Suli Sun,Zhongyi Wang,Haining Fu,Canxing Duan,Xiaoming Wang,Zhendong Zhu*

National Key Facility for Crop Gene Resources and Genetic Improvement,Institute of Crop Science,Chinese Academy of Agricultural Sciences, Beijing 100081,China

Resistance to powdery mildew in the pea cultivar Xucai 1 is conferred by the gene er1

Suli Sun1,Zhongyi Wang1,Haining Fu,Canxing Duan,Xiaoming Wang,Zhendong Zhu*

National Key Facility for Crop Gene Resources and Genetic Improvement,Institute of Crop Science,Chinese Academy of Agricultural Sciences, Beijing 100081,China

A R T I C L E I N F O

Article history:

Accepted 6 August 2015

Available online 17 August 2015

Erysiphe pisi er1-2 Pea Powdery mildew Xucai1

Powdery mildew,caused by Erysiphe pisi D.C.,is a major constraint to pea production worldwide.The pea cultivar Xucai1 has shown high resistance to E.pisi under greenhouse and field conditions.The objectives of this study were to identify and characterize genes conferring resistance to powdery mildew in Xucai 1.Three crosses,Qizhen 76×Xucai 1, Bawan 6×Xucai 1,and Xucai 1×Bawan 6,were made to generate populations for genetic analysis.The resistance to E.pisi and segregation ratios in the F1,F2,and F2:3populations suggested a single recessive gene conferring the resistance of Xucai 1.Bulked segregant analysis was used to map the resistance gene using two F2populations.The resistance gene was close to markers AD60 and c5DNAmet on linkage group VI with genetic distances of 9.9 cM and 15.4 cM in the Xucai 1×Bawan 6 F2population and 8.7 cM and 8.1 cM in the Qizhen 76×Xucai1 F2population,respectively,suggesting that the resistance gene was aner1 allele.This hypothesis was confirmed by comparison of the cDNA sequences of the

PsMLO1 gene between the parents and the PsMLO1 wild type.Three distinct types of transcripts in Xucai 1,characterized by a 129-bp deletion and 155-and 220-bp insertions, were detected,consistent with the structure of the er1-2 allele.We concluded that resistance in Xucai 1 was conferred by er1-2 and that its linked markers will be useful in pea breeding programs.

©2015 Crop Science Society of China and Institute of Crop Science,CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

Pea(Pisum sativum L.)is widely distributed and has been cultivated in China for more than two thousand years[1]. According to the latest report,China ranks second only to Canada in dry pea production[2].Pea frequently suffers from biotic and abiotic stresses throughout the lifecycle of the plant.Powdery mildew,caused by Erysiphe pisi D.C.,is one of the most important pea diseases worldwide.The disease severely affects yield and quality of peas[3,4].It is of serious concern in temperate and subtropicalregions with warm,dry days and cool nights[4].Powdery mildew caused by E.pisi results in 25-50%yield losses in pea production[5-7].Severe infection by E.pisi can result in yield loss of up to 80%in susceptible cultivars[8].In pea production,agronomic practices,chemical prevention,and genetic resistance strategieshave been applied to controlthe disease.However,the control efficacy of chemical and agronomic practices is limited by many factors.Genetic resistance has become the first choice not only for its efficiency,low cost,and environmental friendliness[8,9]but also for qualitative disease resistance that are relatively easy to use in breeding programs.

As early as 1948,Harland[10]identified powdery mildew resistance in the pea landrace Huancabamba and showed that it carried a single recessive gene.Since then,screening and genetic analysis of resistance to pea powdery mildew have been performed for more than 60 years[9].Many resistant pea accessions have been identified and characterized their gene(s)for resistance to E.pisi.Two recessive genes(er1 and er2)and one dominant gene(Er3)have been identified for powdery mildew resistance in pea germplasm[10-12].Genetic analyses of resistance to E.pisi indicated that the vast majority of resistant pea accessions carried the resistance gene er1[13].The resistance gene er2 is harbored by only a few resistant pea accessions[13].Er3 is a newly identified dominant gene from a wild relative of pea(P.fulvum)that has recently been successfully introduced into cultivated pea (P.sativum)[7,12].

With respect to their resistance mechanisms,the modes of action at the cellular level of the two single recessive genes,er1 and er2,have been revealed by histological studies [14,15].er1 confers complete immunity or high levels of durable broad-spectrum resistance to powdery mildew by inhibiting E. pisi invasionofpea epidermalcells.The expression ofresistance gene er2 is influenced primarily by temperature and leaf age. The complete resistance conferred by er2 is revealed only at high temperature(25°C)or in mature leaves and is based mainly on post-penetration cell death,mediated by a hypersensitive response[14].The Er3 gene confers effective resistance to all E.pisi isolates tested to date[7].On Er3 plants,most E.pisi conidia are able to penetrate pea epidermalcells and form secondary hyphae,but growth of these established colonies is prevented by a strong hypersensitive response[12,16].

DNA marker technologies and their application in genetic analysis have developed rapidly over the last decade.In peas, molecular markers linked to the three resistance genes(er1, er2,and Er3)for powdery mildew have been developed by several groups[17-25].The single recessive genes er1 and er2 have been located on pea linkage groups(LGs)VI and III, respectively[18,22,26],whereas Er3 has been mapped between the sequenced characterized amplified region(SCAR)marker Scw4637and random amplified polymorphic DNA(RAPD) marker OPAG05_1240,located on an uncertain pea LG[27].

The recessively inherited resistance gene er1 has been used successfully for decades in pea breeding programs[13]. Recently,the resistance of er1 in pea has been found to be similar to that of MLO(mildew resistance locus O)genes in barley(Hordeum vulgare L.),Arabidopsis thaliana,and tomato (Lycopersicon esculentum Mill.)[28,29].The er1 resistant phenotype is caused by loss-of-function mutations of a pea MLO homolog,known as PsMLO1[28,29].Thus far,five er1 alleles (er1-1 to er1-5)have been characterized in pea resistant accessions,according to the mutation site and pattern of PsMLO1,each corresponding to a different PsMLO1 mutation [28-30].Among the reported five er1 alleles,er1-1 and er1-2 are used commonly in pea breeding programs[28,29].

In China,powdery mildew is one of the most important diseases among more than 20 diseases infecting peas and severely affects the yield and quality ofpeas[31].The incidence of pea powdery mildew has reached 100%in some regions of Sichuan province[32].To date,the pathogen E.pisi is thought to be the only causal agent of pea powdery mildew in China [33-35],although two other species,E.baeumleri and E.trifolii, have been reported to cause powdery mildew on peas and showed the same symptoms as E.pisi in the Czech Republic[36] and the USA[37].Ondřej et al.[36]and Attanayake et al.[37] showed that E.baeumleri and E.trifolii were able to overcome the er1 gene conferring resistance to E.pisi.Fondevilla et al.[38] further confirmed that E.trifolii had also overcome resistance gene Er3,but not er2.In China,screening for resistance to E. pisi in pea germplasms has been performed in greenhouses and in fields in recent years[32-35].Several pea cultivars/ landraces were highly resistant to E.pisi isolates EPBJ(NCBI, accession number KR912079)and EPYN(NCBI,accession number KR957355)[34,35].Both E.pisi isolates were identified by morphological and molecular characterization[39].However,the genetic basis of their resistance and the characteristics of their resistance gene(s)are unknown.

Xucai 1,derived from a cross between Cuipimi and Baofeng 2,is the firstsemi-leafless pea cultivar(cv.)in China.Xucai1 has desirable traits including high yield and resistance to lodging and drought,root rot,and powdery mildew[40].Recently,Zeng et al.[33]showed that Xucai 1 was stably resistant to pea powdery mildew both in the greenhouse and in fields in 3 consecutive years.The present study was designed to clarify the genetic basis of this resistance and identify the resistance gene for powdery mildew present in Xucai1.With this aim,we performed an inheritance study using three crosses between resistant cv.Xucai1 and two susceptible cultivars(cvs.),Qizhen 76 and Bawan 6.We further characterized the resistance gene as e r1 by sequencing cDNA sequences of the PsMLO1 gene in the parents of the crosses and the pea cv.Sprinter[28],which is susceptible to E.pisi and carries the PsMLO1 wild-type allele.

2.Materials and methods

2.1.Plant materials and inoculum

Three pea cultivars,Xucai 1(resistant to E.pisi),Qizhen 76 (susceptible to E.pisi),and Bawan 6(susceptible to E.pisi)were used in the experiment.Xucai 1 was kindly provided by Prof. Fengbao Wang of the Hebei Normal University of Science& Technology,Qinghuangdao,Hebei province,China;Bawan 6 was provided by Mr.Dongxu Xu of the Zhangjiakou Academy of Agricultural Sciences,Zhangjiakou,Hebei province,China; and Qizhen 76 was from Dr.Xin Chen of Jiangsu Academy of Agricultural Sciences,Nanjing,Jiangsu province,China.Three crosses were produced including reciprocal crosses between Bawan 6 and Xucai 1 and Qizhen 76×Xucai 1.Derived F1,F2, and F2:3populations of each cross were subjected to phenotypic evaluation and inheritance analysis.The F1plants from the crosses were self-pollinated to produce F2progeny.Each F2plant produced seeds for the F2:3families by self-pollination and was threshed individually.The two F2populations derived from the crosses of Qizhen 76×Xucai 1,and Xucai1×Bawan 6 and comprising 91 and 161 individuals,respectively,were used for genotypic evaluation and genetic analysis.

The E.pisi isolate EPBJ(NCBIaccession number KR912079),a highly virulent isolate collected from a greenhouse in Beijing, was used as the inoculum in the present study[34,35].The isolate was maintained on the seedlings of susceptible pea cv.“Longwan 1.”Reproduction of inocula was achieved by continuous transfer to new healthy seedlings of pea cv.“Longwan 1”by gently shaking plants containing masses of E.pisi conidia. The inoculated plants were incubated in a growth chamber at 10±1°C with a 12 h light regime.

2.2.Inoculation with E.pisi and disease assessment

2.2.1.Parentalphenotypic evaluation

Seeds of Xucai 1,Bawan 6,and Qizhen 76 were planted in 15-cm-diameter paper pots(six seeds per pot)filled with a mixture of vermiculite and peat moss(1:1).Each parent was planted in five replications using a total of 30 seeds per cultivar.The potted plants were placed under controlled conditions in a greenhouse at 18-26°C.Ten to fourteen days after planting,the plants at the fourth-or fifth-leaf stage were inoculated with E.pisi isolate EPBJ,using conidia shed by shaking heavily infected plants of pea cv.Longwan 1 over them.Ten days after inoculation,disease severity was scored on a 0-4 scale,according to the infected foliage area, macroscopic and microscopic density of mycelia and sporulation on the lower part of the plant[41,42].Plants with scores of 0-2 were classified as resistant(R)and those with scores of 3-4 as susceptible(S)[41,42].

2.2.2.Phenotypic evaluation of F1and F2populations

Planting was performed in a propagation greenhouse to generate offspring seeds.Plants were grown under the same conditions as described above for the parents.Inoculation was conducted under controlled conditions using the detached leaf method,together with susceptible and resistant parents as the controls.This inoculation method is effective for the evaluation of resistance to E.pisi in peas[14,16].The third or fourth leaf along with petiole from the bottom of the plant was detached from each F1and F2seedling at the fourth-or fifth-leaf stage.The cut leaves were inserted into 9-cm-diameter Petri dishes containing 2%water agar and 50 mg L-1benzimidazole to enhance longevity[43].The detached leaves were then inoculated with E.pisi isolate EPBJ using the method described above forthe inoculation ofthe parentalplants.After inoculation, the plates containing the inoculated leaves were sealed tightly with Parafilm and placed in a growth chamber at 20°C with a 14 h photoperiod.The detached leaves of the three parents Xucai 1,Qizhen 76,and Bawan 6 were treated in the same way with and without inoculation as controls.The disease severity was determined using the 0-4 scale 10 days after inoculation.The plants scored as resistant to E.pisi were subjected to repeated inoculation.

2.2.3.Phenotypic evaluation of F2:3populations

Twenty-four seeds selected randomly from each F2:3family were grown as described above.Disease was scored 10 days after inoculation using the 0-4 scale.F2:3families classified as either homozygous resistant or segregating for resistance to E.pisi were subjected to repeated inoculation.A chi-squared (χ2)test was used to determine the goodness-of-fit to Mendelian segregation patterns of phenotypes of all F2and F2:3populations.

2.3.DNA extraction and pooling for bulk segregation analysis (BSA)

Genomic DNAwas isolated fromhealthy young leaves collected from seedlings of the three pea parents,Xucai1,Qizhen 76,and Bawan 6,and of two F2mapping populations derived from Qizhen 76×Xucai 1,and Xucai 1×Bawan 6 using the cetyl trimethylammonium bromide(CTAB)extraction method with minor modifications[44].The DNA concentration was determined using an ND-1000 Nano-Drop instrument(NanoDrop Technologies Inc.,Wilmington,DE,USA).The DNAsolution was diluted to a working concentration of 50 ngμL-1with Tris-EDTA buffer(pH 8.0)and maintained at-20°C.For the bulked segregant analysis(BSA),resistant and susceptible bulks were prepared from equalamounts of DNA from 10 each of resistant and susceptible individual F2plants[45].

2.4.Molecular marker analysis

Initially 148 simple sequence repeat(SSR)markers that were approximately evenly distributed over a high-density pea genetic linkage map[46]were selected to screen for polymorphisms between the resistant and susceptible parents and pooled bulks. PCR amplification of SSR markers was performed in a total volume of 20μL,containing 50 ng of genomic DNA,2.5μL 10× PCR reaction buffer(20 mmol L-1MgCl2),0.2 mmol L-1of each dNTP,1.5 U of Taq DNA polymerase,and 0.2μmol L-1of primer mixture.PCR reactions were performed at 94°C for 5 min; followed by 35 cycles of 94°C for 45 s,49-60°C(depending on the primer-specific annealing temperature according to Loridon et al.[46])for 45 s and 72°C for 1 min;with a final extension at 72°C for 10 min,using a thermal cycler(Biometra,Goettingen, Germany).The amplified PCR products were separated on a 6% polyacrylamide sequencing gel after mixing with 4μL of 6× loading buffer(0.25%bromophenolblue,0.25%xylene cyanol FF, and 40%sucrose).

SSR markers that were polymorphic between the contrasting bulks were further tested in the full F2populations derived from the crosses Qizhen 76×Xucai 1,and Xucai 1×Bawan 6. Based on the results of preliminary genetic linkage analysis, markers near the linked markers were further selected from a consensus functionalmap[47]to identify polymorphisms and analyze genetic linkage and were amplified using the method described above for SSR markers.

Four SCAR markers,ScOPD10-650[22],ScOPE16-1600[23], ScOPO18-1200[23],and ScOPX04-880[25]linked to resistance gene er1,were also used to identify polymorphisms between the two parents and contrasting bulks and to analyze genetic linkage.PCR amplification and reaction were performed as described previously[34].

2.5.Data analysis and genetic linkage map construction

Linkage analysis was performed with MAPMAKER/EXP version 3.0b[48].Genetic distances were computed using the Kosambi mapping function[49].Linkage groups were determined usinga logarithm of odds(LOD)threshold of 3.0,with a maximum distance of 50 cM.The segregation of molecular markers in the F2populations was evaluated for goodness-of-fit to Mendelian segregation ratios byχ2test.The genetic linkage map was drawn with molecular markers linked to the resistance gene using MapDraw[50].

2.6.Sequence analysis of PsMLO1 gene

Total RNA was extracted from the young leaves of three parents Xucai 1,Bawan 6,and Qizhen 76 using an RNAprep pure Plant kit(Tiangen Biotech,Co.,Ltd.,Beijing,China), according to the manufacturer's instructions.

First-strand cDNAs were synthesized using a BioRT Two Step RT-PCR Kit(Hangzhou Bioer Technology Co.,Ltd,Hangzhou, China)including an oligo(dT)primer.The PCRs were performed to amplify the full-length cDNAofthe target gene,PsMLO1,using the PsMLO1-specific primer pair PsMLO1F/PsMLO1R(F:5′-AAAAT GGCTGAAGAGGGAGTT-3′;R:5′-TCCACAAATCAAGCTGCTACC-3′)[29].The amplification reaction was performed under the following conditions:5 minat 95°C;35 cycles of30 s at94°C,45 s at 58°C(annealing temperature),and 70 s at 72°C;with a final extension of 10 min at72°C.Amplicons were purified with a PCR Purification Kit(Qiagen)and ligated into the pEasy_T5 vector (TransGen Biotech,Beijing,China).Sequencing reactions were performed using universal M13F or M13R primers at the Beijing Genomics Institute(BGI).The resulting cDNA sequences of the PsMLO1 gene in the parents were compared with the reported cDNA sequence of the PsMLO1 gene in the susceptible pea cv. Sprinter,having the wild-type PsMLO1 sequence(NCBI,accession number FJ463618.1)[28],using ClustalX2[51].

2.7.Amplification of the molecular marker er1-2/MGB

PCR amplification of marker er1-2/MGB linked to er1-2 in the parents was performed in a total volume of 20μL reaction, containing 50 ng of genomic DNA,2.5μL of 10×PCR reaction buffer(containing 20 mmol L-1MgCl2),0.8 mmol L-1of each dNTP,2.5 U oflong-range TaKaRa LA Taq(TaKaRa Bio Inc.,Otsu, Japan),and 0.2 mmolL-1of primer mixture.PCRreactions were performed in a thermalcycler(Biometra,Goettingen,Germany), at 94°C for 5 min;followed by 35 cycles of 94°C for 45 s,54°C for 3 min,and 72°Cfor 1 min;with a finalextension at 72°Cfor 15 min.The amplified PCR products were separated on a 6% polyacrylamide gel.

3.Results

3.1.Phenotypic evaluation of resistance to E.pisi

3.1.1.Parentalphenotypic evaluation

The two susceptible parents Qizhen 76 and Bawan 6 showed severe infection by E.pisi isolate EPBJ10 days after inoculation under greenhouse conditions.All plants of Qizhen 76 and Bawan 6 were covered by masses of conidia and mycelium of E.pisi,showing a disease severity of 4 and confirming their susceptibility(Fig.1;Table 1).By contrast,there were no symptoms on the resistant parent Xucai1,which appeared to be completely resistant to E.pisi isolate EPBJ(Fig.1;Table 1). These results are consistent with previous observations [31,33].

3.1.2.Phenotypic evaluations of F1,F2,and F2:3populations

The segregation pattern of pea resistance to powdery mildew in the F1,F2,and F2:3populations derived from the three crosses of Qizhen 76×Xucai 1,Bawan 6×Xucai 1,and Xucai 1×Bawan 6 are presented in Table 1.

The cross of Qizhen 76×Xucai 1 generated four F1plants that were susceptible to E.pisi(Table 1).One of four F1plants produced 91 F2plants,of which 17 were resistant and 74 susceptible.Thus,the segregation of susceptibility and resistance in F2population fitted a ratio of 3:1(χ2=1.94;P=0.16), indicating monogenic recessive inheritance.Moreover,the segregation ratio in the F2:3population gave a good fit to a 1:2:1 ratio(χ2=2.41;P=0.30)(Table 1),confirming that the resistance was controlled by a single recessive gene in Xucai1.

The cross between the susceptible parent Bawan 6 and the resistant parent Xucai 1 also generated four F1plants susceptible to E.pisi.The F2population comprised 58 plantsthat segregated into 47 susceptible and 11 resistant,with a good fit to a 3:1 ratio(χ2=1.13;P=0.28),as expected for single recessive gene inheritance(Table 1).

Fig.1–Phenotypic evaluation of the resistant pea cultivar Xucai 1(A)and susceptible cultivars Bawan 6(B)and Qizhen 76 (C)inoculated with E.pisi isolate EPBJ.

Table 1–The segregation patterns of pea resistance to powdery mildew in the F1,F2,and F2:3populations derived from the crosses of Qizhen 76×Xucai 1,Bawan 6×Xucai 1,and Xucai 1×Bawan 6.

The cross between the resistant parent Xucai 1 and the susceptible parent Bawan 6 generated eightsusceptible F1plants. In the F2population of 161 plants,35 were resistant and 126 susceptible,fitting a 3:1 ratio(χ2=0.91;P=0.34).Moreover,in the F2:3population(161 families),35 were homozygous resistant,48 were homozygous susceptible,and 78 segregated in response to isolate E.pisi EPBJ(Table 1),fitting the genetic model ratio 1:2:1 (χ2=2.26;P=0.32)for a single recessive gene.

These results indicated that the observed segregation patterns fitted 3(susceptible):1(resistant)and 1(homozygous susceptible):2(segregating):1(homozygous resistant)ratios in all F2and F2:3populations derived from the three crosses, respectively,suggesting that the resistance to E.pisi in Xucai1 was controlled by a single recessive gene.

3.2.Molecular marker analysis

Among the 148 SSR markers,59 were polymorphic between the parents Qizhen 76 and Xucai1 and 74 between Xucai1 and Bawan 6.These polymorphic SSR markers were further used to screen for polymorphism between the resistant and susceptible DNA pools.Only SSR marker AD60 in pea LG VI was polymorphic between the two contrasting DNA bulks differing in their response to E.pisi[46]and was thus likely to be linked to the resistance gene.SSR marker AD60 was verified as a marker linked to the er1 locus by Ek et al.[19]. This marker was accordingly genotyped in both F2populations.The marker was codominant and its segregation in both F2populations fitted the expected Mendelian ratio of 1:2:1. Further linkage analysis confirmed that the SSR marker AD60 was linked to the resistance gene(Fig.2-A),showing that the gene is located on LG VI,which harbors the er1 locus[19].

In addition,according to this primary mapping result, molecular markers on LG VI adjacent to SSR AD60 were selected from a genetic map[47]and analyzed in the contrasting DNA bulks.The codominant marker c5DNAmet (cytosine-5 DNA-methyltransferase)was polymorphic between the bulks and thus likely to be linked to the resistance gene.Its segregation in both F2populations fitted the expected Mendelian ratio of 1:2:1.Further linkage analysis confirmed that c5DNAmet was linked to the resistance gene(Fig.2-B).

Fig.2–The segregation patterns of molecular markers AD60(A)and c5DNAmet(B)linked to the resistance gene in some of the F2progeny derived from the cross of Qizhen 76×Xucai1.M:100 bp marker ladder;PR:resistant parent Xucai 1;PS:susceptible parent Qizhen 76;R:resistant families;H:families segregating for resistance and susceptibility;S:susceptible families.Arrows indicate recombinants between the markers and the resistance gene.

Table 2–Amplification of four SCAR markers known to be linked to resistance gene er1 in resistant parent Xucai 1 and susceptible parents Bawan 6 and Qizhen 76(“–”indicates no amplification).

The four known SCAR markers Sc OPD10-650[22], ScOPE16-1600[23],Sc OPO18-1200[23],and ScOPX04-880 [25]linked to er1 were used to identify polymorphism between the parents Xucai 1 and Bawan 6 and between Xucai 1 and Qizhen 76.The results of amplification by SCAR markers in the three parents are shown in Table 2.Marker ScOPD10-650 amplified a fragment of expected size(650-bp)in the resistant Xucai 1 and the susceptible Qizhen 76,but there was no amplification in the other susceptible cultivar Bawan 6.Marker ScOPE16-1600 amplified a 1600-bp fragment in the susceptible Qizhen 76,but there was no amplification in either resistant Xucai1 or susceptible Bawan 6.Marker ScOPX04-880 amplified an 880-bp fragment in both resistant Xucai 1 and susceptible Bawan 6,but produced no fragment in susceptible Qizhen 76. Marker ScOPO18-1200 amplified a 1200-bp fragment only in susceptible Bawan 6.Amplifications in the contrasting bulks pooled from F2plants were then performed using the SCAR markers polymorphic in the parents.However,these markers did not form different fragment patterns in the contrasting bulks.

3.3.Genetic mapping of the resistance gene

Linkage analysis of segregation data allowed construction of genetic maps for both F2mapping populations derived from the crosses of Xucai 1×Bawan 6 and Qizhen 76×Xucai 1,respectively.Maps comprising two SSRmarkers and the resistance gene for both F2populations were constructed with MapDraw[50]. Linked markers AD60 and c5DNAmet were located on the same side of the resistance gene.For the F2population derived from Qizhen 76×Xucai1,markers c5DNAmet and AD6 were linked to the resistance gene at genetic distances of 8.1 and 8.7 cM, respectively(Fig.3-A).For the F2population derived from Xucai 1×Bawan 6,the resistance gene was linked to markers AD60 and c5DNAmet at genetic distances of 9.9 and 15.4 cM,respectively (Fig.3-B).These results indicated that the powdery mildew resistance gene in Xucai1 is an allele of er1,in agreement with a previous study[19].

Fig.3–Genetic linkage maps of F2populations derived from Qizhen 76×Xucai1(A)and Xucai1×Bawan 6(B)showing the position ofthe gene conferring resistance to powdery mildew and the molecular markers AD60 and c5DNAmet.Map distances and locus orders were determined with MAPMAKER 3.0(Lander et al.1993).Estimated genetic distances between loci are shown on the left in centiMorgans(cM).

3.4.PsMLO1 cDNA sequence analysis

We determined the nucleotide sequences of homologous PsMLO1 cDNAs of the contrasting parents to confirm which allele of er1 is present in Xucai 1.PsMLO1 full-length coding sequences of the three parents were obtained by cloning and sequencing of PCR products amplified by the specific primers PsMLO1F and PsMLO1R[29].On the basis of 10 clones,the homologous cDNA sequences of the PsMLO1 gene from Bawan 6 and Qizhen 76 were identical to that of PsMLO1 from the wild-type pea cv.Sprinter(susceptible to E.pisi;NCBI accession number FJ463618).Thus,Bawan 6 and Qizhen 76 carry the susceptibility gene Er1(Fig.4).In contrast,the cDNA sequences of PsMLO1 from Xucai 1 were markedly different from that ofwild-type pea cv.Sprinter and the two susceptible parents.The cDNAs from Xucai 1 comprised three distinct types of mis-spliced transcripts that were not present in the wild-type pea or the two susceptible parents.On the basis of 10 clones from Xucai 1,we obtained three PsMLO1 transcripts that were characterized by a 129-bp deletion and 155-and 220-bp insertions,respectively,in the PsMLO1 gene of wild-type Sprinter (Fig.4).Compared to the PsMLO1 transcript of the susceptible cv. Sprinter,Bawan 6,and Qizhen 76,cDNA1 from Xucai1 showed a 129-bp deletion between positions 1171 and 1299 of the PsMLO1 coding sequence,and the cDNA2 and cDNA3 from Xucai 1 had larger insertions of 155-and 220-bp at position 1263 in PsMLO1 coding sequence,respectively(Fig.4).These distinct transcripts were attributed to alternative splicing during transcription.The cDNA1 sequence also showeda 5-bp deletion frompositions 770-774 of the PsMLO1 coding sequence(Fig.5),which was attributed to an aberrant splicing event.The transcripts ofthe PsMLO1 gene in Xucai1 are consistent with those in pea cv.Stratagem(JI 2302) and Franklin which harbor the er1-2 alleles of resistance gene er1, indicating that Xucai 1 carries the er1-2 allele for resistance to powdery mildew[28,29].

4.Discussion

Fig.4–Comparisons of cDNA sequence of the PsMLO1 gene from the wild-type pea cultivar Sprinter(Wild_type_PsMLO1)with those identified in three parents,including two susceptible cultivars,Qizhen 76(QZ76_PsMLO1_cDNA)and Bawan 6 (BW6_PsMLO1_cDNA),and a resistant cultivar,Xucai 1(XC1_PsMLO1_cDNA1,2 and 3).Mutation sites with the deletion or insertion fragments in PsMLO1 coding sequence are boxed in black.

In this study,the pea cv.Xucai 1 was confirmed to have high resistance(immunity)to powdery mildew,and no colonies developed on the whole plants after artificialinoculation with E.pisi(Fig.1,Table 1).The genetic basis of resistance to E.pisi in Xucai 1 was investigated using three F2and two F2:3populations derived from three crosses.Phenotypic observations of the populations showed that resistance in Xucai 1 is conferred by a single recessive gene.

To date,only two recessive genes(er1 and er2)and one dominant gene(Er3)have been identified as conferring resistance to E.pisi[10-12].A majority ofpea germplasm resistant to E.pisi carries the recessive gene er1,which provides durable broad-spectrum resistance to powdery mildew disease caused by E.pisi[13,52].

Fig.5–Alignment showing a 5-bp deletion from 770 to 774 bp,compared to the wild type(Wild_type_PsMLO1),in one of the transcripts derived from Xucai1(XC1_PsMLO1_cDNA1).Transcripts“QZ76_PsMLO1_cDNA”and“BW6_PsMLO1_cDNA”derived from the two susceptible cultivars Qizhen 76 and Bawan 6,respectively.

To aid marker-assisted selection(MAS)in pea breeding programs,studies have been performed to identify different types of molecular markers,including RFLP(restriction fragment length polymorphism),RAPD,SCAR,and SSR, linked to the er1 locus[17-25,53].In these studies,various markers linked to the resistance gene er1 were reported by different researchers;however,the markers were located at different genetic distances in different mapping populations. First,Sarala[17]and Timmerman et al.[22]placed the er1 gene on pea LG VI.Dirlewanger et al.[18]found the er1 gene 9.8 cM distant from a RFLP marker,p236,whereas Timmerman et al. [22]placed the RAPD marker OPD10650at a distance of 2.1 cM from er1.This linked RAPD marker(OPD10650)was converted to a SCARmarker,ScOPD10650,which was mapped at a distance of 3.4 cM from er1[20].However,Tiwari et al.[23]found that ScOPD10650was not useful to identify Canadian pea resistant germplasm and related F3progeny derived from“Highlight (er1)×Radley.”Tiwari et al.[23]obtained three additional linked RAPD markers:OPO181200,OPE161600,and OPL61900.The first two ofthese were converted into SCARmarkers(ScOPE161200and ScOPL61600)and used successfully to identify resistant pea germplasm.Janila and Sharma[20]found that of the three SCAR markers,only ScOPD10650was linked to er1,at a genetic distance of 3.4 cM.Later,Ek et al.[19]discovered five SSR markers(PSMPSAD51,PSMPSA5,PSMPSAD60,PSMPSAA374e, and PSMPSAA369)linked to er1,and PSMPSAD60(named AD60 in this study)was identified as the most closely linked marker,at 10.4 cM distance from the er1 locus.Pereira et al.[21]identified two mutated genes,er1mut1 and er1mut2,that induced powdery mildew resistant mutants in pea,via ethylnitrosourea mutagenesis.The er1mut1 and er1mut2 loci were mapped to a pea LG containing 16 DNA markers including six linked markers.Pereira et al.[21]also attempted to remap all previously reported SCARs and five SSR markers linked to er1.Only the reported SCAR marker ScOPL61600and SSR marker PSMPSA5 were linked to the er1mut2 locus,at a longer distance.Recently, Santo et al.[54]developed specific molecular markers SCAR and a sequence-tagged site(STS)for the mutation er1mut1. Srivastava et al.[25]also developed another coupling-phase SCARmarker,ScOPX04880,linked to er1.In their study,a previous repulsion-phase marker,ScOPD10650,and a coupling-phase marker,ScOPX04880,were mapped flanking er1 at 2.2 and 0.6 cM distance,respectively.Taken together,these studies identified several markers linked to the er1 locus,with various degrees of utility,in different plant materials.In our study,in addition to the previous SCAR marker AD60,gene marker c5DNAmet was newly identified as linked to resistance gene er1.This novel marker can be used in MAS for powdery mildew resistance breeding in pea.

In this study,the resistance gene in Xucai1 was mapped at distances of 8.7 and 9.9 cM from the linked marker AD60 in the respective F2populations(Fig.3-A and 3-B).Our result is comparable with that reported by Ek et al.[19].The parents were then tested with four previous SCAR markers linked to the er1 locus,ScOPD10650,ScOPO181200,ScOPE161600,and ScOPX04880using the methods described by Wang et al.[34]. However,the four markers were useless among the three tested parents because they were unable to distinguish the resistant parent from the susceptible ones and showed the same single band in the contrasting bulks(Table 2).This result was consistent with those of previous studies[20,21,23].We then selected molecular markers distributed in the vicinity of the linked marker AD60 on a pea linkage map[47]to identify additional markers linked to the resistance gene in our populations,and found that gene marker c5DNAmet was linked to the resistance gene in Xucai 1.Thus,we further confirmed that powdery mildew resistance gene in Xucai 1 resides on LG VI of the pea genetic map.The marker c5DNAmet mapped at 8.1 cM and 15.4 cM distances from the resistance gene in the two F2populations derived from Xucai 1×Bawan 6 and Qizhen 76×Xucai 1,respectively(Fig.3). Markers AD60 and c5DNAmet were mapped to the same side of the resistance gene on LG VI in the two F2populations (Fig.3).The mapping of the resistance gene in LG VIlinked to AD60 and c5DNAmet markers suggested that the resistance gene is an er1 allele.

Recently,er1 resistance was demonstrated to be caused by a loss-of-function mutations of a powdery mildew susceptibility gene,PsMLO1,belonging to the MLO gene family[28,29].To date,five er1 alleles,each corresponding to a different PsMLO1 mutation,have been reported in pea accessions resistant to E.pisi [30].Humphry etal.[28]identified four er1 resistance alleles,er1-1 (JI1559),er1-2(JI2302),er1-3(JI210),and er1-4(JI1951),in naturally occurring PsMLO1 loss-of function mutations in resistant pea accessions.Pavan et al.[29]identified another er1 allele,er1-5,in pea line ROI3/02 following a mutagenesis program.Four of the five er1 alleles(excluding er1-2)are caused by point mutations in the PsMLO1 gene.Allele er1-2 comprises a large DNA insertion of unknown size and identities in the PsMLO1 gene,leading to aberrant PsMLO1 transcription[28,29].Allele er1-2 has been identified in a few pea powdery mildew resistant cultivars/lines, including Stratagem(JI2302),Franklin,Dorian,and Nadir[28,29]. Stratagem(JI 2302)is the earliest identified pea cultivar resistantto E.pisi and has been widely used in pea breeding,owing to its stable resistance[52].

In the present study,the mutation mode that occurred in Xucai 1 is consistent with that in the pea cv.Stratagem(JI 2302)carrying the er1 resistance allele,er1-2[28,29].Thus, the resistance gene in cv.Xucai 1 is an er1 allele,er1-2.The inserted fragments of 155-and 220-bp were compared against the NCBI database by BLASTN.The result revealed that,as reported for er1-2 allele by Humphry et al.[28],the 155-and 220-bp insertions were very similar(96%and 95%,respectively)to a sequence repeated five times in the genomic BAC clone(GenBank accession number,CU655882).Interestingly,the 220-bp insertion is also highly similar(～87%identity)to part of the sequence of the pea Ogre retrotransposon(GenBank accession number AY299395).The Ogre is a giant retroelement of more than 22 kb that makes up at least 5%of the pea genome [54,55].

Recently,Pavan et al.[30]developed different types of functionalmarkers corresponding to five er1 alleles,including an STS dominant marker er1-2/MGB targeting the er1-2 allele, according to the sequence difference.er1-2/MGB was used to test our parents in the present study,but showed no amplification in either resistant or susceptible parents.To date,er1-2/MGB has not been tested in other resistance cultivars or in derived populations carrying er1-2,except Franklin[30].Our finding may have been due to the large difference in genetic background between Xucai 1 and Franklin.Such difference can be dissected with obtaintion of complicated genome sequence of pea carrying resistance gene er1-2 after developing a codominant marker for er1-2.Four foreign pea cultivars or lines,including Stratagem (JI2302),Franklin,Dorian,and Nadir,have been shown to harbor the resistance gene er1-2[28,29].In our study,we identified another recessive resistance source harboring er1-2:the Chinese pea cultivar Xucai1.

The desirable agronomic traits of Xucai 1,including high yield and tolerance to lodging and drought[31],would be an advantage for the use of this cultivar in breeding programs.Our study revealed that resistance to E.pisi in the Xucai 1 is conferred by the gene er1-2.This result provides valuable genetic information about Xucai 1 that will improve its use in pea breeding for resistance to E.pisi.

5.Conclusions

Powdery mildew resistance in Xucai 1 was governed by a recessive gene that was mapped in pea LG VI,close to markers AD60 and c5DNAmet.These results suggested that the resistance gene is an allele of er1.The analysis of PsMLO1 coding sequence in Xucai 1 revealed that the resistance gene was generated by a large insertion or deletion in the PsMLO1 gene. Thus,the resistance gene in Xucai 1 was identified as an er1 allele,specifically the er1-2 allele.

Acknowledgements

This study was supported by the Modern Agro-industry Technology Research System(CARS-09),the Crop Germplasm Conservation and Utilization Program(2014NWB030-14)fromthe Ministry of Agriculture of China,and the Scientific Innovation Program of Chinese Academy of Agricultural Sciences.We thank Prof.Fengbao Wang(Hebei Normal University of Science& Technology,Qinghuangdao,Hebeiprovince,China),Dr.Xin Chen (Jiangsu Academy of Agricultural Sciences,Nanjing,Jiangsu province,China),and Mr.Dongxu Xu(Zhangjiakou Academy of Agricultural Sciences,Zhangjiakou,Hebei province,China)for providing the pea cultivars.

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17 March 2015

in revised form13 July 2015

.

E-mail address:zhuzhendong@caas.cn(Z.Zhu).

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science,CAAS.1Suli Sun and Zhongyi Wang contributed equally to this work.