Isolation,Cloning and Characterization of Resistance Gene Analogues in Pearl Millet Based on Conserved Nucleotide‐binding Sites

Sequence analysis of plant disease resistance genes shows similarity among themselves, with the presence of conserved motifs common to the nucleotide‐binding site (NBS). Oligonucleotide degenerate primers designed from the conserved NBS motifs encoded by several plant disease resistance genes were used to amplify resistance gene analogues (RGAs) corresponding to the NBS sequences from the genomic DNA of various plant species. Using specific primers designed from the conserved NBS regions, 22 RGAs were cloned and sequenced from pearl millet (Pennisetum glaucum L. Br.). Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into nine distinct classes. GenBank database searches with the consensus protein sequences of each of the nine classes revealed their conserved NBS domains and similarity to other known R genes of various crop species. One RGA 213 was mapped onto LG1 and LG7 in the pearl millet linkage map. This is the first report of the isolation and characterization of RGAs from pearl millet, which will facilitate the improvement of marker‐assisted breeding strategies.     

Isolation, characterization and evolutionary analysis of resistance gene analogs in annual ryegrass, perennial ryegrass and their hybrid

Recently, a number of disease-resistance genes related to a diverse range of pathogens were isolated from a wide variety of plant species. The majority of plant disease-resistance genes encoded a nucleotide-binding site (NBS) domain. According to the comparisons of the NBS domain of cloned R -genes, it has shown highly conserved amino acid motifs in this structure, which made it possible to isolate resistance gene analogs (RGAs) by PCR using degenerate primers. We have designed three pairs of degenerate primers based on two conserved motifs in the NBS domain of resistance proteins encoded by R -genes to amplify genomic sequences from ryegrass ( Lolium sp.). Sixteen NBS-like RGAs were isolated from turf and forage type grasses. The sequence analysis of these RGAs revealed that there existed a high similarity (up to 85%) between RGA sequences among ryegrass species and other plants. The alignment of the predicted amino acid sequences of RGAs showed that ryegrass RGAs contained four conserved motifs (P-Loop, kinase-2, kinase-3a, GLPL) present in other known plant NBS-leucine rich repeat resistance genes. These ryegrass RGAs all belonged to non-toll and interleukin-1 receptor subclass. Phylogenetic analysis of ryegrass RGAs and other cloned R -genes indicated that gene mutation was the predominant source of gene variations, and the sequence polymorphism was due to purifying selection rather than diversifying selection. We further analyzed the source of gene variation in other monocots, rice, barley, wheat, and maize based on the data published before. Our analysis indicated that the source of RGA diversity in these monocots was the same as in ryegrass. Thus, monocots were probably the same as dicots in the source of RGA diversity. Ryegrass RGAs in the present paper represented a large group of resistance gene homologs in monocots. We discussed the origin and the evolution of R -genes in grass species.     

Isolation,genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine (<Emphasis Type="Italic">Pinus monticola </Emphasis>Dougl. ex. D. Don.)

Western white pine (Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust (Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.Communicated by R. Hagemann     

Identification and characterization of NBS-LRR class resistance gene analogs in faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.).

A PCR approach with degenerate primers designed from conserved NBS-LRR (nucleotide binding site-leucine-rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from 5 faba bean (Vicia faba) lines and 2 chickpea (Cicer arietinum) accessions. Sixty-nine sequenced clones showed homologies to various R genes deposited in the GenBank database. The presence of internal kinase-2 and kinase-3a motifs in all the sequences isolated confirm that these clones correspond to NBS-containing genes. Using an amino-acid sequence identity of 70% as a threshold value, the clones were grouped into 10 classes of resistance-gene analogs (RGA01 to RGA10). The number of clones per class varied from 1 to 30. RGA classes 1, 6, 8, and 9 were comprised solely of clones isolated from faba bean, whereas classes 2, 3, 4, 5, and 7 included only chickpea clones. RGA10, showing a within-class identity of 99%, was the only class consisting of both faba bean and chickpea clones. A phylogenetic tree, based on the deduced amino-acid sequences of 12 representative clones from the 10 RGA classes and the NBS domains of 6 known R genes (I2 and Prf from tomato, RPP13 from Arabidopsis, Gro1-4 from potato, N from tobacco, L6 from flax), clearly indicated the separation between TIR (Toll/interleukin-1 receptor homology: Gro1-4, L6, N, RGA05 to RGA10)- and non-TIR (I2, Prf, RPP13, RGA01 to RGA04)-type NBS-LRR sequences. The development of suitable polymorphic markers based on cloned RGA sequences to be used in genetic mapping will facilitate the assessment of their potential linkage relationships with disease-resistance genes in faba bean and chickpea. This work is the first to report on faba bean RGAs.     

Origin, diversity and evolution of NBS-type disease-resistance gene homologues in coffee trees (Coffea L.)

The majority of plant disease-resistance genes (R-genes) isolated so far encode a predicted nucleotide-binding site (NBS) domain. NBS domains related to R-genes show a highly conserved backbone of amino acid motifs, which makes it possible to isolate resistance gene analogues (RGAs) by PCR with degenerate primers. Multiple combinations of primers with low degeneracy, designed from two conserved motifs in the NBS regions of R-genes of various plants, were used on genomic DNA from coffee trees, an important perennial tropical crop. Nine distinct classes of RGAs of the NBS-like type, representing a highly diverse sample, were isolated from Coffea arabica and C. canephora species. The analysis of one coffee RGA family suggested point mutations as the primary source of diversity. With one exception, coffee RGA families appeared to be closely related in sequence to at least one cloned R-gene. In addition, deduced amino acid sequences of coffee RGAs were identified that showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)-type R-genes. The high degree of similarity between particular coffee RGAs and R-genes isolated from other angiosperm species, such as Arabidopsis, tomato and rice, indicates an ancestral relationship and the existence of common ancestors. The data obtained from coffee species suggests that the evolution of NBS-encoding sequences involves the gradual accumulation of mutations and slow rates of divergence within distinct R-gene families, rather than being a rapid process. Functional inferences drawn from the suggested pattern of evolution of NBS-type R-genes is also discussed.     

Comparative analysis of superfamilies of NBS-encoding disease resistance gene analogs in cultivated and wild apple species

Eleven distinct families of resistance gene analogs (RGAs) with the characteristic nucleotide-binding sequence (NBS) were identified in two wild apple species, Malus prunifolia and M. baccata, and two cultivated apple cultivars, M. domestica cv. Fuji and M. domestica cv. Hong-ok, using PCR approaches with degenerate primers based on two conserved motifs of known NBS-LRR resistance genes. These RGA families were found to be represented in all the apple species tested, including wild and cultivated species. However, their sequences are very divergent from each other. Furthermore, the low level of recombination detected within their RGA families supports the idea that the evolution of NBS-encoding sequences in apple species involves the gradual accumulation of mutations. Despite the high diversity of the RGA families found in all apple species, the apparent lack of differentiation between wild and cultivated forms suggests that other factors, such as the capacity to tolerate pathogens, might play an important role in the survival of wild-type species.     

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.     

Molecular characterization of NBS-LRR-RGAs in the rose genome

To isolate resistance gene analogues (RGAs) from roses we used various degenerate oligonucleotide primers targeting conserved motifs within the NBS region of nucleotide binding site (NBS)-leucine-rich repeat (LRR) resistance genes. A large RGA sublibrary consisting of 7000 clones was constructed. This sublibrary contains at least 40 unique RGA families of the TIR (toll-/interleukin-1 receptor) and the LZ (leucine zipper) type, which were further analysed. Phylogenetic studies revealed close relationships of some rose RGAs to R genes and RGAs from other plants and gave rise to the assumption that rose R genes evolved from different starting points, prior to and subsequent to speciation. Southern blot analyses showed that the RGAs were organized as single, low and multicopy loci in the rose genome. None of the analysed sequences detected any hybridization signal in Prunus cérasus indicating that the analysed RGAs are not conserved across genera. The efficiency and selectivity of the different degenerate primers used for the RGA isolation is discussed in detail.     

Isolation, genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine ( Pinus monticola Dougl. ex. D. Don.)

Western white pine ( Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust ( Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.     

Efficient targeting of plant disease resistance loci using NBS profiling

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50–90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.Communicated by H.F. Linskens     

三浅裂野牵牛NBS类抗病基因同源序列的克隆与分析

NBS类植物抗病基因保守结构域的克隆为利用简并引物扩增抗病基因同源序列提供了可能.根据抗病基因Gro1-4、Gpa2、N等的P-loop和GLPL保守结构域设计简并引物,分离甘薯近缘野生种三浅裂野牵牛NBS类型抗病基因同源序列,共获得6条相关序列,核苷酸序列的相似性为48%~97%,推测氨基酸序列的相似性在25.2%~95.1%之间.系统进化分析表明,6条三浅裂野牵牛RGA序列可分为2个不同的类群:TIR-NBS和non-TIR-NBS.三浅裂野牵牛RGA序列与源自甘薯的RGA序列有很高的相似性,这在一定程度上反映了三浅裂野牵牛与甘薯之间的亲缘关系.分离的6条RGA序列分别命名为ItRGA1~ItRGA6,GenBank登录号分别为DQ849027~DQ849032.     

Genetic Analysis of Resistance Gene Analogues from a Sugarcane Cultivar Resistant to Red Rot Disease

One of the important approaches for disease control in sugarcane is to develop a disease‐resistant variety; this may be accomplished through identification of resistance genes in sugarcane. In this study, PCR primers targeting the conserved motifs of the nucleotide‐binding site (NBS) class and kinase class of the resistance gene analogues (RGAs) were used to amplify the RGAs from a red rot‐resistant sugarcane cultivar (Saccharum spp. hybrid) HSF 240. Upon subcloning and sequencing, fifteen putative RGAs were identified. These RGAs shared 63–98% identity to the reported disease‐resistant genes in the NCBI GenBank database. Deduced amino acid sequences also showed the presence of expected conserved domains characteristic of RGAs. Phylogenetic analysis indicated that these RGAs clustered with R genes from other plant species. The findings will be useful for studying disease‐resistant genes in sugarcane.     

小麦NBS-LRR类抗病基因同源序列的分离与鉴定

根据已知植物抗病基因的保守区域设计引物,从抗锈病小麦品种西农88基因组DNA扩增出3条与植物抗病基因同源的序列,分别为WRGA1、WRGA2和WRGA14。这三条同源片段均含有典型的NBS-LRR类抗病基因所拥有的保守性结构域Kinase-2a、Kinase-3a和疏水结构域(HD)．它们与部分已知NBS-LRR类抗病基因的氨基酸序列同源性为46．0%-9．9%,三个片段间在氨基酸水平上的同源性为80．7%-56．8%。Northern杂交表明WRGA1在小麦中受水杨酸正调控,属诱导型表达。     

Isolation and characterization of nucleotide-binding site and C-terminal leucine-rich repeat-resistance gene candidates in bananas

Commercial banana varieties are highly susceptible to fungal pathogens, as well as bacterial pathogens, nematodes, viruses, and insect pests. The largest known family of plant resistance genes encodes proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for the isolation of candidate genes in banana that may be involved in plant defense. Six degenerate PCR primers were designed to target NBS and additional domains were tested on commercial banana species Musa acuminata subsp malaccensis and the Musa AAB Group propagated in vitro and plants maintained in a greenhouse. Total DNA was isolated by a modified CTAB extraction technique. Four resistance gene analogs were amplified and deposited in GenBank and assigned numbers HQ199833-HQ199836. The predicted amino acid sequences compared to the amino acid sequences of known resistance genes (MRGL1, MRGL2, MRGL3, and MRGL4) revealed significant sequence similarity. The presence of consensus domains, namely kinase-1a, kinase-2 and hydrophobic domain, provided evidence that the cloned sequences belong to the typical non-Toll/interleukin-1 receptor-like domain NBS-LRR gene family.     

Diversity and evolutionary relationship of nucleotide binding site-encoding disease-resistance gene analogues in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> Lam.)

Most plant disease-resistance genes (R-genes) isolated so far encode proteins with a nucleotide binding site (NBS) domain and belong to a superfamily. NBS domains related to R-genes show a highly conserved backbone of an amino acid motif, which makes it possible to isolate resistance gene analogues (RGAs) by degenerate primers. Degenerate primers based on the conserved motif (P-loop and GLPL) of the NBS domain from R -genes were used to isolate RGAs from the genomic DNA of sweet potato cultivar Qingnong no.2. Five distinct clusters of RGAs (22 sequences) with the characteristic NBS representing a highly diverse sample were identified in sweet potato genomic DNA. Sequence identity among the 22 RGA nucleotide sequences ranged from 41.2% to 99.4%, while the deduced amino acid sequence identity from the 22 RGAs ranged from 20.6%to 100%. The analysis of sweet potato RGA sequences suggested mutation as the primary source of diversity. The phylogenetic analyses for RGA nucleotide sequences and deduced amino acids showed that RGAs from sweet potato were classified into two distinct groups--toll and interleukin receptor-1 (TIR)-NBS-LRR and non-TIR-NBS-LRR. The high degree of similarity between sweet potato RGAs and NBS sequences derived from R-genes cloned from tomato, tobacco, flax and potato suggest an ancestral relationship. Further studies showed that the ratio of non-synonymous to synonymous substitution within families was low. These data obtained from sweet potato suggest that the evolution of NBS-encoding sequences in sweet potato occur by the gradual accumulation of mutations leading to purifying selection and slow rates of divergence within distinct R-gene families.     

云南悬钩子蔷薇NBS LRR类抗病基因同源克隆与分析

为研究云南野生蔷薇属中的NBS类抗病基因,根据已知抗病基因NBS LRR序列中的保守区域设计简并引物,利用RT PCR技术从云南悬钩子蔷薇中进行体外扩增,获得了对应区域的cDNA片段,回收、克隆这些特异片段,测序分析,共得到4个含有NBS LRR保守结构域的抗病基因同源序列(RGAs),分别命名为AC9、AC39、AC50和AC68。它们与已报道的11个NBS类抗病基因相应区段的氨基酸序列相似性为5.4%~79.2%,其中这4个RGAs片段与Mi、RPS2、Pib和RPM1基因聚为一类。表明这4条RGAs序列可进一步用作悬钩子蔷薇抗病候选基因的分子筛选及遗传图谱的构建。     

PCR-based approach for mining of resistant gene analogues in taro (Colocasia esculenta)

Primers based on the conserved motifs were used to isolate nucleotide-binding sites (NBS) type sequences in taro (Colocasia esculenta). Cloning and sequencing identified three taro NBS-type sequences called resistance gene analogues (RGAs) that depicted similarity to other cloned RGA sequences. The deduced amino acid sequences of the RGAs detected the presence of conserved domains, viz. P-loop, categorising them with the NBS–leucine-rich repeat class gene family. Phylogenetic characterisation of the taro RGAs along with RGAs of other plant species grouped them with the non-toll interleukin receptor subclasses of the NBS sequences. The isolation and characterisation of taro RGAs have been reported for the first time in this study. This will provide a starting point towards characterisation of candidate resistance genes in taro and can act as a reference guide for future studies.     

花生RGA 片段的克隆及初步分析

目的:通过同源克隆获得了花生闽花6号的RGA片段,为其抗性的研究及抗性育种提供了参考资料。方法:试验分为两组:其一通过利用抗性基因的NBS保守区所设计的简并引物对花生品种闽花六号进行了RGA片段扩增,其二结合已登录的花生RGA片段序列经过多元比对后设计简并引物进行RGA片段的扩增及序列分析;分析比较两组克隆方法的效果。结果:测序分析表明:前者20条随机测序序列中没有一条与已知RGA片段序列相似;后者20条随机测序序列中有18条为RGA片段序列,其登录号为GenBankEU639668-EU639685。结论:前一种方法克隆扩增RGA基因片段的效率很低,而后一种方法克隆扩增效果更好,这为闽花6号花生的遗传改良提供了理论基础。     

Resistance gene analogues of chickpea ( Cicer arietinum L.): isolation,genetic mapping and association with a Fusarium resistance gene cluster

Resistance gene analogues (RGAs) of Cicer were isolated by different PCR approaches and mapped in an inter-specific cross segregating for fusarium wilt by RFLP and CAPS analysis. Initially, two pairs of degenerate primers targeting sequences encoded at nucleotide-binding sites (NBS), which are conserved in plant disease resistance genes such as RPS2, L6 and N, were selected for amplification. Cloning and sequence analysis of amplified products from C. arietinum DNA revealed eight different RGAs. Additionally, five RGAs were identified after characterisation of the presumptive RGA alleles from C. reticulatum. Therefore, a total of 13 different RGAs were isolated from Cicer and classified through pair-wise comparison into nine distinct classes with sequence similarities below a 68% amino acid identity threshold. Sequence comparison of seven RGA alleles of C. arietinum and C. reticulatum revealed polymorphisms in four RGAs with identical numbers of synonymous and non-synonymous substitutions. An NlaIII site, unique in the RGA-A allele of C. arietinum, was exploited for CAPS analysis. Genomic organisation and map position of the NBS-LRR candidate resistance genes was probed by RFLP analysis. Both single-copy as well as multi-copy sequence families were present for the selected RGAs, which represented eight different classes. Five RGAs were mapped in an inter-specific population segregating for three race-specific Fusarium resistances. All RGAs mapped to four of the previously established eight linkage groups for chickpea. Two NBS-LRR clusters were identified that could not be resolved in our mapping population. One of these clusters, which is characterised by RFLP probe CaRGA-D, mapped to the linkage group harbouring two of three Fusarium resistance genes characterised in the inter-specific population. Our study provides a starting point for the characterisation and genetic mapping of candidate resistance genes in Cicer that is useful for marker-assisted selection and as a pool for resistance genes of Cicer.     

Resistance Gene Analogues as a Tool for Rapid Identification and Cloning of Disease Resistance Genes in Plants 3 A Review

Most of the disease resistance genes (R-genes) discovered in plants have conserved functional domains, predominantly among them are nucleotide binding sites (NBS) and leucine rich repeats (LRR). The sequence information of the conserved domains can be invariably used to mine similar sequences from other plant species, using degenerate and specific primers for their amplification in a polymerase chain reaction. Such derived sequences, known as Resistance Gene Analogues (RGAs), can serve as molecular markers for rapid identification and isolation of R-genes. Besides, they can also provide clues about the evolutionary mechanism of resistance genes and the interaction involved in pathogen recognition. In the recent years, this sequence-homology based approach has been used extensively for the cloning and mapping of RGAs in cereals, pulses, oilseeds, coffee, spices, forest trees and horticultural crops. In this article, the current status of cloning of RGAs from different crops has been reviewed. A general method of RGA cloning and its modifications like NBS-profiling and AFLP-NBS have also been discussed along with examples. Further, it has been suggested that the RGAs cloned in various crops would be a useful genomic resource for developing cultivars with durable resistance to diseases in different crop breeding programmes.