Identification And Functional Analysis Of MiRNAs Resistant To Powdery Mildew In Cucumber

Cucumber(Cucumis sativus L.)is one of the main horticultural vegetable crops cultivated in China,and powdery mildew(PM),fusarium wilt and downy mildew are the three prevalent diseases that harm cucumber.lt is known that the occurrence of powdery mildew limt cucumber production worldwild every year.MicroRNAs(miRNAs)are a classis of non-coding endogenous small RNAs with a length of 20-24 nt,which degrade or inhibit mRNA through the complementary pairing principle,thereby regulating the physiological activities of plant growth process.Plants can express some miRNAs under the stresses,and act on target genes related to stress,so that the plants can adapt to stress in physiological response.However,it is still unclear which miRNAs are involved in regulation and how they regulate cucumber resistance to PM.In the early stage of this experiment,a series of chromosomal fragment substitution lines were created with the cucumber highly resistant powdery mildew strain Jin5-508 and the susceptible powdery mildew strain D8 as parent,of which SSSL508-28 is highly resistant to powdery mildew and can be stably inherited.In this study,using D8(high-sensitivity PM)and SSSL508-28(high-resistance PM)as materials,miRNA sequencing technology was used to compare the difference in miRNA transcription levels of the entire genome 48 hours after inoculation with powdery mildew,and the relevant targets were predicted.Using qPCR to verify the differential miRNA expression.The specific experimental results are as follows:1.Based on sequencing analysis,156 known miRNAs and 147 new miRNAs were finally identified.Among them,32 differentially expressed miRNAs were identified in the comparison between ID and NID,and 6 differentially expressed miRNAs were identified in the comparison between IS and NIS.and we found that all 32 DEMs identified in PM susceptible D8 were upregulated,four of the six DEMs identified in the PM-resistant line SSSL508-28 were downregulated.2.Through miRDeep2 software package and degradation group sequencing method identified 517 and 20 targets of DEMs in D8 and SSSL508-28 were predicted respectively.3.The expression of miRNAs may vary greatly depending on the strain or variety,so our study highlights the broader genotype-specific response to PM,rather than the common response to high resistance and susceptibility genotypes.4.MiRNAs such as Csa-miR395d-3p,Csa-miR398d,Csa-miR156j,Csa-miR160f,Csa-miR156g,Csa-miR172c-3p,Csa_novel-19 were up-regulated after inoculation with PM in D8,and down-regulated after inoculation with PM in SSSL508-28.The target genes of them like transcription factors such as GRAS,DoF,eIF2α,PG,UGT and STPKs are also differentially expressed in inoculation5.Five DEMs in the replacement segment,including two upregulated DEMs in D8,Csa-miR172c-3p and Csa-miR395a-3p,and two downregulated DEMs in SSSL508-28,Csa-miR395d-3p and Csa-miR398b-3p.One gene encoding L-aspartate oxidase targeted by Csa-miR162a was also located on the same fragment and was down-regulated in D8 during PM inoculation.Our results identify the application basis of miRNA in the breeding of cucumber varieties with enhanced PM resistance.