Dioguo An *,Guoho Hn ,Jing Wng ,Hnwen Yn ,Yilin Zhou ,Lijun Co ,Yuli Jin,Xiotin Zhng

a Center for Agricultural Resources Research,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Shijiazhuang 050022,Hebei,China

b The State Key Laboratory for Biology of Plant Disease and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China

c The Innovative Academy of Seed Design,Chinese Academy of Sciences,Beijing 100101,China

Keywords:Blumeria graminis f.sp. tritici Disease resistance Ditelosomic line Secale cereale Triticum aestivum

ABSTRACT Rye(Secale cereale genome RR),a close relative of common wheat,possesses valuable resistance genes for wheat improvement.Due to the co-evolution of pathogen virulence and host resistance,some resistance genes derived from rye have lost effectiveness.Development and identification of new,effective resistance genes from rye is thus required.In the current study,wheat-rye line WR56 was produced through distant hybridization,embryo rescue culture,chromosome doubling and backcrossing.WR56 was then proved to be a wheat-rye 2RL ditelosomic addition line using GISH (genomic in situ hybridization),mc-FISH (multicolor fluorescence in situ hybridization),ND-FISH (non-denaturing FISH),mc-GISH (multicolor GISH) and rye chromosome arm-specific marker analysis.WR56 exhibited a high level of adult plant resistance to powdery mildew caused by Blumeria graminis f.sp.tritici(Bgt).This resistance was carried by the added 2RL telosomes and presumed to be different from Pm7 which is also located on chromosome arm 2RL but confers resistance at the seedling and adult stages.WR56 will be a promising bridging parent for transfer of the resistance to a more stable wheat breeding line.A newly developed 2RL-specific KASP (kompetitive allele specific PCR) marker should expedite that work.

1.Introduction

Powdery mildew caused by Blumeria graminis (DC.) E.O.f.sp.tritici(Bgt)is one of the most economically important wheat(Triticum aestivum L.)diseases worldwide[1].Resistant cultivars are the preferred means to control powdery mildew.Although resistance alleles at 68 loci (Pm) have been formally designated,several are duplicates (Pm18=Pm1c,Pm22=Pm1e,Pm23=Pm4c,and Pm31=Pm21),and more than 30 others in wheat and its close relatives carry temporary designations [2–4].However,rapid evolution in the pathogen leads to new virulent isolates that neutralize the resistance in previously resistant cultivars.Consequently,damaging epidemics continue to be a problem despite breeding efforts [5,6].Therefore,it is important to continue the search for new sources of resistance and to introduce them into breeding populations.

Cereal rye(Secale cereale L.,2n=2x=14,RR),a close relative of common wheat,possesses many valuable genes for disease resistance,yield improvement,abiotic-stress tolerance,and wide adaptation [7].Many resistance genes have been introduced from various rye sources.For example,powdery mildew resistance gene Pm8,along with stripe rust resistance gene Yr9,stem rust resistance gene Sr31 and leaf rust resistance gene Lr26 are all present in a chromosome T1BL·1RS introgression with 1RS being from Petkus rye [8];Sr50 is present in a 1RS introgression from Imperial rye;and Pm17 and Sr1RSAmigoare present in a 1RS introgression from Insave rye [9].Other cereal rye introgressions with wheat names include Pm7 and Lr25 in chromosome arm 2RL from Rosen rye;Lr45 in 2R from Petkus rye;Sr59 in arm 2RL from triticale line VTB28041;Sr27 in arm 3RS from Imperial rye;Pm20 in arm 6RL from Prolific rye;Pm56 in arm 6RS from Qinling rye;and Yr83 in arm 6RL from Merced rye [6,9–11].Several of these resistance genes are no longer effective.For example,resistance genes Pm8,Yr9 and Lr26 were rapidly overcome in most countries,and Sr31 was defeated by the well-known Ug99 race group in East Africa after many years of worldwide deployment[12,13].Most cultivars of cereal rye are naturally cross-pollinating,and genetic heterogeneity may be present both within and between cultivars[14,15].Therefore,it is especially essential to continually explore and characterize more effective and durable resistance gene sources from other rye genotypes against new pathogen isolates.

Combined analyses of GISH (genomic in situ hybridization) and mc-FISH (multicolor fluorescence in situ hybridization) along with rye chromosome-specific markers enable accurate identification of rye chromosomes,or chromosome segments,in wheat[6,10].With the increased numbers of rye introgressions involving the same chromosome regions it becomes more important to identify and utilize unique rye-specific markers for efficient tracing of each one.In the absence of recombination,a marker in any part of an introgression within wheat will be completely associated with a gene for any trait of interest within the introduced segment.The only exceptions will be in crosses between wheat lines with overlapping chromosome introgressions that allow pairing between the introduced rye chromosome segments.KASP (kompetitive allele specific PCR) markers are currently preferred for selection in high-throughput resistance breeding [10,16,17].

Winter rye cultivar German White is immune to many wheat diseases,including powdery mildew,at both the seedling and adult growth stages[6]whereas the winter wheat cultivar Xiaoyan 6,an important founder parent in Chinese wheat breeding,is highly susceptible[18].A program of distant hybridization between Xiaoyan 6 and German White has been carried out in our laboratory since the 1990s.A series of wheat-rye derivatives,including chromosome translocation lines WR04-32 [13] and WR41 [19],chromosome substitution line WR02-145 [20] and chromosome addition lines WR35 [6] and WR49 [21] were developed and characterized by molecular cytogenetic methods.Wheat-rye derivative WR56 has shown a high level of adult plant resistance to powdery mildew for 12 years.The objectives of the current study were to (i)identify the chromosome composition of WR56 using molecular cytogenetic methods;(ii) characterize the resistance to powdery mildew using multiple Bgt isolates and determine whether the resistance was derived from the rye parent;and (iii) develop and validate a 2RL-specific KASP marker to identify plants with the powdery mildew resistance gene.

2.Materials and methods

2.1.Plant materials

Wheat-rye derived line WR56 was developed from a cross between wheat cultivar Xiaoyan 6 and rye cultivar German White.The hybrids were recovered by embryo rescue and following chromosome doubling,the amphiploid was backcrossed to the wheat parent with testing for response to a composite of Bgt isolates in each generation.Wheat genotypes with known Pm gene (s) were used as controls when evaluating the powdery mildew response of WR56 at the seedling stage.These included Kavkaz with Pm8,Amigo with Pm17,CI14189 with Pm7,and TAM104/Thatcher with Pm20.Twenty-five single-pustule-derived Bgt isolates were used to test WR56 and the other wheat genotypes in the same way.Wheat cultivar Mingxian 169 with no known resistance gene was used as a susceptible control for both seedling and adult stage tests.F2and F2:3populations of a cross of WR56 and susceptible cultivar Shixin 633 were used to determine the inheritance of adult plant resistance and to evaluate the 2RL-specific KASP marker.A complete set of disomic addition lines(DA1R to 7R),2RS and 2RL ditelosomic addition lines derived from cross Chinese Spring (CS) × Imperial,and CS 4A,4B,and 4D nullisomic lines were used to characterize WR56 by PCR analysis.

2.2.GISH,mc-FISH,non-denaturing FISH (ND-FISH) and multicolor-GISH (mc-GISH) analyses

GISH was performed for detection of rye chromatin in WR56.The chromosomes of root tips cells were prepared and observed as previously described[19].Total genomic DNA of German White was labeled with fluorescein-12-dUTP as probe using nick translation and genomic DNA of CS was used as a blocker for GISH detection with a probe:blocker ratio of 1:50 [16].Chromosomes were counterstained with 4,6-diamidino-2-phenylindole (DAPI),detection and visualization of rye chromatin were performed with an epifluorescence Olympus BX53 microscope fitted with a cooled CCD digital camera.Images were processed and analyzed as described elsewhere [10].

After GISH detection,mc-FISH and ND-FISH were conducted using highly repeated DNA sequences as probes.The probe pSc119.2 (green) combined with pAs1 (red) were used in mc-FISH detection [6].Probes Oligo-pSc119.2-2 (green) and Oligo-pTa535-2 (red) for ND-FISH detection were synthesized by Shanghai Invitrogen Biotechnology Co.,Ltd.(Shanghai),and FISH analyses were performed following Tang et al.[22].

The genomic identity of WR56 was determined by mc-GISH detection.Total genomic DNA of German White and T.urartu(2n=2x=14,AA)were labeled with fluorescein-12-dUTP,and total genomic DNA of Aegilops tauschii (2n=2x=14,DD) was labeled with Texas-red-5-dUTP and total genomic DNA of Ae.speltoides(2n=2x=14,BB) was used for blocking [23].Detection and visualization were performed as previously described [19].

2.3.PCR analysis

EST-SSR (expressed sequence tag -simple sequence repeat)markers CGG62 (F:5′-GCCCTCGACGACATGAAA-3′,R:5′-CGCTTGC CGGTCTTGTAT-3′) and SWES120 (F:5′-CGACGACTACCTCCTCAA GAA-3′,R:5′-GAACAGGCAACGACGACAG-3′) specific for rye chromosome arms 2RS and 2RL[24],respectively,were used to identify rye chromosomes in WR56.SSR markers Xwmc52,Xwmc285,Xwmc720 and Xcfd106 specific for wheat chromosome arm 4DS,and six SSR markers Xwmc331,Xwmc399,Xwmc457,Xwmc622,Xcfd23 and Xcfd84 specific for 4DL,were used to confirm the presence or absence of wheat chromosome 4D in WR56 [25].Xiaoyan 6,CS,and CS 4A,4B and 4D nullisomic lines were used as controls.

2.4.Assessments of powdery mildew reaction

To assess the adult plant reactions of WR56 to powdery mildew,a mixture of Bgt isolates E09,E18 and E20 representing isolates currently prevalent in China was used to infect field nurseries in early spring during 2016– 2020.Tests were performed in three replicates in each year at Luancheng Agro-Ecological Experimental Station,Chinese Academy of Sciences,Shijiazhuang,in Hebei province.Xiaoyan 6,German White,and controls Kavkaz,CI14189,Amigo,TAM104/Thatcher and Mingxian 169 were included.Each genotype was planted in two 1.5 m rows with 20 seeds per row spaced 25 cm apart,and all trials were surrounded with Mingxian 169 as an inoculum spreader.Powdery mildew reactions were scored at heading when Mingxian 169 showed full disease development and again during the grain-filling stage,using a 0–9 scale with 0–4 considered resistant and 5–9 susceptible [26].

Seedling tests with 25 single-pustule-derived isolates were separately assessed in three replicated trials in a greenhouse.The methods of inoculation and disease induction are described in Qie et al.[3].Xiaoyan 6,German White,Mingxian 169,and Kavkaz,CI14189,Amigo,TAM104/Thatcher with the known Pm gene (s)were used as controls.At 15 days post-inoculation,when the pustules were fully developed on Mingxian 169,infection types (IT)were recorded according to a 0–4 scale;plants with IT 0–2 were considered resistant and those with IT 3–4 were susceptible[19,27].

2.5.Genetic analysis of powdery mildew resistance in WR56

To determine whether the resistance of WR56 was derived from its German White rye parent,an F2population of cross WR56 × Shixin 633 was tested in the field.EST-SSR marker SWES120 specific for rye chromosome arm 2RL and GISH analysis were used to detect the presence or absence of 2RL in each F2plant.

2.6.Development and validation of KASP marker

To expedite future transfer of the powdery mildew resistance of WR56 into susceptible wheat cultivars,SLAF(specific-locus amplified fragment)-based marker SW22374 specific for rye 2RL was converted to a KASP marker as previously described[10].The KASP amplification system and thermal cycling conditions were set up as described by Han et al.[10].The reaction was performed in a Bio-Rad CFX real-time PCR system (Bio-Rad Laboratories,Inc.California,USA),and fluorescence was detected using Bio-Rad CFX Manage 3.1 software.

3.Results

3.1.GISH analysis of WR56

GISH results for WR56 showed that the 44 chromosomes included a pair of ditelosomic rye chromosomes (Fig.1A,B).GISH analyses of offspring plants from three consecutive generations of selfing confirmed high cytological stability.Therefore,WR56 was proved to be a genetically stable ditelosomic line with 44 chromosomes.

3.2.FISH analysis of WR56

Fig.1.GISH(Genomic in situ hybridization),mc-FISH(multicolor fluorescence in situ hybridization)and ND-FISH(non-denaturing fluorescence in situ hybridization)analyses of wheat-rye 2RL ditelosomic line WR56.(A) and (B) GISH analyses in WR56.(C) Sequential mc-FISH on the same metaphase after GISH analysis (A) of WR56 using probes pSc119.2(green)and pAs1(red).(D)ND-FISH analysis on the same metaphase cell after GISH analysis(B)of WR56 using probes Oligo-pSc119.2–2(green)and Oligo-pTa535-2 (red).Arrows point to the 2RL telosomes,stars indicate a pair of small,atypical wheat chromosomes.Scale bars,10 μm.

Mc-FISH was conducted to characterize the rye and wheat chromosomes in WR56.There were two strong hybridization sites in the telomere regions of the rye telosomes in WR56 when labeled with both pSc119.2 and Oligo-pSc119.2-2,while there were no hybridization signals on those chromosomes when labeled with pAs1 or Oligo-pTa535-2 (Fig.1C,D).Thereby,the rye telosomes in WR56 were identified as 2RL.Using pSc119.2 and pAs1 as probes,the 20 wheat chromosome pairs in WR56 were distinguished from each other and one pair of wheat 4D chromosomes was replaced by an unidentified pair of small chromosomes(Fig.1C).Detection results using Oligo-pSc119.2-2 and OligopTa535-2 as probes were consistent with those using pSc119.2 and pAs1(Fig.1D).Therefore,WR56 was identified as a 2RL ditelosomic addition line,which also contained one pair of unknown small chromosomes replacing the normal wheat 4D chromosome pair.

3.3.Mc-GISH analysis of WR56

Mc-GISH results identified seven chromosome pairs from each of the A-and B-genomes,six chromosome pairs from the Dgenome,and one pair of ditelosomic rye chromosomes in WR56,respectively (Fig.2).In addition,one pair of small chromosomes showed a deep brown or gray color (4-pointed stars,Fig.2).The absence of the 4D chromosome pair was presumably compensated by an extra pair of unknown B-genome chromosomes.

3.4.Molecular marker analysis of WR56

EST-SSR markers CGG62 and SWES120 specific for rye chromosome arms 2RS and 2RL amplified approximate 290 bp and 110 bp diagnostic fragments in German White,respectively.The corresponding fragments were also detected in Imperial rye 2RS and 2RL ditelosomic lines,and 2R disomic addition line from cross CS×Imperial,but not in Xiaoyan 6,CS,and the other addition lines of CS × Imperial.These results indicated that the two markers could be used for detecting the entire 2R chromosomes or separate 2R telosomes from German White.WR56 could amplify only the 110 bp specific band of CGG62 indicating that it contained DNA regions specific for the German White 2RL chromosome,but not 2RS.In addition,the absence of four and six SSR markers specific for wheat chromosome arms 4DS and 4DL,respectively,confirmed the absence of chromosomes 4D in WR56.These markers amplified in controls including Xiaoyan 6,CS and CS plants that were nullisomic for chromosomes 4A or 4B,but not CS plants nullisomic for 4D.

Fig.2.Multicolor-genomic in situ hybridization (mc-GISH) analysis of WR56.The cell shows two bright-green R-genome telosomes (indicated by arrows),yellow-green Agenome chromosomes,including the well-known T4A-7B translocation pair (5-pointed stars),brown or gray B-genome chromosomes,and 12 pink or red D-genome chromosomes.A pair of unknown small B-genome-like chromosomes are indicated by 4-pointed stars.Scale bar,10 μm.

Fig.3.Assessment of resistance to powdery mildew at the adult stage and molecular detection of rye chromosome 2RL from WR56 using 2RL-specific marker SWES120 in F2 plants from cross WR56×Shixin 633.M,marker pUC19 Msp I;1,Xiaoyan 6;2,German White rye;3,Shixin 633;4,WR56;R and S,10 resistant and 10 susceptible F2 plants from cross WR56 × Shixin 633,respectively.Arrow indicates the diagnostic fragment of marker SWES120 specific for 2RL.

The combined results from the PCR and GISH/FISH showed that WR56 was a wheat-rye 2RL ditelosomic addition line with 44 chromosomes.However,it lacked chromosomes 4D,but contained a pair of small unidentified chromosomes,possibly from the Bgenome.

3.5.Reactions of WR56 to powdery mildew

Although German White showed seedling immunity (infection type (IT) 0) to all 25 Bgt isolates,WR56 along with Xiaoyan 6 and Mingxian 169 was susceptible (IT 4).However,WR56 was highly resistant in adult plant field tests during the 2016– 2020 wheatgrowing seasons whereas control lines were highly susceptible.

3.6.Powdery mildew resistance is conferred by rye chromosome

To determine the inheritance of adult plant resistance in WR56,a population of 127 F2plants from cross WR56 × Shixin 633 was genotyped with rye chromosome arm 2RL-specific marker SWES120,subjected to GISH analysis and phenotyped for adult plant response to powdery mildew.All 48 resistant plants possessed at least one rye chromosome arm 2RL,whereas the remaining 79 susceptible plants lacked rye chromatin.WR56 showed a high level of resistance whereas Xiaoyan 6 and Shixin 633 were highly susceptible.Co-segregation of the resistance phenotype with 2RL indicated that the powdery mildew resistance of WR56 was conferred by rye chromosome arm 2RL (Fig.3).

3.7.Development and validation of the 2RL specific KASP marker

KASP marker WRK2RL (forward 1 with rye allele:5′-gaaggtgac caagttcatgctTTGCACTTCTTTAGGTATGCTC-3′,forward 2 with wheat allele:5′-gaaggtcggagtcaacggattTTGCACTTCTTTAGGTATGCTT-3′,reverse:5′-TGAGGGTTTCCTTTCTTT-3′) was developed and validated to achieve efficient tracing of the 2RL telosome that conferred powdery mildew resistance.

4.Discussion

Rye is a novel source of resistance to fungal diseases of wheat,including powdery mildew,leaf rust,stripe rust(yellow rust),stem rust and sharp eyespot,and is therefore recognized as a species potentially useful for wheat improvement [6].In this study,a new wheat-rye line WR56 developed by distant hybridization and selection was shown to be a wheat-rye 2RL ditelosomic addition line by GISH/FISH analyses and molecular marker assays.The adult plant resistance in WR56 was associated with the presence of the 2RL telosome.All other sources of powdery mildew transferred from rye to wheat confer resistance at both the seedling and adult plant stages (all-stage resistance).Since adult plant resistance is regarded to be more durable than all-stage resistance,further work to transfer the resistance in WR56 to a wheat chromosome should be pursued.

WR56 was determined to be a stable wheat-rye 2RL ditelosomic addition line that lacked chromosome 4D,which was apparently replaced and compensated by an unidentified short chromosome pair that was determined to be B-genome-like by mc-GISH.A similar situation of small chromosomes in the progenies of 4D nullisomic plants derived from Xiaoyan 6 was reported previously[28] and in that case 55 % chromosome pairing was observed between the abnormal chromosome and chromosome 4D.DNA genotyping arrays will be developed and used to determine the identity of this chromosome pair in future work.

The release of high-quality genome assemblies of rye cultivars Weining [29] and Lo7 [30] will enable generation of high-density physical map of chromosome 2R and hence the fine mapping of the APR gene in WR56 and aid its transfer to a wheat chromosome.

CRediT authorship contribution statement

Diaoguo An:Writing–original draft,Conceptualization,Supervision,Funding acquisition.Guohao Han:Writing–review&editing,Resources,Formal analysis,Methodology.Jing Wang:Methodology,Formal analysis,Visualization.Hanwen Yan:Methodology,Formal analysis.Yilin Zhou:Methodology,Formal analysis.Lijun Cao:Formal analysis.Yuli Jin:Investigation.Xiaotian Zhang:Investigation.

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

This research was supported by the National Natural Science Foundation of China (31771793 and 31801358),the National Key Research and Development Program of China (2016YFD0102002),and the Natural Science Foundation of Hebei Province(C2019503064).The authors are grateful to Dr.John Raupp from Kansas State University,Manhattan,KS,for kindly providing the complete set of disomic addition lines and 2RS and 2RL ditelosomic addition lines of ‘Chinese Spring × Imperial rye’,and to Dr.Yiwen Li from Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing,for the 4A,4B and 4D nullisomic lines of Chinese Spring.The authors are grateful to Prof.R.A.McIntosh,Plant Breeding Institute,University of Sydney,for the critical review and language editing of this manuscript.