Identification and characterization of NBS–LRRclass resistance gene analogs in faba bean (Viciafaba L.) and chickpea (Cicer arietinum L.)

C. Palomino, Z. Satovic, J.I. Cubero, and A.M. Torres

Abstract: 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 5faba bean (Vicia faba) lines and 2 chickpea (Cicer arietinum) accessions. Sixty-nine sequenced clones showed homologiesto various R genes deposited in the GenBank database. The presence of internal kinase-2 and kinase-3a motifs in all the se-quences isolated confirm that these clones correspond to NBS-containing genes. Using an amino-acid sequence identitiy of70% as a threshold value, the clones were grouped into 10 classes of resistance-gene analogs (RGA01 to RGA10). Thenumber of clones per class varied from 1 to 30. RGA classes 1, 6, 8, and 9 were comprised solely of clones isolatedfrom faba bean, whereas classes 2, 3, 4, 5, and 7 included only chickpea clones. RGA10, showing a within-classidentity of 99%, was the only class consisting of both faba bean and chickpea clones. A phylogenetic tree, based onthe deduced amino-acid sequences of 12 representative clones from the 10 RGA classes and the NBS domains of 6known R genes (I2 and Prf from tomato, RPP13 from Arabidopsis, Gro1–4 from potato, N from tobacco, L6 fromflax), clearly indicated the separation between TIR (Toll/interleukin-1 receptor homology: Gro1–4, L6, N, RGA05 toRGA10)- and non-TIR (I2, Prf, RPP13, RGA01 to RGA04)-type NBS–LRR sequences. The development of suitablepolymorphic markers based on cloned RGA sequences to be used in genetic mapping will facilitate the assessment oftheir potential linkage relationships with disease-resistance genes in faba bean and chickpea. This work is the first toreport on faba bean RGAs.

Key words: disease resistance, NBS–LRR resistance gene analogs (RGAs), Vicia faba L., Cicer arietinum L., multiple se-quence alignment, genetic mapping.

Resume : Une approche PCR a l’aide d’amorces degenerees, inspirees des regions NBS–LRR (site de liaison nucleoti-dique – repetition riche en leucines) de genes de resistance (R) connus, a ete employee pour amplifier et cloner des se-quences homologues chez 5 varietes de feverole (Vicia faba) et 2 accessions de pois chiche (Cicer arietinum). Soixante-neuf clones sequences montraient de l’homologie a divers genes R presents au sein de GenBank. La presence de motifs in-ternes kinase-2 et kinase-3a chez toutes les sequences a confirme que ces clones correspondent a des genes a motif NBS.En prenant pour seuil une identite de 70 % de la sequence peptidique, les clones ont ete groupes en 10 classes d’analoguesde genes de resistance (RGA01 a RGA10). Le nombre de clones par classe variait entre 1 et 30. Les classes RGA 1, 6, 8et 9 comprenaient uniquement des clones provenant de la feverole tandis que les classes 2, 3, 4, 5 et 7 n’incluaient quedes clones provenant du pois chiche. La classe RGA10 affichait une identite intra-classe de 99 % et elle etait la seule com-prenant des clones de la feverole et du pois chiche. Un arbre phylogenetique fonde sur les sequences peptidiques preditesde 12 clones representatifs au sein des 10 classes RGA ainsi que sur les domaines NBS de 6 genes R connus (I2 et Prf dela tomate, RPP13 d’Arabidopsis, Gro1-4 de la pomme de terre, N du tabac et L6 du lin) a permis d’etablir clairement unedistinction entre les sequences NBS–LLR de type TIR (Toll/Interleukin-1 Receptor : Gro1-4, L6, N, RGA05 a RGA10) etde type non-TIR (I2, Prf, RPP13, RGA01 a RGA04). Le developpement de marqueurs polymorphes a partir des sequencesclonees de RGA permettra de les placer sur une carte genetique et facilitera l’examen de leur liaison potentielle avec desgenes de resistance chez la feverole et le pois chiche. Le present travaille est le premier a decrire des RGA chez la feve-role.

Received 13 January 2006. Accepted 21 May 2006. Published on the NRC Research Press Web site at http://genome.nrc.ca on21 December 2006.

Corresponding Editor: D.J. Somers.

C. Palomino1 and J.I. Cubero. Departamento de Genetica, E.T.S.I.A.M, Universidad de Cordoba, 14071 Cordoba, Spain.Z. Satovic. Department of Seed Science and Technology, Faculty of Agriculture, University of Zagreb, Svetosimunska 25,10000 Zagreb, Croatia.A.M. Torres. Area de Mejora y Biotecnologıa, IFAPA-C.I.C.E. (Junta de Andalucıa) CIFA ‘Alameda del Obispo’, Apdo. 3092,14080 Cordoba, Spain.

1Corresponding author (e-mail: carmen.palomino.ext@juntadeandalucia.es).

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Genome 49: 1227–1237 (2006) doi:10.1139/G06-071 # 2006 NRC Canada

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Mots cles : resistance aux maladies, analogues de genes de resistance (RGA) de type NBS–LRR, Vicia faba L., Cicer arie-tinum L., alignement de sequences multiples, cartographie genetique.

[Traduit par la Redaction]

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IntroductionPlant diseases caused by fungi, viruses, nematodes, bacte-

ria, and insects present severe threats to crop yields world-wide. The fight against plant pathogens is mostly based ona combination of farming practices, chemical treatments,and the use of disease-resistant cultivars. Breeding for dis-ease resistance is one of the top priorities in plant improve-ment. Molecular marker technologies have located andmapped resistance genes in many species, facilitating ge-netic analysis and transfer into desirable lines or cultivars.In recent years, a large number of disease-resistance (R)genes that confer resistance on a diverse spectrum of patho-gens have been isolated from a wide range of plant species(Boyes et al. 1998; Cooley et al. 2000; Gassmann et al.1999; Tai et al. 1999). These genes share striking structuralsimilarities, suggesting that certain signalling events arecommon to all or most plant defense systems. The mostcommon class of plant R genes cloned so far belong to theNBS–LRR group, which contains nucleotide-binding sites(NBS) and a C-terminal leucine-rich repeat (LRR) of varia-ble length. The NBS domain includes a number of amino-acid motifs, most notably the P-loop, kinase-2, kinase-3a,and GLPL motifs, which are highly conserved in most ofthe characterized R-gene products, such Arabidopsis RPS2(Bent et al. 1994; Mindrinos et al. 1994), tobacco N(Whitham et al. 1994), and flax L6 (Lawrence et al. 1995).The R genes encode cell receptors that detect the presenceof specific pathogens (Bent 1996; Hammond-Kosack andJones 1997) and respond by activating signal transductionpathways. Nucleotide triphosphate binding is thought to alterthe interaction between R proteins and other proteins actingdownstream in the cascade (Bent 1996). LRR domains me-diate protein–protein interactions, and are the major determi-nants of recognition specificity (Fluhr 2001).

Plant NBS–LRR proteins can be classified into 2 majorgroups, on the basis of the presence or absence in the com-plete protein of an N-terminal region homologous to theToll and interleukin receptor-like regions (TIR) (Meyers etal. 1999; Pan et al. 2000; Young 2000). The non-TIR pro-teins often contain a N-terminal coiled-coil (CC) or leucinezipper (LZ) motifs, which might play a role in the interac-tion with molecules downstream in the signal transductionpathway (Pan et al. 2000). So far, TIR-type genes have notbeen found in monocot genomes, whereas non-TIR sequen-ces are present in both dicots and monocots (Meyers et al.1999).

A number of studies have used PCR with degenerate oli-gonucleotide primers designed from these short conservedmotifs of cloned plant R genes to amplify multiple DNA se-quences in plant species (Aarts et al. 1998; Deng et al. 2000;Kanazin et al. 1996; Leister et al. 1996; Seah et al. 1998;Shen et al. 1998; Speulman et al. 1998; Wang and Xiao2002; Yaish et al. 2004; Yu et al. 1996). These sequenceshave been called resistance-gene analogs (RGAs) (Kanazin

et al. 1996) or resistance-gene candidates (RGCs) (Shen etal. 1998). Genetic analyses have associated a number ofthese sequences to known genes that confer resistance to vi-ruses, bacteria, fungi, and nematodes (Aarts et al. 1998; Denget al. 2000; Seah et al. 1998; Shen et al. 1998; Speulman etal. 1998; Wang and Xiao 2002; Yaish et al. 2004; Yu et al.1996). Moreover, mapping studies of RGAs have providedevidence that they cosegregate with fungal disease-resistancemarkers (Aarts et al. 1998; Meyers et al. 1999). Conse-quently, this approach provides an attractive strategy to iso-late multiple resistance-gene candidate sequences that can beused to develop molecular markers useful in marker-assistedselection or even lead to molecular cloning of previously un-known disease-resistance genes (Deng et al. 2000).

The faba bean (Vicia faba L.) is the third most importantcool-season food legume in the world, after chickpea andpea. It is grown in many temperate areas and is used as adry bean for food and livestock feed and as a cooked vege-table for human consumption. The chickpea (Cicer arieti-num L.) is mainly cultivated in semi-arid areas of India,East Africa, the Mediterranean countries, North America,Latin America, and Australia. It is a primary human dietaryprotein source in developing countries where it is consumedin various forms. Recent advances in the development of ge-netic maps of faba bean and chickpea have allowed theidentification of genes and quantitative trait loci (QTLs) forresistance to parasitic plants (Orobanche crenata) (Roman etal. 2002, 2003) and fungal diseases, such as ascochytablight, rust, and fusarium wilt (Avila et al. 2003, 2004; Co-bos et al. 2005; Millan et al. 2003; Rubio et al. 2003; Santraet al. 2000; Winter et al. 2000), together with other relevantagronomic traits in both species.

The aim of this study was to isolate and characterize theNBS–LRR class of RGAs sequences from faba bean andchickpea, using degenerate oligonucleotide primers basedon conserved motifs of known R genes. The final objectiveis to design specific primers for these sequences for use indeveloping markers, such as RGC-based cleaved amplifiedpolymorphic sequences, for resistance-gene tagging andmapping. The combined analysis in both crops should facili-tate the saturation of current genetic maps developed by ourgroup and the identification of macrosynthenic relationshipsbetween disease-resistance regions in the genomes of the 2species.

Materials and methods

Plant material and DNA extractionPlant genomic DNA for PCR amplification was extracted

from 5 V. faba lines (Vf6, Vf136, 29H, 2N52, and Vf176)and 2 accessions of C. arietinum (ILC3279 and WR315).All materials are parental lines that have been used in pre-vious mapping studies by our group. Progenies derivedfrom these lines have allowed the detection of genes and

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QTLs for important agronomic traits, such as seed weight orresistance to broomrape, Fusarium, Uromyces, and Asco-chyta (Avila et al. 2003, 2004; Millan et al. 2003; Romanet al. 2002, 2003; Rubio et al. 2003). Total DNA was ex-tracted from young leaf tissue using the extraction methoddescribed by Torres et al. (1993). DNA samples were quan-tified visually with electrophoresis in 0.8% agarose gels, us-ing known concentrations of uncut � DNA as a standard.

Degenerate primers and PCR conditionsPrimers used in this study (Table 1) were synthesized

from the sequences described by Leister et al. (1996). Pri-mers s1 and s2 were designed in the sense direction, corre-sponding to the amino-acid sequence GVGKTT found in theP-loop of N, L6, and RPS2 genes. Primers as1, as3, and as4were designed in the antisense direction, based on the se-quence GLPAL (hydrophobic region in N, L6, and RPS2).PCRs for all parental lines were performed in a total volumeof 25 mL, containing 50 ng of genomic DNA, 1� PCR buf-fer, 2 mmol/L MgCl2, 0.1 mmol/L dNTPs, 0.25 mmol/L ofeach primer, and 1 U Taq polymerase (Biotools, Madrid,Spain). Six combinations of sense and antisense primerswere tested: I, s1/as1; II, s2/as3; III, s2/as4; IV, s1/as3; V,s1/as4; and VI, s2/as1. Amplifications were carried out in agradient thermocycler (T Gradient PCR, Biometra). The op-timum annealing temperature was determined for each pri-mer pair analysed.

Cycling conditions consisted of an initial denaturationstep at 94 8C for 3 min, followed by 35 amplificationcycles at 94 8C for 1 min, 46 8C for 30 s, and 72 8C for30 s. The size of the amplified fragments was determinedusing 1% w/v SeaKem agarose, 1% w/v NuSieve agarosegels in 1� TBE buffer. The amplification products were vi-sualized with ethidium bromide staining.

Cloning and sequencing of the PCR productsPCR products of the expected size (510–550 bp, in ac-

cordance with the R-gene sequences of RPS2, N, and L6)were excised from the gels. DNA was extracted using theQIAquick Gel Extraction kit (Qiagen, Hilden, Germany).PCR purified products were cloned into the pGEM-T vectorsystem (Promega, Madison, Wis.) and transformed intocompetent Escherichia coli JM109 cells, in accordance withthe supplier’s protocol. Between 20 and 40 clones derivedfrom each expected band and parental line were randomlyselected. Clone names consisted of a letter (A to E forV. faba parental lines and F or G for C. arietinum) followedby the primer combination used (I–VI) and the clone num-ber. Positive clones were selected by PCR amplification ofthe inserts, using universal forward and reverse primers.

Clones were then characterized, using restriction analyseswith 3 enzymes (RsaI, HaeIII, and HpaII), and grouped bytheir restriction fragment patterns. After visual inspectionon 1% agarose, 1% NuSieve gels, at least 3 clones represen-tative of each group were selected for sequencing. Plasmidswere extracted using a lysis-by-boiling miniprep protocol.Double-stranded plasmid DNA was sequenced (forward andreverse) using a BigDye terminator cycle sequencing version3.1 kit (PE Biosystems, Foster City, Calif.) on an ABI Prism3100 Genetic Analyzer apparatus (Applied Biosystems, Fos-ter City, Calif.) at the Servicio de Secuenciacion Automaticade DNA, SCAI (University of Cordoba, Spain). Sequenceediting and analysis were conducted with the BioEdit ver.7.0.1 software program (Hall 1999).

Sequences reported here have been assigned GenBank ac-cession Nos. DQ276887 to DQ276955.

Sequence alignment and phylogenetic analysisSequences with conserved NBS domains and homology to

resistance genes or resistance-gene analogs were identifiedusing the BLAST web page (http://www.ncbi.nlm.nih.gov/blast) of the National Center of Biotechnology Information(NCBI, Bethesda, Md.).

Multiple alignment of nucleotide and translated protein se-quences was carried out using the ClustalW system (Higginset al. 1994), as implemented in MEGA ver. 3.0 (Kumar et al.2004). MEGA3 was also used to calculate p-distances be-tween sequences at the amino-acid level and to construct theUPGMA tree. A threshold value for amino-acid identities of70% was used to group cloned sequences into classes. Phy-logenetic analysis was performed to evaluate the relation-ship between RGAs from V. faba and C. arietinum andplant R genes. Amino-acid sequences of 12 representativeclones from 10 RGA classes, as well as the NBS domainsof 6 R genes — I2 (Simons et al. 1998) and Prf (Salmeronet al. 1996) from tomato; RPP13 (Bittner-Eddy et al. 2000)from Arabidopsis; Gro1–4 (Paal et al. 2004) from potato;L6 (Lawrence et al. 1995) from flax; and N (Whitham etal. 1994) from tobacco — were aligned, and a neighbor-joining tree was generated using the PAM matrix (Dayhoff1979). The bootstrap method was applied to evaluate thereliability of the tree branching (Felsenstein 1985). Nucleo-tide diversity, Pi (Nei 1987), among 12 representativeclones was calculated in shifting windows of 30 nucleoti-des, with a step size of 2, using the DNAsp 4.0 (Rozas etal. 2003). The ratio of nonsynonymous to synonymous sub-stitutions (Ka/Ks) was estimated for each of the RGAclasses, using the method described by Nei and Gojobori(1986), with the MEGA3 program.

Table 1. Degenerate primers used to amplify resistance-gene analogs in Vicia faba and Cicerarietinum (Leister et al. 1996). I: ATA, ATC, ATT; R: G or A.

Concensus motif nucleotide binding site Primer Oligonucleotide sequence (5’?3’)

s1 GGTGGGGTTGGGAAGACAACGGGV/IGKTT (P-loop) s2 GGIGGIGTIGGIAAIACIAC

as1 CAACGCTAGTGGCAATCCGLPLAL (hydrophobic domain) as3 IAGIGCIAGIGGIAGICC

as4 ARIGCTARIGGIARICC

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Results and discussion

PCR amplification and molecular cloning of RGAsequences

A degenerate oligo-primed PCR approach was chosen asa first step in the identification of putative resistance genesin V. faba and C. arietinum. The 2 most critical factors indegenerate oligo-primed PCR are the design of the primersand the PCR conditions. The primers should be designedfrom an amino-acid region with minimal degeneracy in co-don usage (Compton 1990), and PCR conditions should beoptimized to give a balance between efficiency and specific-ity. To isolate RGAs in V. faba and C. arietinum, we as-sayed 6 combinations of degenerate primers, designed byLeister et al. (1996), on genomic DNA from the parentallines described in Materials and methods (Tables 1 and 2).

It was necessary to optimize the PCR conditions (e.g., an-nealing temperature and MgCl2 concentrations) and cyclingfor each primer combination. The optimum annealing tem-perature, determined after testing temperatures ranging from40 to 55 8C, were as follows: 44 8C for primer combinationsI and III; 46 8C for II and VI; and 55 8C for IV and V. Theproducts differed considerably among primer combinations(Table 2). Thus, whereas primer combination I yielded 4amplification products, ranging from 310 to 1016 bp, primercombinations IV and V failed to produce bands of the ex-pected size (389 bp) and were thus excluded from furtherstudy. Finally, combinations II, III, and VI generated 2 ma-jor fragments each, from which the bands of the expectedsize (510–550 bp) were selected for further sequence analy-sis.

In initial experiments, some of the fragments obtainedfrom primer combination I that differed in size from thoseexpected were also cloned. Nevertheless, sequencing datarevealed a complete lack of homology with R genes (datanot shown); these clones were disregarded. The main prod-ucts of primer combination III were also cloned and se-

quenced. However, the BLASTX database search showedthat all of the sequence-characterized inserts contained retro-transposon-like sequences (data not shown); these were dis-carded from the analysis as well. Thus, all the informativeamplicons considered in the analysis were obtained by clon-ing the major band from primer combinations II and VI(Fig. 1).

Restriction analysis of positive clones from primer combi-nations II and VI showed that the 510–550 bp DNA bandscontained heterogeneous fragments, indicating amplificationof more than 1 RGA by the degenerate primers used. Clonesshowing different restriction patterns of the insert and, occa-sionally, several clones of identical or similar patterns werechosen and further characterized by sequencing and se-quence analysis.

Sequence analysisA total of 149 putative V. faba and C. arietinum RGA

clones (excluding that from primer combination III) were se-quenced. Searches of the GenBank database, using theBLASTX algorithm, revealed that 14 clones contained retro-

Table 2. PCR products obtained with the differentprimer combinations.

Primer combination Size (bp)a No.b

I (s1/as1) 1016/710/420/310 —II (s2/as3) 540/409 14/73III (s2/as4) 550/409 —IV (s1/as3) 389 —V (s1/as4) 389 —VI (s2/as1) 584/396 55/76

aSize of major fragments from the nucleotide binding siteregion amplified by each primer combination.

bNumber of resistance-gene analog sequences vs. totalnumber of clones sequenced.

Fig. 1. PCR amplification products from genomic DNA of Vicia faba and Cicers arietinum parental lines, using primer combinations II (s2/as3)and VI (s2/as1). Arrows indicate the PCR products of expected size.

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transposon-like sequences, 21 were highly similar to poly-proteins or zinc finger proteins of several species (e.g.,C. arietinum, Oryza sativa, and Sorghum bicolor), and 41clones did not have significant hits on GenBank entries.Four sequences showed similarity to cloned R genes, but 2of them contained 1 or more stop codons and the other 2 in-cluded a sequence of limited size. These clones were ex-cluded from the analysis. The remaining 69 ampliconscould be translated into polypeptides containing single openreading frames and read. A search of the NCBI database in-dicated that their deduced amino-acid sequences showed ahigh level of identity with several plant R genes, such as I2and Mi1–1 from tomato, TMV and RPP13 from Arabidopsisthaliana, Gro1–4 from potato, and a number of RGA se-quences recently isolated using similar PCR-based ap-proaches from genera such as Lens, Pisum, Cicer,Phaseolus, and Citrus (Deng et al. 2000; Ferrier-Cana et al.2003; Huettel et al. 2002; Timmerman-Vaughan et al. 2000;Yaish et al. 2004). Primer combination VI was the most suc-cessful in obtaining NBS sequences, with 55 of 149 (36.9%)sequences showing homologies in the database. In contrast,only 14 (9.4%) NBS sequences were derived from primercombination II.

BLAST analysis detected homology to the internal con-served motifs typical of the NBS–LRR type of R genes(kinase-2 and kinase-3a). The presence of an internalkinase-2 (LXXLDDVX) and kinase-3a (GSRI/VIITTR) mo-tif in all the sequences isolated from V. faba and C. arieti-num, independent of the primer sequences used, confirmthat these clones correspond to NBS-containing genes.Meyers et al. (1999) reported that more than 1% of the A.thaliana genome might correspond to the NBS type of Rgenes, and genetic mapping studies of RGAs provided evi-dence that they cosegregate with fungal disease resistancemarkers (Aarts et al. 1998). Our results indicate that a largenumber of NBS-like sequences is also present in the V. fabaand C. arietinum genomes. Although some RGAs in C. arie-tinum have been mapped (Huettel et al. 2002), no reports inthe literature or GenBank were found on RGAs in V. faba.Therefore, this study is the first report on RGAs in this spe-cies. All V. faba and C. arietinum RGAs detected wereclosely related to sequences of known R genes and RGAsfrom others species. Thus, some of them might encoderesistance-gene products of unknown specificity. Future re-search will be directed toward the identification of associa-tions between the RGAs obtained from V. faba and C.arietinum and some diseases evaluated in our segregatingpopulations.

Multiple alignment of the deduced amino-acid sequencesof the 69 clones and a number of known R genes confirmedthe presence of all the motifs characteristic of the NBS–LRR gene class (Traut 1994). No variants in the GLPL mo-tif were obtained using the degenerate antisense primers as3and as1 deduced from this conserved motif (Fig. 2). Theonly hydrophobic motif obtained from our sequences wasGLPLAL. The consensus XGXGKTT, corresponding to theP-loop motif, is present in almost all NBS–LRR-type plantresistance genes as well, and the corresponding DNA se-quences were derived from primers s1 and s2. In our study,primer s2 always generated amplification products of the ex-pected size in combination with the other primer. In con- F

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trast, no amplicons of the expected size were ever obtainedwith primer s1 (Table 2).

As with the soybean (Yu et al. 1996), the most commonP-loop motif in V. faba and C. arietinum was GGVGKTT.Nevertheless, the variation WGGGKTT was also found inboth species (Fig. 2). In other species, such as the commonbean or lentil, the most common P-loop motifs reportedwere GGLGKTT (Ferrier-Cana et al. 2003) and GGMGKTT(Yaish et al. 2004), respectively.

NBS motifs of R-gene products can be categorized into 2major groups: the TIR and non-TIR linked sequences. Theformer presents an aspartic-acid residue at the end of thekinase-2 motif (LLVLDDVD), whereas the latter has a char-acteristic tryptophan residue (LLVLDDVW) (Meyers et al.1999). TIR and non-TIR types of RGAs have been identi-fied in, among other plants, apple (Lee et al. 2003), alfalfa(Cordero and Skinner 2002), common bean (Lopez et al.2003), and chickpea (Huettel et al. 2002), using similarpairs of degenerate primers matching the P-loop and GLPLmotifs. In this study, primer combination VI (s2/as1) ampli-fied both TIR and non-TIR types of sequences, whereas pri-mer combination II (s2/as3) only amplified TIR-typesequences. Yaish et al. (2004) suggested an association be-tween the primer combination and the type of sequenceamplified. If so, subtle changes in primer combinationscould be used to amplify specific sets of RGA sequences.In legumes, primers derived from P-loop and GLPL motifsappear to amplify preferentially TIR-NBS rather than non-TIR-NBS encoding regions (Bertioli et al. 2003; Yu et al.1996). This study produced similar results with V. faba andC. arietinum (see the Sequence alignment and phylogeneticanalysis section, below).

We observed differences in the kinase-2 and kinase-3a mo-tifs with respect to the TIR and non-TIR group. The kinase-2motif of the TIR group was X(L/I)(I/V)(I/V)(L/I)D(D/N)VD,whereas that of the non-TIR group was FL(F/V)VLDD(L/V)W.All the non-TIR sequences had the characteristic tryptophanresidue (W), whereas the kinase-2 motifs found in the TIRgroup of sequences contained either aspartic acid (D), as-paragine (N), or serine (S) residues at the last position. Inboth species, more variations in the TIR-group were ob-served. In the kinase-3a motif of the non-TIR group, a sin-gle sequence was found in all the faba bean clones(GSSVIITTR), in contrast to the variations observed in thechickpea (GS(R/K)(V/I)(I/L)(I/V)TTR). In the TIR group,the same variations in this motif were observed in fababean and chickpea GS(R/K)(I/V)(I/V)I(T/I)(T/S)R.

In accordance with Pan et al. (2000), short conservedamino-acid residues in the amino-acid motifs NBS-II andNBS-V were also detected (Fig. 2). In NBS-II, a phenylala-nine was invariably present in both TIR and non-TIR groups

Fig. 3. UPGMA tree for RGA amino-acid sequences isolated fromV. faba and C. arietinum. RGA classes were established at anamino-acid identity of 70%. Underlined sequences were chosen asRGA class representatives. Clone names consist of a letter (A–Efor V. faba parental lines and F or G for C. arietinum) followed bythe primer combination used (II or VI) and the clone number.Numbers between brackets on the right side correspond to the 3last digits of the respective accession No. in the NCBI database.

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and, 5 residues downstream, a phenylalanine or a tryptophanresidue was highly conserved in the TIR group and non-TIRgroup, respectively. Conserved motifs in the NBS domainmight play an important role in the function of resistancegenes, because several loss-of-function alleles in R genesare due to point mutations within conserved blocks insidethe NBS domain (Pan et al. 2000). These small regions ofconservation, although poorly understood, are likely to rep-resent functionally relevant sites and useful landmarks inthe isolation of R-gene homologues (Bent 1996). The align-ment of the deduced amino-acid sequences of representa-tives of all RGA classes and the sequences of N, L6, Gro1–4, I2, Prf, and RPP13 in the homologous regions is shownin Fig. 2.

Sequence alignment and phylogenetic analysisA UPGMA tree, based on alignment of the 69 nucleotide

and deduced amino-acid sequences, was constructed usingMEGA3(Kumar et al. 2004). DNA sequences correspondingto the P-loop (GGVGKTT) and hydrophobic motifs(GLPAL) could be contributed by primers and, thus, mightnot correspond to the exact genomic sequences. However,we still considered it appropriate to include them in the cal-culation of the percentages of identity, given the stringentPCR conditions and the degeneracy of the oligonucleotideprimers used.

Ten classes (RGA1–RGA10) could be defined using athreshold level of amino-acid sequence identity of 70%, cor-responding to a nucleotide sequence identity of approxi-mately 80% (Fig. 3). The number of sequences representingeach class varied from 1 for RGA03 and RGA07 to 30 forRGA01. RGAs were distributed in 2 major clades: 46RGAs showed amino-acid sequence similarity to the non-TIR-type NBS and 23 to the TIR type. The non-TIR-typesequences were grouped into 4 classes and the TIR-type se-quences into 6 classes. Classes 1, 6, 8, and 9 were com-prised only of clones isolated from the faba bean, whereasclasses 2, 3, 4, 5, and 7 included only chickpea clones.RGA 10, showing a within-class identity of 99%, was theonly class that contained both faba bean and chickpeaclones. The number of classes of RGAs amplified from agiven plant species seems to depend both on the type of oli-gonucleotide primers used and on the variety/cultivar of aparticular species. In legume species, such as the commonbean, 8 classes of RGAs were reported (Rivkin et al.1999), whereas 9 to 11 soybean classes have been reportedby Kanazin et al. (1996) and Yu et al. (1996), respectively,using different sets of degenerate oligonucleotides and ex-perimental conditions.

Pairwise comparisons of the deduced amino-acid sequen-ces indicated that the percentage of identity within each ofthe classes ranged from 88% to 100%, whereas the identitiesbetween classes ranged from 22% to 68% (Table 3). Two ofthe 10 classes consisted of a single RGA sequence (RGA03and RGA07) from C. arietinum, whereas the remainingclasses contained multiple members. Classes 1 to 4, groupedas non-TIR sequences, were more similar to the I2 and Prfgenes of tomato and the RPP13 gene of Arabidopsis (31%to 51% identity) (Table 3) and, as expected, showed lowerlevels of identity to sequences of the TIR-type R genes(Gro1–4, N, and L6). Classes RGA05 and RGA06 were sim-

Tab

le3.

Perc

ent

amin

o-ac

idid

entit

ies

ofth

e10

clas

ses

ofR

GA

sfr

omV

.fa

ba(V

f)an

dC

.ar

ieti

num

(Ca)

whe

nco

mpa

red

with

each

othe

ran

dth

e6

know

nR

gene

s.

Seq.

No.

No.

RG

AV

fC

aR

GA

01R

GA

02R

GA

03R

GA

04R

GA

05R

GA

06R

GA

07R

GA

08R

GA

09R

GA

10

1R

GA

0130

96.7

42

RG

A02

667

.99

99.4

23

RG

A03

150

.21

47.3

7n/

c4

RG

A04

935

.11

37.5

337

.15

99.4

25

RG

A05

528

.18

28.7

728

.52

29.4

988

.54

6R

GA

064

25.3

927

.95

26.0

929

.18

64.3

510

0.00

7R

GA

071

27.3

128

.91

24.6

929

.61

40.1

237

.28

n/c

8R

GA

086

22.4

024

.22

24.8

426

.99

38.9

638

.66

52.5

598

.55

9R

GA

092

22.9

624

.69

22.8

427

.85

35.0

934

.34

49.4

063

.15

100.

0010

RG

A10

23

21.7

723

.98

24.6

025

.36

33.5

131

.72

48.5

961

.08

69.6

499

.06

11I2

47.2

451

.08

49.4

040

.35

31.6

831

.87

26.0

927

.19

26.7

124

.25

12P

rf32

.02

37.4

531

.01

39.0

925

.91

23.4

223

.27

22.7

821

.94

19.4

913

RP

P13

31.0

733

.63

34.3

442

.38

25.8

223

.78

23.6

424

.09

21.7

422

.44

14G

ro1–

423

.08

27.9

526

.71

27.4

745

.53

46.4

739

.64

38.1

737

.35

33.6

115

N27

.20

29.3

828

.12

26.4

344

.12

41.2

136

.14

35.9

835

.40

32.0

716

L6

24.6

428

.22

22.0

921

.95

39.5

337

.87

34.5

234

.23

33.3

329

.76

Not

e:D

iago

nal,

with

in-c

lass

amin

o-ac

idid

entit

ies;

n/c,

not

calc

ulab

le;

Seq.

No.

,se

quen

cenu

mbe

r.

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ilar to the Gro1–4 gene of potato (46% average identity) andthe N gene of tobacco (42.6% average identity), whereasclasses 7 to 10 revealed similar percentages of identity withthe 3 TIR-type resistance genes (Gro1–4, N, and L6), rang-ing from 29.7% to 39.6%.

The amino-acid sequences of 12 representative clonesfrom the 10 RGA classes were chosen to generate aneighbor-joining tree, based on the multiple alignment ofamino-acid sequences of these representative RGAs and theNBS domains of 6 known R genes (I2 and Prf from tomato,RPP13 from Arabidopsis, Gro1–4 from potato, L6 from flax,N from tobacco) (Figs. 2 and 4). The 2 major branches re-solved in the phylogenetic tree (Fig. 4) include groups of

R genes that can be differentiated on the basis of the pres-ence or absence of the TIR region, supporting previous re-sults (Meyers et al. 1999; Pan et al. 2000). Most of thefaba bean and chickpea RGA sequences (RGA05, RGA06,RGA07, RGA08, RGA09, and RGA10) formed a majorcluster with N, L6, and Gro1–4. Most of these sequencesshared an aspartic-acid residue (D) at the final position ofthe kinase-2 motif, as frequently observed in the TIRgroup. The only exception was RGA06, which presentedan asparagine (N) residue at this position (Fig. 2). Theother major cluster comprised classes RGA01, RGA02,RGA03, and RGA04, which clustered together with I2,Prf, and RPP13. In these classes, the end of the kinase-2

Fig. 4. Unrooted neighbor-joining tree, based on the alignment of amino-acid sequences of RGAs from V. faba and C. arietinum and NBSdomains of cloned R genes. Clone naming is described in Fig. 3. Numbers above branches indicate bootstrap support percentages over 50%in 1000 pseudoreplicates.

Fig. 5. Nucleotide polymorphism of 12 representative RGAs from V. faba and C. arietinum. Lower polymorphism rates indicate the con-served regions of NBS (represented by arrows).

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motif contained a tryptophan residue (W), as expected forthe non-TIR group.

The analysis of the nucleotide polymorphism and the diver-sity of the 12 representative clones showed lower polymor-phism rates at the kinase-2 and kinase-3a motifs comparedwith the rest of the sequence (Fig. 5). The identification of di-vergent sequence regions will facilitate the future design ofspecific primers for the development of molecular markersthat might be useful in marker-assisted selection.

We calculated the ratio of nonsynonymous (Ka) to synon-ymous (Ks) nucleotide substitutions (Ka/Ks ratio) among trip-lets encoding the amino acids of faba bean and chickpea RGAsfor each class to determine either diversifying (Ka/Ks > 1) orpurifying (Ka/Ks < 1) selection. The Ka/Ks ratio for each groupranged from 0.0 to 0.84. These ratios are similar to those of theNBS domains from cloned R genes (Michelmore and Meyers1998), suggesting that the faba bean and chickpea NBS regionis subject to purifying selection. Similar results have been re-cently obtained with the first RGAs isolated from strawberries(Martınez Zamora et al. 2004). However, the faba bean NBSdomain within group RGA06 showed a ratio value higherthan 1, which could indicate that diversifying selection waspredominant between the sequences included in that group.Ferrier-Cana et al. (2003) did not find any evidence of diver-sifying selection in the NBS region of 7 common beanRGA sequences, but they did find diversifying selection inthe LRR and spacer regions of these sequences.

In this paper, we report the amplification, cloning, andcharacterization of NBS–LRR class resistance-gene candi-date sequences from V. faba and C. arietinum, using degen-erate primers based on conserved motifs of known R genes.This is the first report on the isolation of RGAs in V. fabausing a PCR-based approach.

Currently, we are carrying out a phylogenetic analysis,which includes these faba bean and chickpea RGA sequen-ces and the RGAs of other legume species, as well as clonedR genes, to determine the phylogenetic relationships and tostudy the diversity of the RGAs obtained. Furthermore, spe-cific primers for each RGA class, in combination with anumber of restriction enzymes, are being assayed to detectpolymorphic cleaved amplified polymorphic markers thatwill be genotyped in our faba bean and chickpea recombi-nant inbred line populations segregating for resistance toparasitic plants and different fungal diseases (Avila et al.2003, 2004; Cobos et al. 2005; Millan et al. 2003; Romanet al. 2002, 2003; Rubio et al. 2003). Because RGAs tendto cluster in close proximity to disease-resistance loci(Leister et al. 1996), these PCR-based markers might pro-vide a method for selecting resistant germplasm or progenyin faba bean and chickpea breeding programs. The com-bined approach might further identify macrosyntenic rela-tionships between disease resistance regions in the genomesof the 2 legume species.

AcknowledgementsThis study was supported by the European Community

project FP6–2002-FOOD-1–506223 GRAIN LEGUMES In-tegrated Project. The authors are grateful to Dr. Antonio DiPietro for critical revision of the manuscript.

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