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Mapping of adult plant stripe rust resistance genes in diploid A genome wheat species and their transfer to bread wheat

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Stripe rust, caused by Puccinia striiformis West. f.sp. tritici, is one of the most damaging diseases of wheat worldwide. Forty genes for stripe rust resistance have been catalogued so far, but the majority of them are not effective against emerging
  Theor Appl Genet (2008) 116:313–324 DOI 10.1007/s00122-007-0668-0  1 3 ORIGINAL PAPER Mapping of adult plant stripe rust resistance genes in diploid A genome wheat species and their transfer to bread wheat Parveen Chhuneja · Satinder Kaur · Tosh Garg · Meenu Ghai · Simarjit Kaur · M. Prashar · N. S. Bains · R. K. Goel · Beat Keller · H. S. Dhaliwal · Kuldeep Singh Received: 10 July 2007 / Accepted: 20 October 2007 / Published online: 8 November 2007 ©  Springer-Verlag 2007 Abstract Stripe rust, caused by Puccinia striiformis West. f.sp. tritici , is one of the most damaging diseases of wheat worldwide. Forty genes for stripe rust resistancehave been catalogued so far, but the majority of them arenot e V  ective against emerging pathotypes. Triticum mono-coccum  and T. boeoticum  have excellent levels of resis-tance to rusts, but so far, no stripe rust resistance gene hasbeen identi W ed or transferred from these species. A set of 121 RILs generated from a cross involving T. monococcum (acc. pau14087) and T. boeoticum  (acc. pau5088) wasscreened for 3years against a mixture of pathotypes under W eld conditions. The parental accessions were susceptibleto all the prevalent pathotypes at the seedling stage, butresistant at the adult plant stage. Genetic analysis of theRIL population revealed the presence of two genes forstripe rust resistance, with one gene each being contributedby each of the parental lines. A linkage map with 169 SSRand RFLP loci generated from a set of 93 RILs was used formapping these resistance genes. Based on phenotypic datafor 3years and the pooled data, two QTLs, one each in T.monococcum  acc. pau14087 and T. boeoticum  acc.pau5088, were detected for resistance in the RIL popula-tion. The QTL in T. monococcum  mapped on chromosome2A in a 3.6cM interval between  Xwmc407   and  Xwmc170 ,whereas the QTL from T. boeoticum  mapped on 5A in8.9cM interval between  Xbarc151  and  Xcfd12  and thesewere designated as QYrtm.pau-2A  and QYrtb.pau-5A ,respectively. Based on W eld data for 3years, their  R 2  valueswere 14 and 24%, respectively. T. monococcum  acc.pau14087 and three resistant RILs were crossed to hexaploid Communicated by B. Friebe. Electronic supplementary material The online version of this article (doi:10.1007/s00122-007-0668-0) contains supplementary material, which is available to authorized users.P. Chhuneja · S. Kaur · T. Garg · M. Ghai · S. Kaur · N. S. Bains · R. K. Goel · H. S. Dhaliwal · K. Singh ( & )Department of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University, Ludhiana 141 004, Indiae-mail: kuldeep35@yahoo.comP. Chhunejae-mail: pchhuneja@redi V  mail.comS. Kaure-mail: satink88@yahoo.comT. Garge-mail: gargtosh@gmail.comS. Kaure-mail: S. Bainse-mail: nsbains@redi V  mail.comM. PrasharDirectorate of Wheat Research, Regional Station, Flowerdale, Shimla, Indiae-mail: dwrfdl@hotmail.comB. KellerInstitute of Plant Biology, University of Zurich, Zurich, Switzerlande-mail: Present Address: H. S. DhaliwalIndian Institute of Technology, Roorkee, Uttarakhand, Indiae-mail:  314Theor Appl Genet (2008) 116:313–324  1 3 wheat cvs WL711 and PBW343, using T. durum  as a bridgingspecies with the objective of transferring these genes intohexaploid wheat. The B genome of T. durum  suppressedresistance in the F 1  plants, but with subsequent backcross-ing one resistance gene could be transferred from one of theRILs to the hexaploid wheat background. This gene wasderived from T. boeoticum  acc. pau5088 as indicated byco-introgression of T. boeoticum  sequences linked to striperust resistance QTL, QYrtb.pau-5A . Homozygous resistantprogenies with 40–42 chromosomes have been identi W ed. Introduction Stripe rust, caused by the fungal pathogen Puccinia striifor-mis  West. f.sp. tritici  ( Pst  ), is one of the most damagingdiseases of wheat. Worldwide, 43million ha (46%) and inIndia about 9.4million ha (>35%) area under wheatcultivation is prone to stripe rust (Singh etal. 2004). Striperust propagates in cool and moist environments and caninfect the wheat crop in the early growth stages leading toyield losses as high as 50% (Roelfs etal. 1992). The devel-opment and deployment of cultivars with genetic resistanceis the most economical and environment friendly approachto reduce yield losses due to rusts. For stripe rust, race spe-ci W c seedling and adult plant resistance as well as race-non-speci W c adult plant resistance have been reported (Johnson1988). In the long term, the major resistance genes haveturned out to be non-durable as virulence in the pathogenpopulation was selected or it rapidly evolved following theintroduction of such resistance genes. Consequently, a con-stant search and transfer from novel and e V  ective sources of resistance is necessary to counterbalance the continuousevolution of rust pathogens. So far, 40 stripe rust resistance( Yr  ) genes have been catalogued and designated and 11 of these have been transferred from alien species (McIntoshetal. 2005; Uauy etal. 2005; Marais etal. 2005a, 2005b, 2006; Kuraparthy etal. 2007). Except for Yr28 , Yr35  and Yr36  , all other alien genes have been transferred from non-progenitor species. Yr28  has been transferred from  Ae. tau-schii  (Singh etal. 2000) and Yr35  and Yr36   have beentransferred from T. dicoccoides  (Marais etal. 2005b; Uauyetal. 2005). Most of the designated Yr   genes confer ahypersensitive reaction and the majority of these are nolonger e V  ective due to evolution of new virulences in thepathogen (Ma etal. 1997). Genes Yr5 , Yr10 , Yr15 , Yr26  and Yr40  are e V  ective against the predominant Pst   patho-types in India, and all of them, except Yr10 , are of alien ori-gin and provide seedling resistance. Yr27  , which had beenthe basis of resistance in the widely grown Indian cultivarPBW343 (an Attila sib), has now become ine V  ectiveagainst a new virulence designated as 78S84 (Prashar etal.2007).Many genes conferring resistance to rusts, powdery mil-dew and insect pests have been transferred from  Aegilops species into cultivated wheat (Jiang etal. 1994; Friebe etal.1996; Marais etal. 2005a, 2006). Some of the genes trans- ferred from distantly related species have been exploitedcommercially, but others seem to be associated with yieldpenalty due to linkage drag (Friebe etal. 1996). The alienchromosome segments from distantly related species, onceretained in early generations, are di Y cult to eliminate evenafter repeated backcrosses (Young and Tanksley 1989).The diploid “A” genome progenitor gene pool of wheat,comprising three closely related species T. monococcum ssp monococcum  ( T. monococcum ), T. monococcum  ssp aegilopoides  ( T. boeoticum ) and T. urartu , harbours usefulvariability for many economically important genes,including resistance to diseases (Feldman and Sears 1981;Dhaliwal etal. 1993; Hussien etal. 1997). These species have served as a valuable source for leaf rust (Dyck andBartos 1994; Kerber 1983; Valkoun etal. 1986; Hussien etal. 1997), stem rust (Kerber and Dyck 1973; McIntosh etal. 1984; The and Baker 1975; Valkoun etal. 1989; Ma etal. 1997) and powdery mildew resistance genes (Shietal. 1998; Yao etal. 2007 ) for hexaploid wheat improve- ment. Their genomes share considerable homology with theA genomes of cultivated tetraploid and hexaploid wheat(Dvorak etal. 1993) enabling the transfer of desirable alle-les from the “alien” A genome chromosomes into their“cultivated” homologues without any signi W cant linkagedrag. However, the transfers to hexaploid wheat generallyrequire the use of T. durum  as bridging species and thepresence of suppressor loci on the A and/or B genome of  T.durum  may present a major hurdle in transferring usefulvariability from diploid to hexaploid wheats (Knott2000; Ma etal. 1997; Qiu etal. 2005). Identi W cation of DNA markers linked to the desirable genes at the diploidlevel can facilitate their transfer to hexaploid wheat(Yao etal. 2007). Some of the race-speci W c stripe rustresistance genes such as Yr15  (Chague etal. 1999), YrH52 (Peng etal. 1999), Yr17   (Robert etal. 1999), Yrns-B1 (Börner etal. 2000), Yr28  (Singh etal. 2000) and Yr40 (Kuraparthy etal. 2007) and race non-speci W c genes suchas Yr18-Lr34  complex (Bossolini etal. 2006) and Yr39  (Linand Chen 2007) have been tagged with DNA markers.Genes for stripe rust resistance from the diploid Agenome species, unlike genes for resistance to other dis-eases as mentioned above, have not been catalogued ortransferred to hexaploid wheat. A large number of Agenome germplasm accessions have been screened overmany years at our institute. Most of the T. monococcum accessions showed moderate to complete resistance; mostof the T. boeoticum  accessions showed complete resistanceand majority of the T. urartu  accessions were highly sus-ceptible. A spring type T. monococcum  acc. pau14087 has  Theor Appl Genet (2008) 116:313–324 315  1 3 maintained a high level of resistance to a number of wheatdiseases including stripe rust in Punjab (India) over years(Dhaliwal etal. 2003). In this accession, at the stage of infection initiation, compatible stripes developed at the tipsof the leaves and the reaction could be categorized as sus-ceptible. However, within a few days of infection, necroticareas developed around stripe rust infection sites and thereaction was categorized as moderate resistant. The striperust reaction in this accession conforms to the criteria of slow rusting, as argued by Singh etal. (2005). This type of potentially durable resistance is less studied for stripe rustas compared to leaf rust. The identi W ed T. monococcum accession was crossed with T. boeoticum  acc. pau5088 togenerate an RIL population, which besides stripe rust resis-tance showed segregation for leaf rust, powdery mildew,Karnal bunt and cereal cyst nematode (Dhaliwal etal.2003; Singh etal. 2007a). This population was used for generating a linkage map of the diploid A genome of wheat(Singh etal. 2007b) and the mapping of several diseaseresistance genes. Here, we provide the W rst report of map-ping of adult plant stripe rust resistance genes in T. mono-coccum  and T. boeoticum  and their transfer to hexaploidwheat using T. durum  as a bridging species. Materials and methods Plant material The plant material used for studying inheritance and map-ping of the stripe rust resistance genes consisted of a set of 121 recombinant inbred lines (RILs) derived from the cross T. boeoticum  acc. pau5088/  T. monococcum  acc. pau14087(hereafter referred to as Tb5088 and Tm14087, respec-tively) through single seed descent. Detailed information onthese accessions and a molecular linkage map generatedusing this population was described by Singh etal. (2007b)and is available at GrainGenes ( ggpages/map_summary.html). Of the 121 RILs, 93 wereused for generating the linkage map, whereas all theRILs were screened against three stripe rust pathotypes atthe seedling stage and against a mixture of three or fourpathotypes at the adult plant stage under W eld conditions.For the transfer of resistance, T. monococcum  and threeRILs (designated as RIL86, RIL101and RIL130) were usedas donors and two hexaploid spring wheat cultivars WL711and PBW343 were used as the recipients. N59, a stripe rustsusceptible T. durum  cultivar served as a bridging cross parent.Screening against stripe rust pathotypesFour stripe rust pathotypes, 46S102, 47S103, 46S119 and78S84 with known avirulence/virulence formula (Table1),were used for screening parental accessions and the RILpopulation at the seedling stage. Screening at the seedlingstage was done against individual pathotypes following theprocedure of Nayar etal. (1997) in controlled conditions.Brie X y, seedlings at the two-leaf stage were inoculatedthrough atomization with rust urediospores immersed inlight mineral oil (Pegasol). The inoculated material wasplaced in humid chambers at 10°C for 48h. After incuba-tion, the trays were shifted to a glass house maintained at16 § 2°C. Infection types (ITs) of seedlings were scored14days after inoculation, according to the modi W ed scaleof Stakman etal. (1962). Since the parents and the RILpopulation turned out to be susceptible to all pathotypes atthe seedling stage, further screening was done against amixture of these pathotypes at the adult plant stage under W eld conditions. The RIL population and the parents werescreened for four consecutive crop seasons 2004, 2005,2006 and 2007. For adult plant screening, the RILs wereplanted in 2m rows, with a row-to-row distance of 70cm.The RILs, being longer in duration, were planted in the lastweek of October, whereas the F 1  plants and the backcrosspopulations were planted in the second fortnight of November. Spreader rows were planted all around the pop-ulation to ensure an e V  ective disease spread. Inoculationswere done by spraying the mixture of pathotypes thrice aweek, starting from the W rst week of January until secondweek of February when the crop was in Zadoks growthstages 23–33. The inoculum consisted of three striperust pathotypes, 46S102, 47S103 and 46S119, during2004, 2005 and 2006 and a fourth pathotype, 78S84, whichis virulent on Yr27   (Prashar etal. 2007) was added to the Table1 Avirulence/virulence formulae of stripe rust ( Pst  ) pathotypes used for screening of the Tb5088/Tm14087 RILs and the introgression lines a Determined at the seedling stage in a glasshouse under standard conditions (modi W ed from Nayar etal. 1997) b Most of the genes tested were in Avocet background except for Yr3  (Vilmorin23), Yr4  (Hybrid46), Yr6  (Heines Kolben), Yr7  (Lee), Yr8  (Compair) Yr10  (Moro) and Yr40 (WL711)PathotypeAvirulence a Virulence a 46S102 Yr1 b ,  Yr5 ,  Yr9 ,  Yr10 ,  Yr15 ,  Yr17  ,  Yr24 ,  Yr25 ,  Yr26  ,  Yr27  ,  Yr40Yr2 ,  Yr3 ,  Yr4 ,  Yr6  ,  Yr7  ,  Yr8 47S103 Yr5 ,  Yr9 ,  Yr10 ,  Yr15 ,  Yr17  ,  Yr24 ,  Yr25 ,  Yr26  ,  Yr27  ,  Yr40Yr1 ,  Yr2 ,  Yr3 ,  Yr4 ,  Yr6  ,  Yr7  ,  Yr8 46S119 Yr1 , Yr5 ,  Yr10 ,  Yr15 ,  Yr24 ,  Yr25 ,  Yr26  ,  Yr27  ,  Yr40Yr2 ,  Yr3 ,  Yr4 ,  Yr6  ,  Yr7  ,  Yr8 ,  Yr9 , Yr17  78S84 Yr1 , Yr5 ,  Yr10 ,  Yr15 ,  Yr17  ,  Yr24 ,  Yr25 ,  Yr26  ,  Yr40Yr2 ,  Yr3 ,  Yr4 ,  Yr6  ,  Yr7  ,  Yr8 ,  Yr9 ,  Yr27   316Theor Appl Genet (2008) 116:313–324  1 3 mixture in 2007 as its W eld-testing was allowed only from2007.Disease reaction was recorded twice in the season at theadult plant stage (Zadoks stage 69–83), W rst when striperust reaction of the susceptible check reached 40–60S andthe second when the disease reaction of the susceptiblecheck reached 100S. The disease data was recorded follow-ing modi W ed Cobb’s scale (Peterson etal. 1948) thatincludes disease severity (percentage of leaf area coveredwith rust urediospores) as well as disease response (infec-tion type). The infection types were recorded as zero(immune); TR (traces of severity); MR (moderately resis-tant), MS (moderately susceptible); S (susceptible) anddisease severity was recorded as percent leaf area infected.Data recorded during second scoring was used for geneticanalysis. The RILs were characterized as resistant orsusceptible depending upon the maximum disease severityobserved in the parental lines, Tm14087 and Tb5088, ina particular crop season. Chi square analysis was usedto estimate the number of genes governing resistancein Tm14087 and Tb5088. Backcross populations andintrogression lines were screened only at the adult plantstage and data were recorded as described above for RILpopulation.QTL mappingDisease severity recorded as percentage of leaf area cov-ered with rust urediospores as well as coe Y cient of infec-tion were used for detection and localization of QTL in theparental lines, Tm14087 and Tb5088. The coe Y cient of infection (CI), which weighs the modi W ed Cobb scale ratingby disease response (R, MR, MS, S), was calculated bymultiplying the percentage infection with response values0.2, 0.4, 0.8, and 1.0 assigned for the infection types TR,MR, MS and S, respectively as per Loegering (1959). Boththe scores were used because the CI is probably moreclosely correlated with crop loss than is either scale fromwhich it is calculated, but it does have a problem in that thetwo variables from which it is estimated are not indepen-dent (McIntosh etal. 1995). As the RIL population did notshow a normal distribution, the data were transformed formaking the distribution near normal, a prerequisite for QTLanalysis using likelihood ratio statistics (Mao and Xu 2004;Yang etal. 2006). The data for stripe rust in a set of 93RILs (for which genotypic data was available; Singh etal.2007b) was used for detection and localization of QTLs forresistance in Tm14087 and Tb5088. QTL were detectedand localized by single marker regression (SMA) and com-posite interval mapping (CIM) using MapManagerQTXb20 (Manly etal. 2001). In this analysis, the data of the RILs for individual years and the pooled data wereentered along with the genotypic data of the RILs. The“marker regression” function ( P =0.01) was used to detectpossible single marker loci associated with the QTL. Thelocus with the highest likelihood ratio statistic (LRS) foreach set of data was added to the background and compos-ite interval mapping used for localization and estimation of e V  ects of each QTL after correcting the e V  ect of back-ground loci. Signi W cant ( P <0.05) and highly signi W cant( P <0.01) threshold levels were determined by the permu-tation test function of MapManager, which is based on thestatistical methods developed by Churchill and Doerge(1994). A likelihood ratio statistic (LRS) value of 4.6 isequivalent to one logarithm of the odds (LOD). Phenotypicvariance explained by each QTL (  R 2 ) was estimated foreach data set as di V  erence between the total variance andthe residual variance expressed as the percent of the totalvariance. The data was also analysed with software QTL-Network-2.0 (Yang etal. 2007) for detecting interactionsbetween the QTL.Transfer of stripe rust resistance to hexaploid wheatTm14087 was crossed as male with a stripe rust suscep-tible T. durum  cultivar N59. The triploid F 1  (N59/ Tm14087, 2 n =21) plants were completely male sterileand susceptible to the stripe rust. The F 1  plants werebackcrossed to durum parent N59. The BC 1 F 1  and BC 1 F 2 plants thus obtained were screened under W eld condi-tions at the adult plant stage against a mixture of threepathotypes 46S102, 47S103, and 46S119. The F 1  of N59/Tm14087 was also crossed to susceptible hexaploidwheat cultivars, WL711 and PBW343. WL711 was sus-ceptible to all the four pathotypes, whereas PBW343was resistant to pathotypes 47S103, 46S102, and46S119, but susceptible to 78S84. The F 1  plants of thecross N59/Tm14087//WL711 were backcrossed toWL711 to generate BC 1 F 1  progenies. These BC 1 F 1  prog-enies were screened under W eld conditions at the adultplant stage against a mixture of three pathotypes46S102, 47S103, and 46S119. Resistant plants obtainedin the BC 1 F 1  progenies of the cross N59/Tm14087// 2*WL711 were either selfed or backcrossed to the recur-rent hexaploid parent WL711. In addition to Tm14087,three resistant RILs, viz. RIL86, RIL101 and RIL130,were also crossed to WL711 and PBW343 using N59 asthe bridging species and the crosses were followed asdescribed above for Tm14087. Transfer of stripe rustresistance in PBW343 could not be followed in earliergenerations because PBW343 was resistant to patho-types 46S102, 47S103 and 46S119, and pathotype78S84 became available for W eld evaluation in 2007.However, transfer of leaf rust resistance from Tm14087and RIL101 to PBW343 was followed separately. Theleaf rust resistant progenies were screened for stripe rust  Theor Appl Genet (2008) 116:313–324 317  1 3 during 2007. Screening against stripe rust and datarecording were done as described in the precedingsection.Cytological analysisChromosome number was analysed in F 1  of N59/Tm14087and N59/Tm14087//WL711 and BC 1 F 1 and BC 2 F 2  proge-nies of N59/Tm14087//n*WL711 and N59/Tm14087// n*PBW343. The chromosome number was analysed frompollen mother cells (PMCs) of individual plants usingstandard acetocarmine squashing technique. The spikeswere W xed at the pre-booting stage in Carnoy’s solution II(6 ethanol: 3 chloroform: 1 glacial acetic acid) and trans-ferred to 70% ethanol after 48h. Squash preparations weremade in 2% acetocarmine. Results Inheritance of stripe rust resistance At the seedling stage, Tm14087 showed susceptible reac-tion against all four stripe rust pathotypes. At maximum til-lering stage under W eld conditions, it showed a susceptiblereaction at the initiation of the disease with compatiblestripes con W ned to the tips of the leaves. The terminal dis-ease severity, however, was recorded as moderately resis-tant with necrotic areas developing around the rust stripes(Fig.1), thus indicating the presence of adult plant resis-tance in Tm14087. Tb5088 was also susceptible to all thefour pathotypes at the seedling stage, but at the adult plantstage, it showed complete resistance, displaying diseaseseverity 0-TR. Tb5088, thus, also had adult plant resistance(APR) for stripe rust. The F 1  of Tm14087/Tb5088 showedstripe rust development similar to that of Tm14087, withsmall stripes at the tips of the leaves. The RIL populationalso showed susceptible reaction at the seedling stage to allthe pathotypes.At the adult plant stage, under W eld conditions, data wasrecorded for four consecutive years, but disease develop-ment during 2006 was poor and is not included for furtherdiscussions. At the initial stages of disease establishment inTm14087, the W rst uredinia to appear induced susceptiblereaction and disease severity of 10S, 20S and 10S wasrecorded during 2004, 2005 and 2007, respectively. Subse-quent fungal mycelial growth, as is often observed in slowrusting, caused necrosis and the terminal disease severityrecorded was 5MR-10MR (Fig.1). Disease severity in theRIL population ranged from 0 to 80S (Fig.1). As diseaseseverity varied during 2004, 2005 and 2007 (Table2), thestripe rust reaction of Tm14087 and Tb5088 in a particularyear was taken into consideration for classifying a RIL asresistant or susceptible. During 2004 and 2007, Tb5088showed complete resistance (0-TR), whereas Tm14087developed stripe rust up to a maximum of 10S. Thus, theRILs with terminal stripe rust severity of 0–10S were clas-si W ed as resistant and the ones with disease severity of 20Sor more were classi W ed as susceptible. In 2005, Tm14087 Fig.1 Stripe rust reaction of Tm14087 (1–2), Tb5088 (3) and a set of RILs (4–9). Samples 1 and 2 show stripe rust reaction in Tm14087 atthe initial stage of infection and a week later, respectively Table2 Distribution of Tb5088  /  Tm14087 RILs for rust severityagainst mixture of stripe rust pathotypes under W eld conditionsStripe rust reactionNo. of RILs200420052007Tm1408710S-10MR20S-10MR10S-5MRTb5088–0TR018510TR1816185MR1717265MS2531110MR06810MS11121710S714620S15231340S720760S24180S111Total121121118  2  (3:1)1.21 ( P <0.27)1.21 ( P <0.27)2.54 ( P <0.11)
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