Identification And Validation Of MiRNAs Associated With Paulownia Witches Broom In Paulownia Seedlings

Paulownia is one of the important fast-growing timber tree species in China.However,Paulownia is vulnerable to Paulownia Witches Broom(Pa WB)during its growth process,causing great losses to the economic development of Paulownia industry.Pa WB is a destructive plant disease caused by phytoplasma.Because it is difficult to culture phytoplasma in vitro,the molecular mechanism of Pa WB occurrence is still unclear so far.As an important regulator,mi RNAs regulate gene expression at the post-transcriptional level.In this study,Pa WB-related mi RNAs in Paulownia fortunei,P.tomentosa and P.fortunei × P.tomentosa were investigated for the first time.Mi RNAs associated with Pa WB in Paulownia were identified by high-throughput sequencing,and their target genes were determined by Target Finder and degradome sequencing.Quantitative real-time PCR(q RT-PCR)was used to validate the accuracy of sequencing,and the expression patterns between mi RNAs and their target genes was also analysed.The results of this study provides new clues to uncover the molecular mechanism of Pa WB occurrence and provides important reference values for the research in Pa WB prevention and Paulownia breeding.In this study,healthy seedlings,Pa WB phytoplasma-infected seedlings and Pa WB phytoplasma-infected seedlings treated with MMS of P.fortunei,P.tomentosa and P.fortunei × P.tomentosa were used to construct nine small RNA libraries and nine degradome libraries,respectively.The differential expression profiles of mi RNAs after phytoplasma infection had been analyzed.The main results of this study are as follows:(1)In P.fortunei,a total of 417 mi RNAs were identified in PF,PFI and PFI-20 libraries including 252 known mi RNAs(40 mi RNA families)and 165 novel mi RNAs.In P.tomentosa,a total of 392 mi RNAs were identified in PT,PTI and PTI-20 libraries including 277 known mi RNAs(40 mi RNA families)and 115 novel mi RNAs.In P.fortunei × P.tomentosa,a total of 398 mi RNAs were identified in PTF,PTFI and PTFI-15 libraries including 281 known mi RNAs(42 mi RNA families)and 117 novel mi RNAs.(2)113,159 and 165 differential expressed mi RNAs were identified in P.fortunei,P.tomentosa and P.fortunei × P.tomentosa,respectively.Among these mi RNAs,20 common Pa WB-related mi RNAs including 17 known mi RNAs and 3 novel mi RNAs were found.(3)Target Finder and degradome analysis were used to determine the target genes of differential expressed mi RNAs.In P.fortunei,237 target genes for 63 differential expressed mi RNAs were identified(213 targets for 55 known mi RNAs and 26 targets for 8 novel mi RNAs).In P.tomentosa,265 target genes for 82 differential expressed mi RNAs were identified(233 targets for 72 known mi RNAs and 32 targets for 10 novel mi RNAs).While 260 target genes for 83 differential expressed mi RNAs were identified(226 targets for 73 known mi RNAs and 34 targets for 10 novel mi RNAs)in P.fortunei × P.tomentosa.These mi RNAs were mainly involved in regulation of transcription,DNA-dependent,apoptosis,auxin mediated signaling pathway,ATP binding,DNA binding and metal ion binding based on GO functional analysis.KEGG pathway analysis showed that these mi RNAs were mainly involved in nitrogen metabolism,starch and sucrose metabolism,inositol phosphate metabolism,purine metabolism and aminoacyl-t RNA biosynthesis.(4)Seven mi RNAs and four target genes were selected randomly for qRT-PCR validation.The results showed that the expression patterns obtained from q RT-PCR data were obviously consistent with those obtained from Illumina sequencing analysis,indicating the reliability of the RNA-seq analysis.