncatula and Arabidopsis, once the genes responsible are cloned. Moreover, the male-sterile, female-fertile nature of the mtapetala mutant provides a convenient tool for genetic analyses in M. truncatula.
Gene duplication has generally been viewed as a necessary source of material for the origin of evolutionary novelties, but
it is unclear how often gene duplicates arise and how frequently they evolve new functions. Observations from the genomic
databases for several eukaryotic species suggest that duplicate genes arise at a very high rate, on average 0.01 per gene
per million years. Most duplicated genes experience a brief period of relaxed selection early in their history, with a moderate
fraction of them evolving in an effectively neutral manner during this period. However, the vast majority of gene duplicates
are silenced within a few million years, with the few survivors subsequently experiencing strong purifying selection. Although
duplicate genes may only rarely evolve new functions, the stochastic silencing of such genes may play a significant role in
the passive origin of new species.
Phylogenetic relationships among the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance gene homologues (RGHs) from 30 genera and nine families were evaluated relative to phylogenies for these taxa. More than 800 NBS-LRR RGHs were analyzed, primarily from Fabaceae, Brassicaceae, Poaceae, and Solanaceae species, but also from representatives of other angiosperm and gymnosperm families. Parsimony, maximum likelihood, and distance methods were used to classify these RGHs relative to previously observed gene subfamilies as well as within more closely related sequence clades. Grouping sequences using a distance cutoff of 250 PAM units (point accepted mutations per 100 residues) identified at least five ancient sequence clades with representatives from several plant families: the previously observed TIR gene subfamily and a minimum of four deep splits within the non-TIR gene subfamily. The deep splits in the non-TIR subfamily are also reflected in comparisons of amino acid substitution rates in various species and in ratios of nonsynonymous-to-synonymous nucleotide substitution rates ( K(A)/ K(S) values) in Arabidopsis thaliana. Lower K(A)/ K(S) values in the TIR than the non-TIR sequences suggest greater functional constraints in the TIR subfamily. At least three of the five identified ancient clades appear to predate the angiosperm-gymnosperm radiation. Monocot sequences are absent from the TIR subfamily, as observed in previous studies. In both subfamilies, clades with sequences separated by approximately 150 PAM units are family but not genus specific, providing a rough measure of minimum dates for the first diversification event within these clades. Within any one clade, particular taxa may be dramatically over- or underrepresented, suggesting preferential expansions or losses of certain RGH types within particular taxa and suggesting that no one species will provide models for all major sequence types in other taxa.