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Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp

BACKGROUND: Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain
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  Pest Management Science Pest Manag Sci   64 :48–56 (2008) Isolation of nucleotide bindingsite-leucine rich repeat and kinaseresistance gene analogues from sugarcane(  Saccharum  spp.) Neil C Glynn, 1 ∗ Jack C Comstock, 1 Sushma G Sood, 1 Phat M Dang 2 andJose X Chaparro 3 1 USDA-ARS, Sugarcane Field Station, 12990 US Hwy 441N, Canal Point, 33438 FL, USA 2 USDA-ARS, National Peanut Research Laboratory, 1011 Forrester Drive, Dawson, 39842 GA, USA 3 Horticultural Sciences Department, University of Florida, 1301 Fifield Hall, PO Box 110690, Gainesville, 32611 FL, USA Abstract BACKGROUND: Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers.Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximalto R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs asmarkers for disease resistance is isolation and characterization of the sequences.RESULTS: Sugarcaneclone US01-1158was identifiedasresistant toyellow leafcaused bythe sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by  Puccinia melanocephala  Sydow & Sydow. Degenerateprimers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean wereused to amplify DNA from sugarcane ( Saccharum spp.) clone US-01-1158. Sequences generated from 1512positiveclones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuseswith the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear bindingsite and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acidsequences with similar sequences fromthe NCBI databaseallowed the identificationofseveralconserveddomains.The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequencesfrom other species than to one another.CONCLUSIONS: The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinasegene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.Published in 2007 by John Wiley & Sons, Ltd. Keywords:  resistance gene analogues; NBS-LRR; kinase; sugarcane;  Puccinia melanocephala ; yellow leaf; SCYLV 1 INTRODUCTION Sugarcane ( Saccharum  spp.) is an economicallyimportant crop that is cultivated in all tropical andsubtropical regions. Several agents of crop loss affectsugarcane productivity, including viruses, bacteria,fungi and invertebrates. Two of the most severediseases to affect production in South Florida areyellow leaf [caused by the sugarcane yellow leaf virus(SCYLV)] 1 and brown rust caused by the fungus Puccinia melanocephala  Sydow & Sydow. 2 The onlyviable form of protection against many sugarcanediseases is through varietal resistance. As a result, thegreatest losses due to disease occur when resistancebreaks down owing to more virulent forms of thepathogen. Economic losses due to rust caused by P. melanocephala  are estimated to be $40million and$3.5million to the Florida and Australian industriesrespectively. 2 , 3 SugarcaneplantsinfectedwithSCYLVhave been estimated to suffer a yield loss of 4–10% incane weight and sucrose. 1 Cultivated sugarcane ( Saccharum  spp.) is a complexpolyploid resulting from interspecific hybridizationprimarily between  S. officinarum  L. and  S. spontaneum L. Generally the  S. officinarum  genome contributesdesirable yield and processing traits, whereas the  S.spontaneum  genome contributes disease and stressresistance traits to the hybrid progeny. 4 Typically,the time from the initial cross to the release of a commercial sugarcane variety is 8–10years andinvolves several stages of selections and advancementsbased on field testing. 5 Small-scale inoculationtechniques for rust and YLS have been reported; 6 , 7 ∗ Correspondence to: Neil C Glynn, USDA-ARS, Sugarcane Field Station, 12990 US Hwy 441N, Canal Point, 33438 FL, USA E-mail:  Received 28 September 2006; revised version received 14 June 2007; accepted 2 July 2007   )Published online 15 October 2007 ;  DOI: 10.1002/ps.1469This article is a US Government work and is in the public domain in the USA.  Pest Manag Sci   1526–498X/2007/$30.00  NBS-LRR and kinase resistance gene analogues from sugarcane however, the absence of field-scale inoculationmethods means that accurate selection for diseaseresistance is particularly difficult and relies on theabsence of infection in field experiments wheresusceptible clones are infected by naturally occurringinoculum. Marker-assisted selection has the potentialto improve breeding for disease resistance. 8 As wellas the use of generally arbitrary markers such as SSR and AFLP, useful markers for disease resistance canbe developed by examining variation in genes knownto be important. 9 , 10 The first step towards developingmarkers using this candidate gene approach is theisolation of genes encoding sequences from materialwith a known phenotype.In recent years, much emphasis has been placedon the identification of gene sequences that conferresistance to plant diseases. Many resistance genesshare common structural features between oftendistantly related species of both dicotyledon andmonocotyledon plants, 11 but features that are uniqueto each have also been identified. 12 The majority of resistance genes to date fall into one of five categories:those containing nucleotide binding site and leucinerich repeat (NBS-LRR) domains, extracellular LRR,serine/threonine kinase, a transmembrane type thatcarriesanLRRandintracellularproteinkinasedomainor a single domain of the coiled coil type. 13 Inspite of their similarities, these genes have beenreported as conferring resistance to viral, 14 bacterial, 15 fungal 16 and invertebrate pests. 17 Sequence motifswhich show a high degree of conservation betweendifferentplantspecieshavebeensuccessfullyexploitedfor cross-species isolation of R-genes through the useof degenerate primers designed on the amino acidsequences they encode. 18 , 19 The activation of diseaseresistance genes occurs either from direct binding of theplantresistancegenetopathogenavirulencefactorsor through recognition by intermediates, and usuallyresults in a hypersensitive response (HR) and celldeath. 12 The complete set of NBS-LRR resistance genes hasbeen characterized in the whole genome sequencesof   Arabidopsis thaliana  (L.) Heynhoe 20 and rice. 21 The Arabidopsis genome was found to contain 149NBS-LRR genes, 20 12 of which were predicted to bepseudogenes. In contrast, the rice genome was foundto contain approximately 500 NBS-LRR genes, over100 of which were predicted as being pseudogenes. 21 Resistance genes occur in clusters in the genomesof several plant species, 20 , 22 , 23 occurring either asallelic series of the same genes, as reported forthe  Pm3  locus conferring powdery mildew resistancein wheat, 24 or as series of tightly linked genesforming complex loci, as is the case for the Cf4/9loci in tomato 25 and the Rp1 locus of maize. 26 In these cases, individual genes in a cluster conferdifferent recognition specificities that confer resistanceto separate pathogen isolates. The clustering of disease resistance genes has potential advantages inbreeding strategies, as selection for one R-gene duringvarietal evaluations has a greater chance of resultingin resistance to multiple pathotypes. 27 Conversely,selecting for resistance using RGA sequences has apotential disadvantage when a resistant allele for onepathogen is linked to that for susceptibility to anotherpathogen. Resistance genes have been reported toboth locate close to kinase genes 28 and to share severalkinase domains, 29 making kinase genes of additionalpotential value for the development of markers fordisease resistance.The present paper reports on the isolation of NBS-LRR RGA and kinase analogue sequences fromsugarcane ( Saccharum  spp.) clone US01-1158. CloneUS01-1158 is an F 1  hybrid of   S. spontaneum  and S. officinarum  that is resistant to yellow leaf caused bySCYLVandmoderatelyresistanttobrownrustcausedby  P. melanocephala . 2 MATERIALS AND METHODS2.1 Source of plant material and diseaseevaluations An interspecific cross between Green German ( Sac-charum officinarum ) and IND81-146 ( S. spontaneum )wasmadeatthe SugarcaneField Station,CanalPoint,FL, USA (Tai PYP, unpublished). The resultant seedfrom the F 1  progeny was harvested and grown in smalltrays (20 × 10 × 5cm) in a glasshouse under standardmanagement practices. After 35days, seedlings wereplanted into the field at the Canal Point Field Station(Canal Point, FL) according to a randomized design.After the first year of cultivation, stalks from 76 sep-arate plants were randomly selected and replanted infieldplots(1.5mplotswiththreereplications).Maturestalks of the two parents were planted in the same fieldat the same time. Plots were maintained in the fieldfrom 2002 to 2006 and were ratooned at the end of each growing season.Plants were evaluated multiple times for susceptibil-ity to brown rust and SCYLV according to previouslydescribed disease rating methods 30 and results fromtissue blot immunoassay tests 31 respectively. CloneUS01-1158 was selected for the present study on thebasis of its high level of resistance to rust and SCYLV. 2.2 DNA Isolation DNA was isolated from sugarcane clone US-01-1158 by adding 600 µ L of extraction buffer (Tris-HCl pH 8.0100m M , EDTA pH 8.050m M , sodiumchloride 500m M , sodium dodecyl sulfate 20g L  − 1 ,polyvinylpyrrolidone 10g L  − 1 and 2-mercaptoethanol1g L  − 1 ) to 100mg of inner whorl tissue in a1.5mL Eppendorf tube and macerated using aFastPrep FP120 (Thermo Savant). The sample wasincubated at 65 ◦ C for 20min, potassium acetatesolution (pH 6.5, 5  M ; 200 µ L) was added andit was incubated on ice for 5min. Then it wascentrifuged (12000 ×  g  ) for 20min, the supernatantwasremovedtoafreshtubeand400 µ Lofisopropanol(4 ◦ C) was added. Following incubation overnight at Pest Manag Sci   64 :48–56 (2008)  49 DOI: 10.1002/ps  NC Glynn  et al  . − 20 ◦ C and centrifugation (12000 ×  g  ) for 20min,the supernatant was removed and the remainingDNA pellet was dissolved in 70 µ L TE (Tris-HClpH 8.010mM, EDTA 1mM)  +  water (10 + 90 byvolume), incubated at room temperature for 2hrs,centrifuged(12000 ×  g  )for5minandthesupernatantremoved to a fresh tube. Sodium acetate solution(pH 7.6, 3M; 7 . 5 µ L) was added followed by 50 µ L isopropanol. The sample was washed twice more byadding 50 µ L ethanol  +  water (70 + 30 by volume)and centrifuging (12000 ×  g  ), the supernatant wasdiscarded and the process was repeated using ethanol +  water (95 + 5 by volume). The pellet was airdried and resuspended in 100 µ L TE containingRNase (0 . 1mg µ L  − 1 ) prior to storage at  − 20 ◦ C.The concentration of DNA was determined byspectrophotometry, diluting to a fixed concentrationof DNA prior to analysis. 2.3 Sequence isolation To isolate NBS-LRR RGAs, several degenerateprimers targeting the conserved P-loop (kinase1a) amino acid motif in NBS-LRR resistancegenes were used as forward primers (Table 1)in combination with degenerate primers targetingthe FMHYAL or GLPLA amino acid motifs asreverse primers (Table 1). Forward and reversedegenerate primers targeting the FGSVYKG andDVYSFG conserved motifs, respectively, were usedto isolate kinase analogue sequences (Table 1). Ninecombinations of the forward and reverse primerswere used in separate PCR reactions (Table 1).Reactions consisted of 20ng of each primer, 2.5m M magnesium chloride (Promega), 0.2m M  each dNTP(Promega), 8ng template DNA, 1 U  Taq  (Promega),12 µ g BSA (Promega) and 1 . 2 µ L Mg-free buffer(Promega), made to a final reaction volume of 12 µ L with molecular biology grade water (Sigma).PCR amplifications were performed in a PTC-200thermocycler (Bio-Rad Laboratories) and consistedof 40 cycles of 92 ◦ C for 1min followed by anannealing temperature of 40, 48 or 55 ◦ C dependingon the primer combination (Table 1) for 1min andextension at 72 ◦ C for 1min with a final extensionof 72 ◦ C for 5min. Amplification products fromeach of the nine reactions were confirmed onagarose gels (2% w/v) following electrophoresis. PCR products were purified using the Wizard SV Gel &PCR clean-up system (Promega) according to themanufacturer’s recommendations. Purified productswere transformed and cloned using pCR4 ® TOPO-TA cloning kit for  Taq  amplified PCR products(Invitrogen). Positive inserts were identified throughblue/white colony screening on amended LB agarplates. White (positive) colonies were selected andusedtoinoculate1.7mLofamendedSOCmedia,andinoculated cultures were grown for 3h at 37 ◦ C withaeration (150rpm). DNA was purified from culturesand sequenced on an ABI3730 DNA analyzer usingM13 primers. 2.4 Sequence analysis Sequences were vector trimmed and assembledinto contigs using Sequencher ™ version 4.5 (GeneCodes Corporation, MI). For contig assembly usingSequencher ™ , the rigorous data algorithm optionwas chosen with the settings of a minimum matchpercentage of 94% in a 50 bp overlap. Alignmentswere examined visually for base inconsistencies. Usingthe Sequencher ™ software, the peaks for individualbase calls were examined, and poor or ambiguousbases were changed according to plurality consensuswith the other sequences in the contig. Wheresequence variations were the result of normal basecall peaks, base variations were left unchanged. Aconsensus sequence based on plurality was generatedfor each contig, herein referred to as sugarcane geneanalogue consensus (SGAC), and used for GenBankcomparison using the BLASTx analysis algorithm. 32 Sequences that produced expectation scores of less than e − 10 and alignments with NBS-LRR orkinase-type sequences were classified into one of three groups: NBS-LRR1 (isolated using GLPLAmotif reverse primer), NBS-LRR2 (isolated usingFMHYAL motif reverse primer) or kinase (isolatedusing kinase motif primers). The translated sequencealignments from the BLASTx analyses were used toproduce a deduced amino acid sequence from the Table 1.  Details of primers and combinations used for gene isolation Primername PCR reaction a Primer sequence b Motif/domain ReferenceNBSfor1 1,4 GGIGGIGTIGGIAAIACIAC P-loop (kinase 1a) 22NBSfor2 5,6 GGIGTIWSIGGIWSIGGIAA P-loop (kinase 1a) 18NBSfor3 2,3 GGNGGNGTHGGIAAGACNAC P-loop (kinase 1a) 18NBSfor4 9 GGNTYNGGIAARACWACIC P-loop (kinase 1a) 18NBSrev1 1,2,5,9 ARIGCTARIGGIARICC GLPLA 22NBSrev2 3,4,6 ARIGCRTARTGCATRAA FMHYAL 18Kinasefor1 7 TTYGGNAARGTITAYAARGG FGSVYKG 18Kinasefor2 8 TTYGGNWSNGTITAYAARGG FGSVYKG 18Kinaserev2 7,8 ATICCRAAISWRTANACRT DVYSFG 18 a  Annealing temperature used in primer reactions 4,6,7 and 8: 40 ◦ C; in reactions 3, 5 and 9: 48 ◦ C; in reactions 1 and 2: 55 ◦ C. b CODEHOP notation used for degeneracies. I = inosine. 50  Pest Manag Sci   64 :48–56 (2008)DOI: 10.1002/ps  NBS-LRR and kinase resistance gene analogues from sugarcane DNAsequence.Conserveddomainswereidentifiedbysearching the position-specific score matrix (PPSM)NCBI conserved domain database (CDD) (version2.05). 33 Translated sequences were aligned using theMUSCLE algorithm 34 in Jalview (version 2.07) 35 with sequences retrieved from GenBank; NBS-LRR1sequences with tomato resistance complex proteinI2C-1 (gi | 2258315), and PRF (gi | 1513144),  Ara-bidopsis thaliana  RPS2 (gi | 30173240) and RPP8-like protein 1 (RPP8L1, gi | 29839442). NBS-LRR2sequences were aligned with  Aegilops tauschii   cystnematode resistance gene like protein (CNR-GLP, gi | 21536933) and  Zea mays  rust resis-tance protein Rp1-dp8 (gi | 12744963). Kinase-type sequences were aligned with wall-associatedkinase-2 (WAK-2, gi | 34915126), wall-associatedkinase-4 (WAK-4, gi | 58737172), S-receptor-likekinase (S-RLK, gi | 50934777), serine/threonine-likekinase (ser/thr-LK, gi | 51090884) and kinase-R-like protein (Kinase-R, gi | 17981564). Translatedsequences for SGAC1241, SGAC1578, SGAC1613and SGAC1726 showed disrupted open readingframes and were excluded from the alignment. Thesequences used in the alignment were submitted toGenBank and are numbers EF656335 to EF656358.AlignmentswereconvertedintoPHYLIPformatusingReadSeq ( Thethree alignments (NBS-LRR1, NBS-LRR2 andkinase) were analyzed using PHYLIP version 3.6 36 programs SEQBOOT (1000 iterations), PROTDIST,NEIGHBOR and CONSENSE, and finally trees wereproduced using DRAWGRAM. 3 RESULTS3.1 Isolation and identification of resistancegene sequences A total of 1512 positive clones were sequenced(1368 from NBS-LRR primer amplification productsand 144 from kinase primer amplification products).Assembly of the sequences using parameters of 94%match percentage in a 50 bp overlap resulted in 134separate contigs. The SGAC contigs ranged in sizefrom 1640 to 57 bp and were produced from thesequencing products of between two and 105 separatepositive clones.A summary of BLASTx results is given in Table 2.Analysis of the 134 SGAC sequences showed 107 tohave no similarity to protein sequences accessionedin the NCBI database or to have produced e valuesgreater than 10 − 10 . Twenty SGACs showed similarity(e values  < 10 − 10 ) to NBS-LRR type resistance genes.Of these, the greatest homology (4.0 e − 95 , 99%identity) was observed for SGAC1301 to a NBS-LRR resistance protein isolated from a  Saccharum  hybrid,and the least homology (5.0 e − 14 , 70% identity) wasobservedforSGAC1726toahypotheticalriceprotein.Eight sequences showed similarity to kinase genes,and, of these, the greatest similarity (1.0 e − 91 , 93%identity) was observed by SGAC1475 to Kinase Lr10protein from wheat, and the least homology (2.0 e − 61 ,79% homology) was observed by contig SGAC1556to kinase-R-type protein from wheat. Twelve contigsshowed homology to unknown protein sequences orto sequences of proteins not known to be related todisease resistance.Alignment of the deduced amino acid sequencesfor the SGAC’s with other NBS-LRR or kinase-type disease resistance protein sequences allowed theidentification of several conserved domains (Figs 1and 2). Sequences within the NBS-LRR1 groupshowed regions similar to kinase 2a, kinase 3a andRNBS-C motifs, as well as the P-loop (kinase 1a)and hydrophobic domain motifs that the degenerateprimers used for gene isolation were based on (Fig. 1).Sequences within the NBS-LRR2 group showedthe same regions as group 1 sequences, with theexception that they lacked RNBS-C and hydrophobicdomains (Fig. 1). The kinase-type sequences showedconservation with the tyrosine and serine/threonineprotein kinase catalytic domains (Fig. 2). Conservedmotifs for the catalytic loop, ATP binding pocket,activation loop and peptide substrate binding werealso evident (Fig. 2).The relative similarity of the SGAC obtained usingthe NBS-LLR1 and 2 and kinase degenerate primersto previously characterized resistance genes is shownin Fig. 3. Two clusters are evident in the dendrogramgenerated from the alignment of the NBS-LRR1sequences with the five resistance gene sequencesobtained from the NCBI database. One clusterincorporated SGAC1365, 1253, PRF and RPP8L1,and the other SGAC1323, 1301 and I2C (Fig. 3).Three clusters were evident from the dendrogram of sequences generated using NBS-LRR2 primers, onewith SGAC sequences 1269, 1369, 1608 and 1628,another with sequences CNRGLP and Rp1-dp8 andSGAC sequences 1441, 1331, 1462 and 1745 andthe third incorporating SGAC sequences 1547 and1548. Analysis of the NBS-LRR nucleotide sequencesby tBLASTx revealed that none of the sequencesobtained using the primer designed to the FMYHAL motif gave significant alignments to other NBS-LRR sequences previously isolated from sugarcane.Of the six consensus sequences examined fromprimers designed to the GLPLA motif, three(SGAC1253, SGAC1301 and SGAC1365) gave sig-nificant alignments with previously accessioned  Sac-charum  spp. NBS-LRR RGA sequences; SGAC1253and SGAC1365 yielded significant alignments toRGA sequences Q15 (accession gi | 56694176) andQ16 (accession gi | 56694177) isolated from the Aus-tralian cultivar Q117. 37 In spite of the high BLASTresultobtained,amultiplesequencealignmentofQ15,Q16, SGAC1253 and SGAC1365 showed an approx-imately 100 bp insert in SGAC1253 and SGAC1365not present in Q15 and Q16, and BLASTx analysisof the insert sequence produced no significant results.Consensus sequence SGAC1301 gave a significantalignment with RGA755 isolated from  Saccharum Pest Manag Sci   64 :48–56 (2008)  51 DOI: 10.1002/ps  NC Glynn  et al  . Table 2.  Summary of BLASTx results for SGACs showing homology (e  <  10 − 10  ) to NBS-LRR, LRR or kinase-type sequences SGACnumberNumber of sequencesin contig ScoreGI numberof highestscoreDescription of highestBLASTx result SpeciesPositives(%) Sequence type1241 18 4e-30 33302329 Resistance proteinCAN RGA1 Triticum aestivum 79 NBS-LRR1253 105 2e-44 50931801 Hypothetical protein  Oryza sativa  58 NBS-LRR1269 41 5e-47 50921301 OSJNBb0060M15.1  Oryza sativa  77 NBS-LRR1293 16 1e-66 17981563 Kinase-R-like protein  Triticum aestivum  83 Kinase R1300 3 5e-85 50926484 OSJNBa0073E02.11  Oryza sativa  94 Kinase II, IV 1301 4 5e-95 33357781 NBS-LRR resistanceprotein Saccharum hybrid   99 NBS-LRR1323 12 2e-67 10303024 Resistance  Avena strigosa  82 NBS-LRR1330 8 3e-31 50921301 OSJNBb0060M15.1  Oryza sativa  73 NBS-LRR1331 12 1e-23 34912524 Putative cystnematoderesistance protein Oryza sativa  56 NBS-LRR1365 15 6e-28 50931801 Hypothetical protein  Oryza sativa  75 NBS-LRR1369 6 9e-43 50921301 OSJNBb0060M15.1  Oryza sativa  74 NBS-LRR1441 10 3e-50 26986268 DW-RGA2 protein  Triticum turgidum subsp. durum 81 NBS-LRR1462 25 6e-48 34912524 Putative cystnematoderesistance protein Oryza sativa  78 NBS-LRR1475 2 1e-91 1680686 Rust resistance kinaseLr10 Triticum aestivum  93 Kinase Lr101538 4 3e-75 15387663 Pollen signallingprotein  Zea mays  87 NBS-LRR1540 3 5e-77 17981564 Kinase-R-like protein  Triticum aestivum  90 Kinase R1547 4 4e-52 50921301 OSJNBb0060M15.1  Oryza sativa  83 NBS-LRR1548 3 2e-51 50921301 OSJNBb0060M15.1  Oryza sativa  79 NBS-LRR1556 3 2e-61 17981564 Kinase R-like protein  Triticum aestivum  79 Kinase R1578 5 4e-18 50941629 Putative NBS-LRRdisease resistanceprotein Oryza sativa  75 NBS-LRR1608 4 7e-38 50921301 OSJNBb0060M15.1  Oryza sativa  74 NBS-LRR1613 4 7e-22 62734623 Leucine-rich repeat,putative Oryza sativa  70 LRR1628 8 2e-46 50921300 OSJNBb0060M15.1  Oryza sativa  76 NBS-LRR1633 3 5e-90 50926484 OSJNBa0073E02.11  Oryza sativa  95 Kinase II, IV 1649 2 2e-80 34899524 Putative receptor-typeprotein kinase LRK1 Oryza sativa  88 Kinase1679 2 3e-85 50934777 Putative S-receptorkinase Oryza sativa  92 Ser/Thr protein kinase1726 2 5e-14 50931801 Hypothetical protein  Oryza sativa  70 NBS-LRR1745 3 2e-46 34912524 Putative cystnematoderesistance protein Oryza sativa  78 NBS-LRR spp. hybrid N11 by Rutherford and Brune, accessiongi | 33357780 (private communication). Two clusterswere evident from the dendrogram of kinase sequencealignment, one that incorporated SGAC sequences1300 and 1633 and WAK-2 and WAK-4, and a sec-ond that incorporated SGAC sequences 1540, 1293and 1556 and kinase-R (Fig. 3). 4 DISCUSSION Previous studies reporting the isolation of RGAs andcharacterizationofsequencedifferenceshavegenerallyfocused on the analysis of multiple individuals. Owingto the highly variable nature of the sugarcane genomeas well as the existence of multiple NBS-LRR RGAsand kinase analogue sequences in the genomes of other crop species, in the present study sequenceswere isolated from the individual hybrid clone US01-1158. This clone was classified as resistant toSCYLV and moderately resistant to rust causedby  P. melanocephala , based on the results of fieldtrials performed over several years in which sensitivecontrols had become infected. US01-1158 mayprovide a breeding source for resistance to these twoimportant sugarcane pathogens.ThedegenerateprimersusedforisolatingNBS-LRR sequences had previously proved useful for isolating 52  Pest Manag Sci   64 :48–56 (2008)DOI: 10.1002/ps
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