Ruiqi Zhng *,Chunxi Xiong ,Hunqing Mu ,Ruonn Yo ,Xingru Meng ,Lingn Kong ,Liping Xing ,Jizhong Wu ,Yigo Feng ,Aizhong Co

a College of Agronomy/JCIC-MCP/National Key Lab of Crop Genetics and Germplasm Enhancement,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China

b Institute of Germplasm Resources and Biotechnology/Provincial Key Lab of Agrobiology,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,Jiangsu,China

ABSTRACT Powdery mildew,caused by the biotrophic fungus Blumeria graminis f.sp. tritici (Bgt),is a global disease that poses a serious threat to wheat production.To explore additional resistance gene,a wheat-Dasypyrum villosum 1V#5 (1D) disomic substitution line NAU1813 (2n=42) with high level of seedling resistance to powdery mildew was used to generate the recombination between chromosomes 1V#5 and 1D.Four introgression lines,including t1VS#5 ditelosomic addition line NAU1815,t1VL#5 ditelosomic addition line NAU1816,homozygous T1DL?1VS#5 translocation line NAU1817,and homozygous T1DS?1VL#5 translocation line NAU1818 were developed from the selfing progenies of 1V#5 and 1D double monosomic line that derived from F1 hybrids of NAU1813/NAU0686.All of them were characterized by fluorescence in situ hybridization,genomic in situ hybridization,1V-specific markers analysis,and powdery mildew tests at different developmental stages.A new powdery mildew resistance gene named Pm67 was physically located in the terminal bin(FL 0.70–1.00)of 1VS#5.Lines with Pm67 exhibited seedling stage immunity and tissue-differentiated reactions at adult plant stage.The sheaths,stems,and spikes of the Pm67 line were still immune,but the leaves showed a low degree of susceptibility.Microscopic observation showed that most penetration attempts were stopped in association with papillae on the sheath,and colonies cannot form conidia on the susceptible leaf of Pm67 line at adult plant stage,suggesting that the defence layers of the Pm67 line is tissue-differentiated.Thus,the T1DL?1VS#5 translocation line NAU1817 provides a new germplasm in wheat breeding for improvement of powdery mildew resistance.

Keywords:Wheat Dasypyrum villosum Powdery mildew Pm67

1.Introduction

Wheat (Triticum aestivumL.) is a widely produced grain crop that contributes to global food security.Existing and new races of fungal pathogens constantly threaten the high and stable yields of wheat.Among them,powdery mildew,caused byBlumeria graminisf.sp.tritici(Bgt),occurs in many wheat-growing regions,especially in highly productive areas where semi-dwarf cultivars are planted under high input conditions [1].Powdery mildew interferes with photosynthesis,thereby reducing plant growth,heading,grain filling,and end-use quality.When weather is favorable and the mycelium occurs at flag leaf emergence or during heading,grain yield losses of up to 40% can occur [2].Utilization of resistant cultivars is the most practical and cost-effective means to control this disease.

The traditional powdery mildew resistance breeding strategy involves the use of a single major resistance gene in the major wheat production where powdery mildew usually causes significant yield losses.Genotypes produced by this strategy provide strong selection pressure for pathogen variants with an increased ability to reproduce upon them.Consequently,the effectiveness of most deployed resistance genes is rapidly lost as the pathogens acquire virulence.AlthoughPm2,Pm4,Pm6,Pm8,Pm13,Pm21,andPm30genes are frequently detected in winter wheat cultivars in China[3–6],onlyPm21provides sufficient protection from powdery mildew in the field currently.Varieties with the T6AL?6VS translocation containingPm21are planted on more than 4 million hectares annually [6,7].Moreover,more than 40% of elite lines carryingPm21were detected in the main wheat production region Yangze River,China,where powdery mildew can cause significant yield losses and most of the knownPmgenes have lost their effectiveness.So far due to good protection ofPm21the use of this gene continues,which will increase the selection pressure for virulent variants of the pathogen.Thus,the discovery and utilization of additional sources of powdery mildew resistance to address this vulnerability is necessary for wheat improvement programs in China and elsewhere.

Wild relatives of wheat are an important source of genetic variation for variety improvement [8].Most species belonging to the tertiary gene pool are not common hosts forBgtand their resistance appears to be broad-spectrum and effective over long periods of time [9,10].Among themDasypyrum villosum(2n=14,VV) is recognized as an excellent species of new resistance genes because it is a near-nonhost to many wheat pathogens and has widespread immunity to multiple diseases,such as stripe rust,leaf rust and powdery mildew[11].TwoD.villosumaccessions,91C43(#4,originally introduced from the Cambridge University Botanical Garden,UK),and 01I140 (#5,collected in the Pisa region of Italy),have been used to develop wheat-D.villosumintrogression lines at the Cytogenetics Institute of Nanjing Agricultural University (CINAU) [12,13].Three formally designated powdery mildew resistance genes,Pm21,Pm55,andPm62,have been transferred fromD.villosumto wheat by means of compensating Robertsonian translocations (RobTs),confirming thatD.villosumis indeed an important source of powdery mildew resistance genes for wheat [13–15].

A set of 1V#5 to 7V#5 disomic addition/substitution lines have been developed in wheat variety NAU0686 background from the progenies of NAU0686/NAU1802 (AABBVV (#5)) at CINAU.Their responses to stripe rust,leaf rust,and powdery mildew were assessed in field disease nurseries and the greenhouse.Results indicated that wheat-D.villosum1V#5 (1D) disomic substitution line NAU1813 conferred effective resistance againstBgtat both seedling and adult stages.The objectives of this study were to:(1)characterize the response of NAU1813 and its chromosome 1V#5 to powdery mildew,(2) introgress the chromosome 1V#5 powdery mildew resistance gene from NAU1813 into the wheat genome through the breakage-fusion mechanism,and(3)physically map a new powdery mildew resistance gene on chromosome 1V#5.

2.Materials and methods

2.1.Plant materials

Durum variety ZY1286 introduced from CIMMYT was initially crossed withD.villosumaccession 01I140 (#5) to generate the amphiploid NAU1802 (2n=42,AABBVV).The 1V#5 (1D) disomic substitution line NAU1813 and 1V#5 deletion line NAU1814 were obtained from BC4F2progenies of the cross NAU0686/NAU1802//4*NAU0686.The T1DL?1VS#4 translocation line NAU1811 and T1DS?1VL#4 translocation line NAU1812 derived fromD.villosumaccession 91C43 (#4) [16] were used as positive controls in molecular marker analyses.An elite T5DL?5VS#4 translocation line TF5V-1 (pedigree: NAU0686/NAU415(T5DL?5VS#4)//4*NAU0686) harboringPm55was used to pyramid powdery mildew resistance genes.All the genetic stocks developed and maintained at CINAU were summarized in Table S1.

All the 1V#5 introgression lines were planted in the field powdery mildew nursery at the Jiangpu Experiment Station of Nanjing Agricultural University for examination of powdery mildew response at different developmental stages.Following inoculation,the materials were covered with plastic sheeting for protection and enhancing disease development during the winter season.Ten plants were grown in each 1.0 m row with 25 cm spacing between rows.Each line with the exception of F2segregating individuals was grown in four rows with three replications.The recurrent parent NAU0686 planted on both sides of each experimental row served as an inoculum spreader ofBgtand susceptible control.

2.2.Cytogenetic analysis

Root tips from the germinating wheat seedlings were soaked in 0.2 μmol L-1amiprophos-methyl (APM) solution for 2 h at 24and then treated with nitrous oxide for 1.5 h and 90% acetic acid for 10 min following Komuro et al.[17].Slides were prepared for genomicin situhybridization (GISH) and fluorescencein situhybridization (FISH) as described by Du et al.[18].Total genomic DNA ofD.villosumwas labeled with fluorescein-12-dUTP (green)as the GISH probe.FISH was performed using mixed oligo-pAs1-2 and oligo-pAs1-5 probes that preferentially paint tandem repeats on D genome chromosomes of common wheat.The oligo-probes labeled by 6-carboxytetramethylrhodamine (TAM) producing red signals were synthesized by the Tsingke Biological Technology Company (Nanjing,China) following the procedures described by Du et al.[18].After hybridization,chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and colored blue (Invitrogen Life Science,Carlsbad,CA,USA),and slides were examined under an Olympus BX60 microscope (Olympus Co.).Images were captured with a SPOT Cooled Color Digital Camera(Diagnostic Instruments,Sterling Heights,MI,USA) and processed using Adobe Photoshop CS 7.0(Adobe Systems,San Jose,CA,USA).

2.3.Molecular marker analysis

Genomic DNA of the genetic stocks were isolated from seedling leaves using a DNA Secure Plant Kit (Tiangen Biotech Co.,Ltd,Nanjing),according to the manufacturer’s instructions.Four chromosome 1V-specific markers,including1V-207,1V-253,1EST-493,andP1/P5developed previously [19,20] were used to screen the progenies of NAU0686/NAU1802//4*NAU0686 for lines carrying chromosome 1V#5 or parts thereof.Markers1V-207and1V-253were specific for chromosome arm 1VS,and1EST-493andP1/P5were specific for 1VL (Table S2).PCR amplification was conducted using a T100 Thermal Cycler (Bio-Rad Laboratories,Hercules,USA)in 10 μL reaction volumes containing 40 ng of genomic DNA,2.5 mmol L-1of each dNTP,2 μmol L-1of each primer,2.5 mmol L-1MgCl2,1× PCR buffer (10 mmol L-1Tris-HCl,50 mmol L-1KCl,pH 8.5),and 0.2 UTaqDNA polymerase.Ampli-fication of DNA samples was programmed at 95for 4 min fol-lowed by 32 cycles at 94,58for 45 s,and 72 s of elongation at 72,with a final extension at 72for 10 min.PCR products were separated on 8% nondenaturing polyacry-lamide gels (Acr:Bis=39:1) and visualized by silver staining.

2.4.Evaluation of powdery mildew response

Seedlings of chromosome 1V#5 introgression lines and recurrent parent NAU0686 were planted in a greenhouse at 18/22(night/day) with 80% relative humidity and photoperiod of 16 h light/8 h darkness.Bgtisolates E26 and E31,and a mixed field sample collected from Yangze River region were used in separate tests under controlled greenhouse conditions.Infection types(ITs)were recorded on a 0–4 scale at 10 days post inoculation when susceptible NAU0686 control plants were heavily diseased [3].

In addition,a mixture ofBgtisolates (collected from Yangze River region) was used to infect NAU0686 and 1V#5 introgression lines at the three-leaf growth stage in the field powdery mildew nursery.Reactions of 1V#5 introgression lines evaluated at the tillering,stem elongation,booting,heading,and grain-filling stages were recorded on a 0–9 IT scale [21].

2.5.Microscopic evaluation

For evaluation of the difference in mycelium development,the leaf and sheath segments of the resistant line NAU1813 and susceptible line NAU1814 at the grain-filling stage were examined by an endogenous peroxidase-dependentin situhistochemical staining procedure.About 5 cm segments of leaf and sheath were transferred to tubes to distain by a solution of acetic acid–ethanol(1:3,v/v)for 24 h.After that,about 1.5 cm segments were cut and immersed into a solution containing 0.6%Coomassie Brilliant Blue,15%trichloroacetic acid in 99%methanol(1:1 v/v)for 25 min[10].Microscope slides were prepared by embedding the stained segments of leaf and sheath in 100% glycerol,with the adaxial side up.Slides were screened using an Olympus BX60 microscope(Olympus Co.) under a white light.

2.6.Evaluation of agronomic traits

The plants with and without T1DL?1VS#5 translocated chromosome were evaluated for plant height (PH,cm),main spike length(SL,cm),grains per spike (GPS) and 1000-kernel weight (TKW,g).Differences in main agronomic traits between the genotypes were evaluated by means of Tukey’s post hoc test (SPSS 16.0) at the P <0.05 significance level.

3.Results

3.1.Development and identification of the 1V#5 introgression lines

Two wheat-D.villosum1V#5 introgression lines in the BC4F2progeny of NAU0686/NAU1802//4*NAU0686 were firstly identified by GISH/FISH and molecular markers.NAU1813 had 40 wheat chromosomes and twoD.villosumchromosomes with one strong hybridization band in the proximal centromere region,and one strong band each in the subtelomeric and telomeric regions of the short arm (Fig.1a).FISH patterns produced with the oligopAs1-2 and oligo-pAs1-5 probes and four 1V-specific marker analysis confirmed that the wheat chromosome 1D pair had been substituted by a pair ofD.villosumchromosomes 1V#5(Fig.1a and 2).NAU1814 had 42 wheat chromosomes and twoD.villosumchromosomes with one strong hybridization band in the proximal centromere region but missing the bands in the subtelomeric and telomeric regions of the short arm (Fig.1b).The specific bands for molecular markers1V-207,1EST-493,andP1/P5were present but the 1VS-specific band of1V-253was missing (Fig.2a and b),suggesting that NAU1814 had a deletion of the distal region of the short arm of chromosome 1V#5 with the breakpoint at FL 0.70.Seedling stage powdery mildew tests showed that NAU1813 was immune (IT 0) whereas NAU1814 was susceptible(IT 4),indicating that a powdery mildew resistance gene(s) was located in the terminal region of chromosome arm 1VS.

D.villosumgenomic DNA labeled with fluorescein-12-dUTP(green) as probes was used for GISH.Probes for FISH were OligopAs1-2 and Oligo-pAs1-5 labeled with TAM (red).Wheat chromosomes were counterstained with DAPI (blue).

(a)GISH/FISH patterns of NAU1813(1V#5(1D),2n=42);Chromosome 1V#5 shows one strong hybridization band at the proximal centromere region,and one strong band each in the subtelomeric and telomeric regions of the short arm.(b) GISH/FISH patterns of NAU1814(2n=44),which has a spontaneous deletion of distal part of the short arm of chromosome 1V#5 with the breakpoint at FL0.70.(c)GISH/FISH patterns of NAU1815(2n=44),which was a t1VS#5 ditelosomic addition line.(d) GISH/FISH patterns of NAU1816(2n=44),a t1VL#5 ditelosomic addition line.(e)GISH/FISH patterns of NAU1817(2n=42),a homozygous T1DL?1VS#5 translocation line.(f) GISH/FISH patterns of NAU1818 (2n=42),a homozygous T1DS?1VL#5 translocation line.Bars,10 μm

1VS-specific markers1V-207(a) and1V-253(b);1VL-specific markers1EST-493(c) andP1/P5(d).Marker,DL2000.The 1DL?1VS lines NAU1811 and NAU1817 have 1VS-and 1DL-specific bands but lack the 1DS-specific band.The 1DS?1VL lines NAU1812 and NAU1818 have 1VL-and 1DS-specific bands but lack the 1DLspecific band.

In order to identify Robertsonian whole-arm translocations of chromosome 1V and a wheat chromosome,the progeny of double monosomic 1V#5 and 1D hybrids of NAU1813 crossed with NAU0686 were screened.Among 250 selfing progenies of 1V#5 and 1D double monosomic plant Bx-4b-13 screened with four 1Vspecific markers,15 plants(6%) had only the 1VS-specific markers1V-207and1V-253.GISH/FISH analysis showed that 14 of these plants had a 1VS#5 telosome and one plant (YL-2b-11) was heterozygous with a normal 1D chromosome and a T1DL?1VS#5 translocation chromosome.Other 13 plants (5.2%) had only the 1VL-specific markers1EST-493 andP1/P5;12 had a 1VL#5 telosome and one(YL-2b-3)was heterozygous for a T1DS?1VL#5 Robertsonian translocation.Lines derived from these various plants included t1VS#5 ditelosomic addition line NAU1815(Fig.1c),t1VL#5 ditelosomic addition line NAU1816 (Fig.1d),homozygous T1DL?1VS#5 translocation line NAU1817(Fig.1e),and homozygous T1DS?1VL#5 translocation line NAU1818(Fig.1f).

In addition,100 seedlings among the selfing progenies of YL-2b-11 (heterozygous T1DL?1VS#5) were examined by GISH;20 were disomic for a T1DL?1VS#5 recombinant chromosome pair,58 were heterozygous and 22 lacked alien chromatin indicating normal gametic transmission of the translocated chromosome relative to its intact 1D homologue (=2.56,Pgreater than 0.05).Measures of agronomic traits shown in Table S3 revealed no obvious differences in plant height,1000-kernel weight,main spike length,and seeds per main spike between plants with and without the T1DL?1VS#5 recombinant chromosome.

3.2.Powdery mildew reactions

Seedlings of above YL-2b-11 selfing-plants were tested for reaction to the field isolatesBgt.All 78 plants harboring 1VS#5 chromosome arm were immune (IT 0–0;),whereas the remaining 22 plants lacking 1VS#5 chromatin were susceptible (IT 3–4)(Table S3).Thus,the powdery mildew resistance gene(s) located on chromosome arm 1VS#5 was dominant and formally designatedPm67.

For characterization ofPm67resistance at different growth stages NAU1813,NAU1814,NAU1815,NAU1816,NAU1817,and NAU1818 were tested separately for reactions toBgtisolates E26 and E31 and a field collection at two-to three-leaf stage.NAU1813,NAU1815,and NAU1817 had IT of 0 (Fig.3a),whereas NAU1814,NAU1816,and,NAU1818 were susceptible (IT 4) as was the recurrent parent NAU0686.In field nursery evaluations,NAU1813,NAU1815,and NAU1817 were highly resistant (IT 0) at the tillering stage(Fig.3b),but showed differences in leaf and leaf sheath reactions from the booting stage.The sheaths of these lines continued to be near immune at all growth stages but the leaves showed a low degree of susceptibility (IT 1–2) (Fig.3c and d).The other three lines were highly susceptible on all tissues similar to those of NAU0686.As the terminal deletion in chromosome 1VS in line NAU1814 led to a highly susceptible response at all growth stages(Fig.4a),Pm67was therefore physically located in the distal region (FL 0.70–1.00) of chromosome 1VS#5.

(a) Seedling (three-leaf) stage,NAU1817 shows immune response to Bgt.(b) Tillering stage,NAU1817 still shows immune response to Bgt.(c) Stem elongation stage,the sheath of NAU1817 shows immune but leaf shows mild necrotic reaction phenotype.(d) Heading stage,the sheath of NAU1817 shows immune but leaf shows a low degree susceptibility.

(a) Sheathes and leaves of NAU1813,NAU1814,and NAU0686,the sheath of NAU1813 shows immune toBgtand limited colonies are formed on the leaf of NAU1813;whereas,Bgtforms large colonies on the leaf and sheath tissues of NAU1814 and NAU0686.(b)Sheath and leaf tissues ofPm55line TF5V-1,Pm67line NAU1817 and their F1hybrid,suggesting pyramidingPm55andPm67genes could provide complete protection from powdery mildew across all tissues.

3.3.Microscopic evaluation of Bgt infected lines

Leaf and sheath infection onPm67line NAU1813 and susceptible line NAU1814 were distinctively different under high inoculum density(Fig.4a).Microscopic observation showed that most of the infection attempts ofBgtwere stopped in association with papilla formation on the sheath of NAU1813 (Fig.5c) and no microcolonies were observed.AlthoughBgtis able to form a lower degree of colony on the leaves of NAU1813 (Fig.4a),the established micro-colonies were not able to form conidiophores on the leaves (Fig.5a).Meanwhile,plenty of conidiophores were formed on leaf and sheath tissues of susceptible line NAU1814 (Fig.5b and d).

(a) Established micro-colony without conidia on the leaf of NAU1813.Micro-colony is indicated with arrow.(b) Established micro-colony with large conidiophores on the susceptible NAU1814 leaf.Conidiophores are indicated with arrows.(c)Stopped penetration attempt with papilla formation on the sheath of NAU1813.Papilla is indicated with arrow.(d)Established microcolony with large conidiophores on the sheath of NAU1814.Conidiophores are indicated with arrows.

3.4.Pyramiding of Pm55 and Pm67

Fig.2.PCR amplification patterns of representative 1VS-and 1VL-specific markers in the wheat-D.villosum introgression lines.

Previously,we reported a T5DL?5VS#4 translocation line NAU415 harboringPm55that confers a near immune response on leaves after the 5-leaf stage but sheaths and spikes tend to develop some disease [15],which is somewhat opposite to the symptoms described here for plants carryingPm67.An elite T5DL?5VS#4 translocation line TF5V-1 (pedigree:NAU0686/NAU415//4*NAU0686) harboringPm55was used to cross withPm67line NAU1817 for pyramiding the two powdery mildew resistance genes.Interestingly,all the F1plants exhibited nearimmune adult plant responses on leaves,sheaths (Fig.4b) and spikes (data not shown),suggesting that pyramiding these two genes could provide complete protection from powdery mildew across all tissues.

4.Discussion

In the present study,six new 1V#5 introgression lines,including disomic substitution line DS1V#5 (1D) (NAU1813),del-1VS?1VL(#5) disomic addition line NAU1814,t1VS#5 ditelosomic addition line NAU1815,t1VL#5 ditelosomic addition line NAU1816,homozygous T1DL?1VS#5 translocation line NAU1817,and homozygous T1DS?1VL#5 translocation line NAU1818 were developed (Fig.1;Table S1).All the 1V#5 introgression lines allowed mapping the alien gene on a chromosome arm.Followed by theBgtevaluation,a dominantPmgene was physically located on the 1VS#5 terminal fragment (FL 0.70–1.00).Previously,three powdery mildew resistance genes,includingPm21,Pm55,andPm62,were introgressed into wheat fromD.villosumchromosomes 6 V,5 V,and 2 V,respectively,and effective againstBgtraces in the Yangze River area [13–15].However,no resistance gene derived from chromosome 1V has been documented.Thus,this resistance gene located on 1VS#5 is new and permanently namedPm67.The compensating Robertsonian translocation (RobT) T1DL?1VS#5 in NAU1817 carryingPm67will be useful as a new germplasm in breeding for resistance.

Fig.3.Powdery mildew reactions of NAU1817 (T1DL?1VS#5) and its recurrent parent NAU0686 at different developmental stages.

Fig.4.Powdery mildew reactions on sheath and leaf tissues at the heading stage planted in greenhouse under high inoculum density.

The powdery mildew resistance sources of wheat were traditionally described as seedling resistance and adult-plant resistance(APR).Seedling resistance genes are generally race-specific and expressed throughout the plant life cycle[22].Whereas,APR genes come into effect at post seedling stages when disease damage is likely to occur under field conditions [23,24].Lines withPm67exhibited immunity at seedling stage and tissue-differentiated reactions at adult plant stage (Fig.3).The sheath,stem and spike tissues of thePm67line were still immune,but the leaves showed a low degree of susceptibility (Fig.4a,b).Histology analyses showed that most penetration attempts were stopped in association with papilla formation on the sheath (Fig.5c),and colonies cannot form conidia on the susceptible leaf ofPm67line (Fig.5a).Thus,the defence ofPm67should be on independent layers on the leaf and sheath tissues at adult plant stage.Interestingly this differential tissue response was opposite to that shown in plants withPm55(Fig.4b).An F1hybrid between lines possessingPm67andPm55showed complete immunity on all tissues at whole stage(Fig.4b).Therefore,Pm55andPm67might represent genes with distinct defense mechanisms at adult plant stage and our findings extend the current understandings of the genes against the wheat fungal pathogens.

Chromosome 1V ofD.villosumis homoeologous to the group 1 chromosomes of bread wheat and has been described as a source for the improvement of various end-use quality of wheat flour[11,25–30].The introgression lines involving 1VS carryingD.villosumstorage protein lociGlu-V1,Gli-V1,andGlu-V3significantly increased wheat gluten strength and protein content,and enhanced flour end-use quality [16,25].Thus,the multiple genes(Glu-V1/Glu-V3/Gli-V1/Pm67) on 1VS#5 arm could represent an ideal combination for modern wheat varieties with good end-use quality and disease resistance if 1VS#5 has no negative effect on agronomy traits.Although the detailed effect of T1DL?1VS#5 translocation chromosome on yield related traits is unknown at present,the important agronomic traits,such as plant height,fertility,kernel weight and anthesis are similar to background parent NAU0686 in the field (Fig.S1;Table S3).Additional studies are in progress to transferPm67into different wheat cultivars and to have a yield trial in order to assess whether the 1VS#5 introgression is associated with any deleterious effects on yield or quality,and whether a smaller 1VS segment will be required for agronomic exploitation.

D.villosumis a near-nonhost to many pathogens of wheat,including rust and powdery mildew [11,13].Although the genetic base of powdery mildew resistance in differentD.villosumaccessions is not clear,this research extends our knowledge of the polygenic mode of inheritance inD.villosumto powdery mildew.Genetic mapping and complementation analysis are necessary to isolate the underlying gene.The fundamental problem in studying the inheritance of alien resistance gene is lack of recombination between the homoeologous chromosomes of wheat and its relatives [8].To date,chromosome 1V from at least five accessions ofD.villosumhas been introgressed into wheat represented by a range of addition,substitution and translocation lines[16,25,30,31].The powdery mildew reactions of wheat-D.villosumdisomic addition line DA1V#1 [31] and disomic substitution line DS1V#2(1B)[26]are unknown,but translocation lines T1DL?1VS#3[25]and T1DL?1VS#4[16]were susceptible.Fifty-two 1VS-specific molecular markers were identified by Zhang et al.[19,20],and 11 of them showed codominant polymorphic differences between the susceptible T1DL?1VS#4 line NAU1811 and resistant T1DL?1VS#5 line NAU1817 as exampled for1V-207and1V-253showed in Fig.2a and b.Thus,the polymorphic markers together with genetic segregation in crosses of translocation lines NAU1811 and NAU1817 will permit genetic mapping of thePm67gene if chromosome arms 1VS#4 and 1VS#5 normally pair and recombine at meiotic phase in the hybrids of NAU1811/NAU1817.Research is currently underway to genetically map and clonePm67for revealing its defense mechanisms.

Fig.5. Bgt hyphal development on sheaths and leaves of NAU1813 and NAU1814 at the heading stage planted in greenhouse under high inoculum density.

CRediT authorship contribution statement

Ruiqi Zhangconceived and designed the experiments.Chuanxi Xiong,Ruonan Yao,and Xiangru Mengdeveloped introgression lines;Chuanxi Xiong and Lingna Konganalyzed molecular markers;Liping Xing,Jizhong Wu,and Yigao Fengidentified the Bgt reaction;Ruiqi Zhang and Aizhong Caowrote the paper.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Prof.Robert McIntosh and Prof.Peidu Chen for critical reading of the manuscript.This work was supported by the National Natural Science Foundation of China(31971938),the Natural Science Foundation of Jiangsu Province (BK20181316),the Special Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu,China(CX(19)1001),and Fundamental Research Funds for the Central Universities (KYZ201809).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.09.012.