Phylogenetic Relationships Of Fragaria Genus And Salicylic Acid Regulation Mechanism Of Wild Fragaria Species In Response To Powdery Mildew

Strawberry is an important economic crop in China.Cultivated strawberry(Fragaria × ananassa)is delicious and has great nutritional value.Now,it has been planted all over the world.However,cultivated strawberries have poor disease resistance and are easily attacked by pathogens,especially powdery mildew.Compared with F.× ananassa,wild Fragaria species has stronger disease resistance.Wild Fragaria has a close relationship with F.× ananassa,and it is an important germplasm resource.Therefore,exploring relatives of F.× ananassa and studying the disease resistance mechanism of F.× ananassa is of great significance for cultivating F.× ananassa with stronger disease resistance.In this study,we firstly analyzed the phylogenetic relationships of Fragaria based on the whole chloroplast genomic sequences to explore the more closely related species to F.× ananassa.Then the disease resistance of five diploid wild strawberries,including F.vesca,F.nilgerrensis,F.viridis,F.daltoniana,and F.pentaphylla,were determined.Finally,the regulation mechanism of salicylic acid(SA)in wild strawberry under powdery mildew stress was analyzed through transcriptome.The main results of this study are as follows:(1)27 complete chloroplast genomes of 11 wild Fragaria species were sequenced using the Illumina platform.Then,the variation among complete chloroplast genomes of Fragaria was analyzed,and phylogenetic relationships were reconstructed from those genome sequences.There was an overall high similarity of sequences,with some divergence.According to analysis with m VISTA,non-coding regions were more variable than coding regions.Inverted repeats(IRs)were observed to contract or expand to different degrees,which resulted in different sizes of chloroplast genomes.Additionally,five variable loci,trn S-trn G,trn R-atp A,trn C-pet N,rbc L-acc D,and psb E-pet L,were identified that could be used to develop DNA barcoding for identification of Fragaria species.Phylogenetic analyses based on the whole chloroplast genomes supported clustering all species into two groups(A and B).Group A species were mainly distributed in western China,while group B contained several species from Europe and Americas.These results support allopolyploid origins of the octoploid species F.chiloensis and F.virginiana and the tetraploid species F.moupinensis and F.tibetica.(2)The tolerance of the five wild strawberries to powdery mildew was determined through investigating the incidence.The incidence rates of F.vesca,F.nilgerrensis,F.viridis,F.daltoniana,and F.pentaphylla were 17.95%,59.84%,37.82%,30.00% and 33.71%,respectively;disease index were 12.90%,33.66%,22.15%,22.33% and 18.48% respectively.The results showed that F.vesca was the most disease resistant,and F.nilgerrensis had the weakest disease resistance.(3)The leaves of F.vesca and F.nilgerrensis infected with powdery mildew were collected from 0,3,7,and 10 days for enzyme activity determination.In addition to phenylalanine ammonia-lyase,the activities of superoxide dismutase,peroxidase,catalase and polyphenol oxidase showed a trend of increasing first and then decreasing,which was the normal stress responses for pathogenic bacteria.In addition,except for polyphenol oxidase,the activity of other enzymes in F.vesca increased faster than that in F.nilgerrensis,which reflected the stronger resistance of F.vesca response to powdery mildew.(4)Through transcriptome analysis of F.vesca and F.nilgerrensis we found that compared with the control group,there were 1,657 differentially expressed genes in F.vesca infected by powdery mildew,of which 1,442 genes were up-regulated;while F.nilgerrensis had 993 differentially expressed genes,of which 265 were up-regulated.Twenty-nine differentially expressed genes related to SA synthesis and signaling transduction pathway were identified.When infected by Podosphaera aphanis,wild Fragaria may mainly promote the ICS pathway-dependent SA synthesis by up-regulating ICS(Fv2339＿4g32070)expression;and the up-regulated expression of PAL(Fv2339＿6g16470,Fv2339＿7g20020)promote PAL-dependent SA synthesis simultaneously.And the wild strawberries mainly synthesize SA through ICS pathway.NPR1(Fv2339＿3g11320)may be a key gene of SA signal transduction in wild strawberry under powdery mildew stress.Sixty-seven transcription factors related to SA synthesis and signal transduction were identified by correlation analysis,belonging to 10 transcription factor families including WRKY,NAC,MYB,b ZIP,TCP,ERF,HSF,GRAS,and B3.When attacked by P.aphanis,wild strawberry may regulate ICS expression through transcription factors such as TCP9(Fv2339＿5g01380)and ANAC072(Fv2339＿3g19980),and regulate PAL expression of through WRKY70(Fv2339＿6g10060,Fv2339＿6g10050),thereby affecting the synthesis of SA.In the SA signal transduction pathway of wild strawberry,WRKY53(Fv2339＿7g31430)may act as a key transcription factor to bind to NPR1 to induce PR-1 expression.The genes related to SA synthesis and signal transduction in F.vesca and the transcription factor genes that play a regulatory role in this process were significantly up-regulated,but not in F.nilgerrensis,which may be the reason why F.vesca is more resistant than F.nilgerrensis.