植物抗病基因同源序列及其在抗病基因克隆与定位中的应用

近10年来已有20多个植物抗病基因被克隆,测序,这些抗病基因所编码的蛋白中大多含有核苷酸结合位点,富含亮氨酸重复序列,蛋白激酶,亮氨酸拉链结构,跨膜结构域,Toll白介素－1区域等保守结构域。利用这些保守结构域合成PCR引物,已扩增出大量的植物抗病基因同源序列（RGA）。对RGA与抗病基因的关系进行了分析,讨论了RGA在研究抗病基因进化中的作用,指出RGA在抗病基因定位和转基因中具有重要意义。     

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.     

Disease resistance gene analogs as candidates for QTLs involved in pepper-pathogen interactions.

Whereas resistance genes (R-genes) governing qualitative resistance have been isolated and characterized, the biological roles of genes governing quantitative resistance (quantitative trait loci, QTLs) are still unknown. We hypothesized that genes at QTLs could share homologies with cloned R-genes. We used a PCR-based approach to isolate R-gene analogs (RGAs) with consensus primers corresponding with conserved domains of cloned R-genes: (i) the nucleotide binding site (NBS) and hydrophobic domain, and (ii) the kinase domain. PCR-amplified fragments were sequenced and mapped on a pepper intraspecific map. NBS-containing sequences of pepper, most similar to the N gene of tobacco, were classified into seven families and all mapped in a unique region covering 64 cM on the Noir chromosome. Kinase domain containing sequences and cloned R-gene homologs (Pto, Fen, Cf-2) were mapped on four different linkage groups. A QTL involved in partial resistance to cucumber mosaic virus (CMV) with an additive effect was closely linked or allelic to one NBS-type family. QTLs with epistatic effects were also detected at several RGA loci. The colocalizations between NBS-containing sequences and resistance QTLs suggest that the mechanisms of qualitative and quantitative resistance may be similar in some cases.     

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.     

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.     

Resistance Gene Analog Polymorphism (RGAP) Markers Co-Localize with Disease Resistance Genes and QTL in Common Bean

Resistance (R) genes containing nucleotide-binding site (NBS)-leucine rich repeats (LRR) are the most prevalent types of R gene in plants. The objective of this study was to develop PCR-based R-gene analog polymorphism (RGAP) markers for common bean (Phaseolus vulgaris L). Twenty degenerate primers were designed from the conserved kinase-1a (GVGKTT) and hydrophobic domains (GLPLAL) of known NBS-LRR type R-genes and from EST databases. Sixty-six of the 100 primer combinations tested yielded polymorphism. Thirty-two RGAP markers were mapped in the BAT 93/Jalo EEP558 core mapping population for common bean. The markers mapped to 10 of 11 linkage groups with a strong tendency for clustering. In addition, the RGAP markers co-located, on six linkage groups, with 15 resistance gene analogs (RGAs) that were previously mapped in other populations of common bean. The distance between the priming sites in NBS-LRR type R-genes is around 500 bp. Of the 32 RGAP markers, 19 had sizes larger and 13 less than 500 bp. RGAP markers mapped close to known R-genes on B11, and to QTLs for resistance on B1, B2, B6, B7, B8, B10, and B11. RGAP appears to provide a useful marker technique for tagging and mapping R-genes in segregating common bean populations, discovery of candidate genes underlying resistance QTL, and future cloning of R-genes in common bean.     

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.     

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.     

Resistance gene analogs are candidate markers for disease-resistance genes in grape ( Vitis spp.)

A set of NBS-containing sequences was isolated from genomic DNA of two grape species ( Vitis amurensis and Vitis riparia) and characterised in a panel of Vitis genotypes carrying different levels of resistance against downy mildew and other diseases. A PCR-mediated approach made use of degenerate primers designed on conserved regions encoding known R-genes, and provided the source for cloning grape analogous sequences. Cloned sequences were digested with ten endonucleases and 29 out of 71 putative recombinant clones, which showed unique restriction patterns, were sequenced. Using a threshold value of 40% identity, at least 12 grape NBS-sequences had a high overall similarity with known R-genes, such as the Arabidopsis gene RPS5 and the tobacco gene N. The presence of internal conserved motifs provided evidence that sequences isolated from grape may belong to the NBS-LRR gene family. A cluster analysis based on the deduced amino acid sequence and carried out on grape NBS-sequences, together with several analogous domains of known R-genes, classified grape sequences into three major groups. A grape sequence of each group was used as a probe on Southern blots with digested genomic DNA from resistant and susceptible grapes. One of the NBS-containing probes showed a clear-cut separation between resistant species and susceptible varieties. This evidence makes the probe a candidate marker for disease resistance genes in Vitis germplasm.     

Isolation and characterization of resistance gene analogues from Psilanthus species that represent wild relatives of cultivated coffee endemic to India

Biotic or abiotic stress can cause considerable damage to crop plants that can be managed by building disease resistance in the cultivated gene pool through breeding for disease resistance genes (R-genes). R-genes, conferring resistance to diverse pathogens or pests share a high level of similarity at the DNA and protein levels in different plant species. This property of R-genes has been successfully employed to isolate putative resistance gene analogues (RGAs) using a PCR-based approach from new plant sources. Using a similar approach, in the present study, we have successfully amplified putative RGAs having nucleotide-binding-site leucine-rich repeats (NBS-LRR-type RGAs) from seven different sources: two cultivated coffee species (Coffea arabica L. and Coffea canephora Pierre ex. A. Froehner), four related taxa endemic to India (wild tree coffee species: Psilanthus bengalensis (Roem. & Schuttles) J.-F. Leroy, Psilanthus khasiana , Psilanthus travencorensis (Wight & Arn.) J.-F. Leroy, Psilanthus weightiana (Wall. ex Wight & Arn.) J.-F. Leroy), and a cDNA pool originally prepared from light- and drought-stressed Coffea arabica L. leaves. The total PCR amplicons obtained using NBS-LRR-specific primers from each source were cloned and transformed to construct seven independent libraries, from which 434 randomly picked clones were sequenced. In silico analysis of the sequenced clones revealed 27 sequences that contained characteristic RGA motifs, of which 24 had complete uninterrupted open reading frames. Comparisons of these with published RGAs showed several of these to be novel RGA sequences. Interestingly, most of such novel RGAs belonged to the related wild Psilanthus species. The data thus suggest the potential of the secondary gene pool as possible untapped donors of resistance genes to the present day cultivated species of coffee.     

甘薯NBS类抗病基因类似物的分离与序列分析

利用已克隆植物抗病基因NBS（Nucleotide binding site）序列中的保守模体（motif）“P-loop”和“GLPL”合成简并引物,以甘薯（Ipomoea batatas）栽培品种青农2号基因组DNA为模板进行PCR扩增,通过T/A克隆、测序和序列分析,共得到15条具有连续ORF的抗病基因类似物（Resistance gene analogues,RGAs）序列,它们之间核苷酸序列间的相似性系数在41.2%-99.4%之间,而相应推测的氨基酸序列间的相似性系数在20.6%-100%之间,同时对分离的RGAs的核苷酸和氨基酸序列进行系统发育树分析,表明甘薯RGAs可分为TIR（Drosophila Toll or human interleukin receptor-like）和nonTIR两类.对甘薯RGAs和5个已克隆植物NBS的氨基酸序列进行结构分析表明,它们包括“P-loop”、“Kinase-2”、“Kinase-3a”、“GLPL”4个抗病基因所共有的保守模体.这些表明甘薯与其它物种的NBS类RGAs可能具有同样的起源和进化机制.     

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     

甜瓜STK类R基因同源序列的克隆与分析

以植物丝氨酸/苏氨酸蛋白激酶类( serine-threonine kinase,STK)抗病基因产物催化结构域I和Ⅸ的保守氨基酸序列( FGK/V/L/SVYK/RG,DY/IYSF/YGV/I/M)设计简并引物,对甜瓜(Cucumis melo L.)基因组DNA进行PCR扩增,得到大约500 bp的目的条带,通过重组质粒克隆并经PCR检测后得到12条不同的DNA序列,命名为tg1～tg12,其中tg2、tg5、tg9和tg12(Genbank登录号为JN646853 ～JN646856)可以编码完整的氨基酸序列.Blast分析结果显示:4条序列均具有ATP结合部位、底物结合部位和激酶结构域的活化环(A-loop)等,属于典型的蛋白激酶基因家族,可能是STK类R基因的同源序列片段;4条序列与蓖麻(Ricinus communisL.)的STK同源性均较高.氨基酸序列比对结果显示tg2、tg5、tg9和tg12均具有R基因的9个保守结构域,为STK类候选抗病基因类序列.分子系统树显示tg2、tg5、tg9和tg12与已知的R基因(Pto、Lr10和Lectin)在氨基酸水平上的相似性仅为33.5％ ～53.4％,且4个甜瓜同源序列的氨基酸相似性也较低,表明甜瓜RGAs标记可能具有较高的特异性.     

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.     

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species.

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.     

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.     

Organization,expression and evolution of a disease resistance gene cluster in soybean

PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar "Williams 82" [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca(2+)-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.     

Characterization and linkage mapping of R-gene analogous DNA sequences in pea (Pisum sativum L.)

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. Received: 4 August 1999 / Accepted: 11 November 1999     

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.     

Targeted isolation,sequence analysis,and physical mapping of nonTIR NBS-LRR genes in soybean

Most cloned plant disease resistance genes (R-genes) code for proteins belonging to the nucleotide binding site (NBS) leucine-rich repeat (LRR) superfamily. NBS-LRRs can be divided into two classes based on the presence of a TIR domain (Toll and interleukin receptor-like sequence) or a coiled coil motif (nonTIR) in their N-terminus. We used conserved motifs specific to nonTIR-NBS-LRR sequences in a targeted PCR approach to generate nearly 50 genomic soybean sequences with strong homology to known resistance gene analogs (RGAs) of the nonTIR class. Phylogenetic analysis classified these sequences into four main subclasses. A representative clone from each subclass was used for genetic mapping, bacterial artificial chromosome (BAC) library screening, and construction of RGA-containing BAC contigs. Of the 14 RGAs that could be mapped genetically, 12 localized to a 25-cM region of soybean linkage group F already known to contain several classical disease resistance loci. A majority of the genomic region encompassing the RGAs was physically isolated in eight BAC contigs, together spanning more than 1 Mb of genomic sequence with at least 12 RGA copies. Phylogenetic and sequence analysis, together with genetic and physical mapping, provided insights into the genome organization and evolution of this large cluster of soybean RGAs. Received: 8 May 2001 / Accepted: 30 June 2001