Xioheng Yu,Shizhong Ren,Lnfei Zho,Jun Guo,Yingung Bo,Yingxue M,Hongwei Wng,Herert W.Ohm,Dzho Yu,Hongjie Li,Lingrng Kong,*

aState Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai'an 271018, Shandong, China

bDepartment of Agronomy, Purdue University, West Lafayette, IN 47907-1150, USA

cHubei Academy of Agricultural Sciences, Wuhan 430064, Hubei, China

dInstitute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Keywords:Marker-assisted selection Ml92145E8-9 Powdery mildew Triticum aestivum L.

ABSTRACT Powdery mildew,caused by Blumeria graminis f.sp.tritici(Bgt),is one of the most devastating diseases of common wheat(Triticum aestivum L.).The wheat line 92145E8-9 is immune to Bgt isolate E09.Genetic analysis reveals that the powdery mildew resistance in 92145E8-9 is controlled by a single dominant gene,temporarily designated Ml92145E8-9.Bulkedsegregant analysis(BSA)with simple sequence repeat(SSR)markers indicates that Ml92145E8-9 is located on chromosome 2AL.According to the reactions of 92145E8-9,VPM1(Pm4b carrier),and Lankao 906(PmLK906 carrier)to 14 Bgt isolates,the resistance spectrum of 92145E8-9 differs from those of Pm4b and PmLK906,both of which were previously localized to 2AL.To test the allelism among Ml92145E8-9,Pm4b and PmLK906,two F2populations of 92145E8-9×VPM1(Pm4b)and 92145E8-9×Lankao 906(PmLK906)were developed in this study.Screening of 784 F2progeny of 92145E8-9×VPM1 and 973 F2 progeny of 92145E8-9×Lankao 906 for Bgt isolate E09 identified 37 and 19 susceptible plants,respectively.These findings indicated that Ml92145E8-9 is non-allelic to either Pm4b or PmLK906.Thus,Ml92145E8-9 is likely to be a new powdery mildew resistance gene on 2AL.New polymorphic markers were developed based on the collinearity of genomic regions of Ml92145E8-9 with the reference sequences of the International Wheat Genome Sequencing Consortium(IWGSC).Ml92145E8-9 was mapped to a 3.6 cM interval flanked by molecular markers Xsdauk13 and Xsdauk682.This study also developed five powdery mildew-resistant wheat lines(SDAU3561,SDAU3562,SDAU4173,SDAU4174,and SDAU4175)using flanking marker-aided selection.The markers closely linked to Ml92145E8-9 would be useful in marker-assisted selection for wheat powdery mildew resistance breeding.

1.Introduction

Powderymildew,causedbyBlumeriagraminis(DC.)E.O.Speerf.sp.tritici(Bgt),is an important disease of common wheat(Triticum aestivum L.),particularly in highly productive areas with a maritime or semi-continental climate,that results in severe grain yield and quality losses[1,2].In addition to fungicides and other biological agents,disease-resistant cultivars are considered the most economically effective and environmentally safe method of controlling powdery mildew[3].There are two types of inherited powdery mildew resistance:qualitative and quantitative.Qualitative resistance to powdery mildew,which is controlled by major genes,has been extensively employed in wheat breeding programs.However,these race-specific resistance genes are effective against only certain Bgt isolates.The durability of qualitative resistance genes is generally limited by the continuous evolution of pathogen populations[4].Consequently,it is necessary to identify novel genes for powdery mildew resistance to newly emerging virulent Bgt isolates.To date,more than 77 genes or alleles for resistance to wheat powdery mildew,including Pm1–Pm54(Pm8 is allelic to Pm17,Pm18=Pm1c,Pm22=Pm1e,Pm23=Pm4c,and Pm31=Pm21)and more than 30 temporarily designated Pm genes,have been identified at 56 loci distributed across all wheat chromosomes[5–7].Ofthesegenes,multipleallelesexistinseveralchromosome loci,including the Pm2a–2c locus on chromosome arm 5DS[8–11],the Pm3a–3j locus on chromosome arm 1AS[12],the Pm4a–4d locus on chromosome arm 2AL[13],and the Pm24a–24b locus on chromosome arm 1DS[14].Although several Pm genes or alleles have been identified,only a handful of these,including Pm2a,Pm4a,Pm4b,Pm6,Pm8,and Pm21,have been successfully used for breeding cultivars[15,16].However,because frequent changes in pathogen populations often overcome the effects of available resistance genes,the identification of diverse resistance genes and clusters of resistance genes is an ongoing process[17–19].

Molecular markers have been successfully used to map powdery mildew resistance genes in wheat[20,21].Simple sequence repeats(SSRs)are easy to handle,inexpensive,highly polymorphic,and reliable.To date,several powdery mildew resistance genes have been successfully tagged using SSR markers.For instance,Pm2b has been tagged and successfully transferred to powdery mildew-susceptible cultivars including Shimai 15,Shixin 828,and Gaocheng 8901 and efficiently selected using closely linked markers for improvement of powdery mildew resistance[8].

92145E8-9 is resistance to powdery mildew during the entire growth period in the field and under controlled conditions.The objectives of the present study were to(1)identify the Bgt resistance gene in line 92145E8-9,(2)perform resistance spectrum comparisons and test the allelism of Ml92145E8-9,PmLK906,and Pm4b,and(3)develop markers closely linked to Ml92145E8-9 for marker-assisted selection(MAS).

2.Materials and methods

2.1.Plant materials

Wheat line 92145E8-9 provided by coauthor H.W.Ohm,was employed as the resistant parent in a cross with a highly susceptible Chinese common wheat cultivar Huixianhong.Sixty F1hybrids,251 F2segregating populations,and 193 F2:3families were evaluated for powdery mildew resistance to Bgt isolate E09.Huixianhong was used as a susceptible control.Cultivars VPM1 carrying Pm4b and Lankao 906 carrying PmLK906 were used as parents to generate two F2populations from crosses with 92145E8-9 for testing allelism among Pm4b,PmLK906,and Ml92145E8-9.

2.2.Evaluation of powdery mildew resistance

The parental lines 92145E8-9 and Huixianhong and the F1,F2,and F2:3families were grown in plastic trays,placed in a growth chamber,and inoculated at the one-leaf stage with Bgt isolate E09 by dusting with newly developed conidia from susceptible seedlings of Huixianhong.Isolate E09 was provided by Prof.Zhiyong Liu of the Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing,China.For each F2:3family,15–20 plants were inoculated with powdery mildew.The plants were scored by infection type(IT)at 15 days after inoculation when Huixianhong was heavily infected and reconfirmed five days later following Liu et al.[22]and Zeng et al.[23],where 0 represents an immune reaction,0;necrotic flecks without uredia,and 1,2,3,and 4 highly resistant,moderately resistant,moderately susceptible,and highly susceptible,respectively.Phenotypes with IT2 or lower were classified as resistant,and those with 3–4 were considered as susceptible[22,23].Observed and expected segregation ratios were compared using the chi-squared(χ2)test for goodness of fit.

2.3.Marker analysis

Genomic DNA was extracted from seedling leaves of the parental lines and F2progeny following the CTAB protocol[24].For bulked-segregant analysis(BSA)[25],equal amounts of DNA from 10 highly resistant and susceptible F2plants were mixed separately to form the resistant(BR)and susceptible(BS)bulks.Wheat genomic SSRs(Xgwm,Xwmc,Xbarc,Xcfa,Xcfd,Xpsp,and Xgpw series,http://wheat.pw.usda.gov/)were used to screen for polymorphisms between the two parents and the two bulks.The polymorphic markers were further used to genotype the mapping populations.PCR was conducted in a 10 μL reaction volume.The PCR program was as follows:one denaturation cycle at 95°C for 5 min,followed by 35 cycles at 94 °C for 30 s,50–60 °C(depending on the specific primers)for 30 s,and 72°C for 50 s,and an extension step of 72 °C for 10 min.The PCR products were mixed with 3 μL of loading buffer(98%formamide,10 mmol L-1EDTA,0.25%bromophenolblue,and0.25%xylene cyanol).ThePCR amplification mixeswerethen loaded into 8% nondenatured polyacrylamide gels (39 acrylamide: 1 bisacrylamide).After electrophoresis,the gels were silverstained and photographed[26].

2.4.Comparative genomic analysis and polymorphic SSR marker development

Chinese Spring reference sequences assembled by the International Wheat Genome Sequencing Consortium(http://www.wheatgenome.org/)and 454 shotgun sequences[27]were both used to identify collinearity of genomic regions of Ml92145E8-9.Using the software SSR Finder (http://fresnostate.edu/csm/faculty-research/ssrfinder/)to identify SSR motifs in these contigs and scaffold sequences,SSR primer pairs were designed with Primer3(Version 0.4.0)(http://bioinfo.ut.ee/primer3-0.4.0/)with the following parameters:amplification product size of 200 bp to 300 bp,primer length of 18 bp to 22 bp,primer Tmof 55 °C to 60 °C,and primer GC content of 40%–60%.

2.5.Genetic map construction

A genetic linkage map of the powdery mildew resistance gene Ml92145E8-9 was constructed using the software JoinMap 4.0[28].Map distances were calculated using the Kosambi function[29].

3.Results

3.1.Genetic analysis of the powdery mildew resistance gene in 92145E8-9

Line 92145E8-9 is resistant(IT=0)to Bgt isolate E09 and Huixianhong is highly susceptible(IT=4)(Fig.S1).Sixty F1plants of 92145E8-9×Huixianhong were all resistant to Bgt isolate E09 with the same infection type(IT=0)as 92145E8-9,indicating that powdery mildew resistance in wheat line 92145E8-9 is controlled by one or more dominant genes.In the F2population,179 plants were resistance and 72 plants were susceptible,fitting the 3:1 segregation ratio expected for control by a single gene.The F2:3families consisted of 54 homozygous resistant:85 segregating:54 homozygous susceptible lines,fitting the expected ratio of 1:2:1 for monogenic resistance(Table 1).These results indicate that the powdery mildew resistance gene in 92145E8-9 is controlled by a single dominant gene,which has been temporarily designated Ml92145E8-9.

3.2.Linkage mapping of Ml92145E8-9

For linkage mapping of the resistance gene in 92145E8-9,the resistant and susceptible DNA bulks of the F2population and the DNA of the parents were initially used to test 486 pairs of SSR primers randomly distributed across 21 wheat chromosomes.Four SSR markers(Xpsp3039,Xwmc658,Xcfa2086,and Xgdm93)were found to be polymorphic between two parents and two bulks and were mapped to chromosome 2AL.Fiftytwo STS and SSR markers on chromosome 2AL were subsequently surveyed for polymorphism.Four further markers(Xgwm356,Xgwm526,Xgpw2046,andXwmc181)revealed polymorphism.Finally,the Ml92145E8-9 gene was mapped to an 18.8 cM interval between SSR markers Xwmc181 and Xgpw2046.Thus,thenewlyidentifiedpowderymildew resistance gene Ml92145E8-9 was localized to the long arm of chromosome 2A.

3.3. Collinearity analysis and polymorphic marker development

ThecorrespondingsequencesofthepolymorphicSSR markers(Table S1)were used as query sequences for BLAST(Basic Local Alignment Search Tool)search against the Chinese Spring contigs,to search for the reference sequences and develop new markers.Using the designed eight polymorphic SSR markers(Xsdauk12,Xsdauk13,Xsdauk638,Xsdauk662,Xsdauk682,Xsdauk782,Xsdauk829,and Xsdauk869),the target resistance gene Ml92145E8-9 was mapped to a 3.6 cM interval flanked by markers Xsdauk13 and Xsdauk682(Fig.1,Table 2).

3.4.Comparison of Ml92145E8-9 with reported powdery mildew resistance genes on chromosome 2AL

Todate,sixresistancegeneshavebeen identifiedon chromosome 2AL:Pm4[30],PmLK906[31],PmPS5A[32],Pm50[33],PmHNK54[34],and PmX[35].Physical bin mapping has localized Pm4 to 2AL1-0.85-1.00[13].However,genetic mapping indicated that the powdery mildew resistance gene Ml92145E8-9 located in chromosome 2AL bin C-2AL1-0.85 together with resistance genes PmHNK54 and Pm50[33,34].By comparing the SSR markers closely linked to PmHNK54 and Pm50,the marker Xgwm312 closely linked to PmHNK54[34]was mapped 35.7 cM from Ml92145E8-9 and Pm50 was found to lie 3.8 cM from Xgwm294[33],which was mapped to 22.7 cM from Ml92145E8-9.Thus,Ml92145E8-9 differs from PmHNK54 and Pm50.

3.5.Testing for allelism among Ml92135E8-9,Pm4b,and PmLK906

To test whether Ml92145E8-9 is an allele of Pm4 or Lankao 906,an allelism test was conducted by crossing 92145E8-9 with VPM1(Pm4b)and Lankao 906(PmLK906).Fourteen Bgt isolateswere used in a pathogenicity test of Ml92145E8-9,Pm4b,and PmLK906 at the seedling stage(Table 3).Ml92145E8-9 and PmLK906 were resistant to Bg74-3,Bg68-2,Bg75-2,and Bg44-5.Bg78-3 was avirulent on PmLK906,but virulent on Ml92145E8-9.Pm4b conferred complete resistance to Bg68-2,Bg75-2,and Bg81-2.

Table 1–Reactions of F2population,F2:3families of 92145E8-9×Huixianhong to isolate E09.

Fig.1–Comparative linkage map of Pm genes on chromosome2AL.CS2AL physicalbin(a),2ALconsensus linkage map(b),linkagemap of Ml92145E8-9(c),linkagemap of PmHNK54(d),linkagemap of Pm50(e),linkage map of Pm4a(f),linkage map of Pm4b(g),linkage map of Pm4c(h),linkage map of Pm4d(i).

Table 2–Description of newly developed SSR markers on chromosome 2AL.

To further clarify the relationships of Ml92135E8-9,Pm4b,and PmLK906,784 F2plants from 92145E8-9×VPM1 and 973 F2plants from 92145E8-9×Lankao 906 were inoculated with Bgt isolate E09.The inoculation results identified 37 susceptible plants from the cross 92145E8-9×VPM1 and 19 susceptible plants from the cross 92145E8-9×Lankao 906.The allelism test confirmed that Ml92135E8-9 is a novel powdery mildew resistance gene that is distinct from Pm4b and PmLK906.

3.6.Application of Ml92145E8-9 in wheat breeding by MAS

To facilitate the use of Ml92145E8-9 in wheat breeding,MAS combined with phenotypic selection was performed to select homozygous progeny carrying the target gene.Line 92145E8-9 was crossed separately with Gaokang 1 and Liangxing 99.A total of 1605 F2plants from the cross Liangxing 99×92145E8-9were screened with codominant markers linked to Ml92145E8-9(Xsdauk13 and Xsdauk682)and MlLX99(Xcfd73 and Xwmc441).A total of905 plants carried Ml92145E8-9 and MlLX99,including 94 homozygotes;311 F2lines of the 905 lines carried only Ml92145E8-9 and 302 of the 905 F2lines carried MlLX99.Marker-assisted selection combined with powdery mildew resistance evaluation and agronomic trait selection was conducted in different generations of Liangxing 99×92145E8-9and Gaokang1×92145E8-9.F5:6progeny derived from Liangxing 99×92145E8-9 and F5:6progeny derived from Gaokang 1×92145E8-9 were tested with Bgt isolate E09 under controlled incubator conditions and separately with a natural Bgt population in the greenhouse(Table S2).Three of the progeny of Gaokang 1×92145E8-9,named SDAU4173,SDAU4174,and SDAU4175,were screened with Xsdauk13 and Xsdauk682 to identify homozygous lines carrying the target gene Ml92145E8-9(Fig.S2-a,c).The three progeny lines showed complete resistance to Bgt isolate E09,indicating that Ml92145E8-9 had been successfully transferred into SDAU4173,SDAU4174,and SDAU4175.SDAU3561 and SDAU3562,derivedfromLiangxing99×92145E8-9,were screened with the markers closely linked to Ml92145E8-9(Xsdauk13 and Xsdauk682)and MlLX99(Xcfd73 and Xwmc441).Both Ml92145E8-9 and MlLX99 had been successfully pyramided in lines SDAU3561 and SDAU3562,which showed complete resistance to Bgt isolate E09(Figs.S2-b,d,S3-a,b;Table S2).Furthermore,the agronomic traits seed protein,plant architecture,and 1000-grain weight in the homozygous progenies were improved in comparison with 92145E8-9(TableS2).Itisnoteworthythattheplantheightsof SDAU4173,SDAU4174,SDAU4175,SDAU4174,and SDAU4175 were lower than that of 92145E8-9,a finding promising for lodging resistance.

Table 3–Infection types of PmLK906,Ml92145E8-9,and Pm4b in response to Bgt isolate E09 and 14 isolates of Blumeria graminis f.sp.tritici from China.

4.Discussion

In this study,Ml92145E8-9 was a single dominant gene localized to chromosome 2AL.To date,Pm4[30],PmLK906[31],PmPS5A[32],Pm50[33],PmHNK54[34],and PmX[35]have also been mapped to chromosome 2AL.Pm4 was assigned to chromosome 2AL by telocentric mapping[30]and localized to asub-telomericregionofchromosome2ALbylinkage mapping[36].Powdery mildew resistance genes mapped to the Pm4 locus include Pm4a,Pm4b[30],Pm4c[37],Pm4d[13],PmPS5A[32],PmLK906[31],PmX[35],and a major QTL for powdery mildew resistance that coincided with Pm4b[38].

In contrast to the linkage maps of the resistance genes at the Pm4 locus(Fig.1),SSR marker Xgwm356 has been linked to all these genes.Among these,Pm4a,Pm4b,and Pm4c show similar genetic distances from marker Xgwm356 and are distal to Pm4d and Ml92145E8-9.The genetic distance between the STS marker BCD1231 that co-segregated with Pm4a[39]and Ml92145E8-9 is 50.3 cM.The STS marker XResPm4[13]appears to be locus-specific for Pm4d,which has been localized 49.3 cM from Ml92145E8-9.The distance between Xgwm356 and Pm4b was 3.7 cM and that between Xgwm356 and Pm4c 3.5 cM,whereas that between Xgwm356 and Ml92145E8-9 was 19.8 cM in this study.Variation in genetic distances and orders of markers or genes among different linkage maps is a common phenomenon.These differences may be attributed to various factors such as population size,mapping parents,chromosome structural variation,and experimental error.Pm4[36],PmPS5A[32],PmLK906[31],and PmX[35]were physically assigned to a distal bin(wheat deletion bin 2AL1-0.85-1.00).Ml92145E8-9 was assigned to bin C-2AL1-0.85 based on the physical location of Xwmc181,Xgpw2046,and Xgwm356,which are closely linked to Ml92145E8-9.The other two resistance genes,PmHNK54[34]and Pm50[33],are also located in chromosome bin C-2AL1-0.85.According to the 2AL consensus linkage map[38],PmHNK54 is flanked by markers Xbarc5 and Xgwm312[34],and Xgwm312 is located 35.7 cM from Ml92145E8-9.Pm50 is located 3.8 cM from marker Xgwm294[33]and 22.7 cM from Ml92145E8-9.Thus,Ml92145E8-9 is not identical to Pm50 and PmHNK54.These results show that Ml92145E8-9 is likely not located at the same position as other powdery mildew resistance genes previously mapped on chromosome 2AL.

In the presentstudy,the resistance spectrum of Ml92145E8-9 differed from those of Pm4b and PmLK906.Allelism testing using F2plants from the cross of 92145E8-9×VPM 1 and 92145E8-9×Lankao 906 was further conducted to clarify the relationships between Ml92145E8-9,Pm4b,and PmLK906.The susceptible recombinants identified in the F2generations indicate that Ml92145E8-9 is distinct from Pm4b and PmLK906.Thus,Ml92145E8-9 is likely to be a novel powdery mildew resistance gene in wheat.

Compared to conventional plant breeding,MAS is more efficient and less time-consuming.The flanking markers Xsdauk13 and Xsdauk682 of Ml92145E8-9 were readily used for MAS of Ml92145E8-9 in the present study.The progeny from MAS with homozygous resistance genes showed that these are immune to Bgt isolate E09 in controlled biological incubator conditions and highly resistant to a natural Bgt population in the greenhouse.The agronomic characters of progeny lines were superior to those of 92145E8-9(Table S2).Thus,the powdery mildew resistance conferred by Ml92145E8-9 has been rapidly and efficiently incorporated into other wheat cultivars and may be useful in wheat breeding.

Acknowledgments

We are grateful to Prof.Zhiyong Liu of Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing,China for providing Bgt isolate E09 and Dr.Robert McIntosh from University of Sydney,Australia for revising the manuscript.The work was financially supported by Genetically Modified Organisms Breeding Major Projects(2016ZX08009003-001-006),the NationalNaturalScience Foundation of China(31471488 and 31520203911),and the National Basic Research Program of China(2014CB138100).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.04.004.