| Wheat powdery mildew is one of devastating fungi diseases which threaten wheat production worldwide.The breeding of resistance cultivars is the most effective and environment-friendly approach to control the disease,which is facilitated by the identification of new resistance genes and elucidation of the resistance mechanism.It has been reported that vacuolar processing enzyme(VPE)gene family play important roles in plant development as well as in response to biotic and abiotic stresses.The release of reference genome sequences makes it possible for genome-wide identification,characterization,and comparative analysis of a gene family from different species.Up to now,VPEs in Triticeae species including Haynaldia villosa,a diploid wild relative of wheat with high resistance to powdery mildew,has not been well studied.In this study,using a bioinformatics approach,genome wide identification of VPEs were performed based on the reference sequences of 14 Gramineae species.The gene number and chromosome distribution,gene and protein structures,and expression profiles were compared among different species.The VPE gene family of H.villosa and VPE3-V,which was one of the interactors of CMPG1-V,was selected for gene cloning and functional analysis.Ae.tauschii is the donor progenitor of wheat D genome.The availability of highly quality reference genome of Ae.tauschii is of great help for gene mining.In previous study,Peng(2017)found one of the Ae.tauschii accessions(accession No.2002I-02)was highly resistant to powdery mildew.A F2 segregating population derived from the cross between 2002I-02(resistant)and 2002I-11(susceptible)was constructed.In the present study,we further mapped the resistance loci by Bulked segregant analysis(BSA)using 55K SNP array and linkage analysis.The main results obtained are as follows:1.Identification and characterization of VPE gene family from Gramineae speciesBy genome-wide screening of the reference genome sequences,a total of 127 VPE family genes were identified from 14 Gramineae species.According to the classification in barley,the VPE genes could be divided into five clades,i.e.VPE1,VPE2,VPE3,VPE4,and VPE5.The VPEs belonging to the same clades had more similarities.Most VPEs had a single copy in the diploid species,gene duplication was observed in Triticeae species.VPE family genes from Triticeae species were mainly distributed on homologous groups 1,2,3,5,and 6.Well conserved collinearity was present among different species.The number and chromosome distributions of VPE1 and VPE5 were most conserved.The VPE4 had an additional member on chromosome 6D,which appeared before the formation of hexaploid wheat.The VPE2s showed a tandemly distributed pattern,which may due to multiple duplication along with the formation of polyploid wheat.The two positions of VPE3s were the distal of the long arm and near the centromere region of chromosome 3,respectively.In which,the distal one is resulted from gene duplication after the formation of hexaploid wheat.2.Structure and expression analysis of the wheat VPEs and their promoter regionsAll wheat VPEs had both exons and introns.The VPE genes from the same clades shared more similar gene structure.The orthologs were more similar,and the sizes and length of introns from the paralogs were more different.All VPEs had the conserved functional domain Peptidase C13.Diverse expression patterns were observed for different VPE genes.VPE1 and VPE2 were dominantly expressed in the seeds,while VPE3 were dominantly expressed in the shoots and leaves.When induced by Pst,Bgt and PAMP(chitin and flg22),only the VPE3(TraesCS3B02G233000)exhibited a down-regulated expression pattern.The expression of VPE3 was also changed under different abiotic stresses.When inoculated by Fusarium pseudograminearum,the expression of VPE4 was down-regulated.Analysis of the VPE promoters showed that promoters of VPE2,VPE1,VPE3,and VPE5 had multiple cis-regulatory elements,such as STRE,ABRE,ABRE3/4a,CGTCC-logo,CGTGG-logo,TATA-box,G-box.The promoters of VPE4 contained AAGTG-logo,GGATC-logo,CAAT-box,Spl,ABRE3a/4 elements,which were different from other VPEs.4.Identification of VPEs in H.villosa,and cloning and functional analysis of VPE3-VReferring to the genome and transcriptome sequences of H.villosa,12 VPE family genes were identified in H.villosa.Among them,VPE1 and VPE4 had only one member,VPE5 had two members,which was consistent with their corresponding homologs in other diploid Triticeae species;VPE3 and VPE2 also had gene duplication because of multiple members.The CMPG1-V is a powdery mildew resistance related gene from H.villosa.By Y2H using CMPG1-V as bait,a vacuole-processing enzyme gene VPE3-V was identified as one of the interactors.The full length of VPE3-V was cloned from H.villosa.VPE3-V had the Peptidase C13 domain and a signal peptide in its N-terminal.The direct interaction between VPE3-V and CMPG1-V was confirmed by bimolecular fluorescence complementation(BiFC).VPE3-V was down-regulated in leaves of H.when inoculated by Bgt.When silencing VPE3 in epidermal cells of Yangmail58 leaves,the haustorium index of Yangmai158(74.44%)was significantly reduced compared with the GUS control(78.82%,p<0.05).Silencing VPE3 in Yangmail58 by RNAi improved the powdery mildew resistance,shown by that at adult stage the infection type of the positive transgenic plants were 5～6,which were significantly lower than the recipient Yangmai158(8～9).5.Molecular mapping of the powdery mildew resistance loci of Ae.tauschii accession 20021-02In previous study,we found that an accession(2002I-02)of Ae.tauschii exhibited high powdery mildew resistance(Peng,2017).In this study,(2002I-02×2002I-11)F2 segregation population was constructed and used for mapping of the powdery mildew resistance loci.The segregation ratio of resistance to susceptible ratio was not 3:1 by genetic analysis,indicating the resistance was not controlled by a single locus.Bulks consisted by 9 extremely resistant or susceptible F2 individuals were constructed and used for bulked segregant analysis(BSA)by the 55K SNP array.A total of 143 polymorphic SNPs were identified,in which 69 were on 7D and 38 were on 2D.The 39 polymorphic SNPs on 7D were mainly distributed within an interval of 4.0-15.5 Mb,and 13 and 16 polymorphic SNPs on 2D were distributed within intervals of 13.5-19.6 Mb and 62.5-82.1 Mb,respectively.These indicated that the powdery mildew resistance of 2002I-02 was contributed by these genome regions.In order to mapping the resistance loci,5 ARMS-PCR and 5 IT markers were developed in regions based on the polymorphic SNPs of 2D and 7D and the intron divergence of genes within the target regions from Ae.tauschii and wheat.Linkage maps were constructed and two major QTLs were identified on 7D and 2D,named as pm-Aet-7DS and pm-Aet-2DS.The pm-Aet-7DS explained about 56.5%of the powdery mildew resistance variation.It was fine mapped to a 2.4Mb interval flanked by two markers on 7D,IT-362 and IT-473.Referring to the reference genome sequence of Ae.tauschii,this interval contains 49 genes,including 10 NB-ARC genes. |
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