Transcriptome And MiRNA Sequencing Analysis Of Sclerotinia Sclerotiorum (Lib.) Resistance-related MiRNAs In Brassica Napus L.

Sclerotinia stem rot?SSR?,caused by Sclerotinia sclerotiorum?Lib.?de Bary is a destructive fungal disease resulting in yield losses and decreases in seed quality in oilseed rape?Brassica napus?worldwide.At present,the research on the disease is mainly focused on the isolation and identification of pathogens,screening of disease resistance resources,resistance breeding and QTL?Quantitative trait loci?mapping,etc.,the study of S.sclerotiorum resistance molecules mechanism at the level of transcription or post-transcriptional is not enought.However,microRNAs?miRNAs?are a class of endogenous,single-stranded and non-coding small RNAs,which is involved in many biological processes such as,growth,development,and response to environmental stresses by negatively regulating target gene expression or inhibiting target mRNA translation at the post-transcript level.So far only 92 B.napus miRNAs were released in the miRBase 21.0 database,which is much less than that of Arabidopsis.In this study,we identified S.sclerotiorum resistance-related miRNAs and genes in B.napus L.from the following two aspects and explored its mechanism in S.sclerotiorum defense system by analyzing the expression patterns of differentially expressed miRNAs and their target genes to reveal complex mechanism of B.napus L.response to the infection of the fungal pathogen S.sclerotiorum and provided new insights about rape disease resistance breeding and cloning of disease resistance genes.1.Five relatively resistant B.napus lines were used as the research materials.Oilseed rape stems inoculated with S.sclerotiorum at 48 h?treatment group,T-48 h?and mock-inoculated?control group,CK-0 h?were collected and used for omics analysis?miRNA,mRNA and degradome sequencing?.After a serious of bioinformatics analysis,we screened miRNAs and genes,which were involved in the interactions between S.sclerotiorum and B.napus.The main findings are following:?1?A total of 77 conserved miRNAs belonging to 30 miRNAs families were identified in the sRNA sequencing,among them 76 and 68 miRNAs were identified inCK-0 h and T-48 h libraries,respectively.Then,176 novel miRNAs were obtained and175 out of them were identified in two libraries except novel_miR₇4,which was only identified in CK-0 h librariy.There were 122 target genes of 51 differentially expressed miRNAs were predicted.Based on the GO?Gene Ontology?? COG?Cluster of Orthologous Groups of proteins?and KEGG?Kyoto Encyclopedia of Genes and Genomes?functional annotations and transcriptome sequencing results,it was found that the predicted target genes of some B.napus miRNAs,such as the target genes regulated by bna-miR156a/156 f are related to the SPL transcription factor and NPH3 family protein,the target genes regulated by bna-miR166 f are related to REVOLUTA,the target genes regulated by novel_miR₁73 are related to TIR-NBS-LRR resistant protein,the target genes regulated by novel_miR₂ and novel_miR₇3 are associated with receptor-like proteins,suggesting that these miRNAs and their target genes are likely to be involved in the response process of S.sclerotiorum in B.napus.?2?A total of 7,371 differentially expressed genes?DEGs?with 2,215 up-regulated and 5,156 down-regulated were detected by transcriptome sequencing.Aligned with the plant transcription factor database?http://planttfdb.cbi.pku.edu.cn/index.php?,602DEGs?168 up-regulated,434 down-regulated?belonging to 39 transgenic families were identified.Then,685 DEGs?216 up-regulated,469 down-regulated?belonging to eight kinds of plant hormones genes were detected after aligned with the Arabidopsis hormone database?http://ahd.cbi.pku.edu.cn/?.Aligned with GO,COG and KEGG database,a total of 1,570 candidate genes related to S.sclerotiorum of B.napus were screened,including 339 protein kinase-related genes,118 transcription factors,43 signal transduction genes,113 transgenic genes,65 resistant genes,187 hormone-related genes,141 secondary metabolic genes,and 592 other functional-related genes.?3?In total,80 cleavage sites with 41 conserved miRNAs and 23 novel miRNAs were predicted using degradome sequencing technologies.The functional analysis showed that target genes were given to Category 0 and 1,and most conserved miRNAs target SBP,ARF,NAC,TOE and other transcriptional regulatory factors,some of the novel miRNAs target genes are DCL1,TCP and MYB transcription factors and disease resistance protein family genes.?4?The Quantitative Real-time PCR?qRT-PCR?verification results of differentially expressed miRNAs and its target genes and disease resistance genes show that the expression patterns of target genes and their corresponding miRNAs were opposite in B.napus,and the expression pattern of disease resistance-related genes wasconsistent with that of transcriptome sequencing.2.Five relatively resistant and susceptible B.napus lines were used as the research materials for differentially expressed miRNAs among genotypes and timepoints.Rapeseed stem non-inoculated and inoculated with S.sclerotiorum were selected 0h,24 h and 48 h after inoculation.Through high-throughput sequencing and bioinformatics analysis,we screened differentially expressed miRNAs and their target genes.The mechanism of differentially expressed miRNAs and its target genes in response to S.sclerotiorum of B.napus was explored.Furthermore,qRT-PCR was used to detect their expression patterns of differentially expressed miRNAs and its target genes.The main results are following:?1?A total of 71 conserved miRNAs were obtained,which belongs to 28 miRNAs families respectively.All known miRNAs were detected in the preceding miRNA sequencing except that bna-miR6034 was detected 24 h after inoculation in the relatively resistant lines.Among 1,402 novel miRNAs,some were highly conserved with known miRNAs family members,suggesting that they may be new members of these miRNAs families.?2?By analyzing the expression patterns of miRNAs between the inoculated stem and non-inoculated stem in the same lines,it was found that there were 569 and 680 differentially expressed miRNAs in resistant and susceptible libraries,respectively,and with the elongation of inoculated time,the number of differentially expressed miRNAs was increased.The number of expressed miRNAs in the resistant lines was higher than that of the susceptible lines at 24 h after inoculation,but slightly lower than that of the susceptible lines at 48 h after inoculation,indicating that the resistant lines responded quicklyto the sclerotinia infection than the susceptible lines;compared with non-inoculated?0h?,the bna-miR156 and bna-miR166 f obtained at 48 h after inoculation were differentially expressed in the previous results,but the fold change was slightly higher than that of the previous study.?3?29 specificially expressed miRNAs were found in non-inoculated lines and 55 in inoculated lines by analyzing the expression patterns of miRNAs between the inoculated stem and non-inoculated stem in the same lines.And the specificially expressed conserved miRNAs?E.g.,bna-miR167 a,bna-miR167 b,bna-miR167 c,bna-miR167 d and bna-miR403?were preferentially expressed in disease-resistant lines after inoculation.?4?941 differentially expressed miRNAs coregulated 5,667 target genes,according to the target gene prediction and annotation results.These genes mainly involved plant hormone signal transduction pathway,secondary metabolic process,stress response,oxidative burst,transcriptional regulation and protein transport.Such as the target genes of bna-miR172,bna-miR403 and novel_mir₃76 are related to TIR-NBS-LRR resistant protein,the target genes regulated by bna-miR164 and bna-miR390 are related to the leucine receptor protein kinase family proteins,the target gene of bna-miR393 is the auxin receptor,the target gene of bna-miR160 and bna-miR167 is auxin response factor,and the target gene of bna-miR168 a is AGO1 protein and ZLL,etc.,suggesting that these miRNAs and their target genes are likely related to Sclerotinia resistance of B.napus.?5?Combined with Pathway analysis,295 metabolic pathways were obtained.The analysis results indicated that miRNAs could improve plants disease resistance by regulating the expression of related genes in the metabolic pathway under adversity stress.For example,bna-miR164a/b/c/d,bna-miR172a/d,bna-miR390a/b/c,bna-miR396 a and bna-miR403 are involved in the plant-pathogen interaction.bna-miR164a/b/c/d,bna-miR390a/b/c,bna-miR393,bna-miR394a/b and bna-miR6028 are involved in the plant hormone signal transduction.bna-miR172a/d are involved in the secondary metabolic process.These further proved that plants disease resistance mechanism,containing multiple metabolic pathways of mutual cooperation,was a very complicated process.?6?The expression of 6 candidate miRNAs was analyzed by qRT-PCR and the results were verified with the sequencing results of the previous small RNA and transcriptome sequencing.We had found that the miRNA expression of resistance and susceptible lines was consistent with sequencing results under sclerotinia stress.And the relative expression of 3 and 5 miRNAs in resistance lines were higher than that in susceptible lines at 24 h and 48 h after inoculation,respectively.The quantitative results of miRNAs target genes also showed that miRNAs and its target genes were negatively regulated.?7?Combining the results of this experiment with other related studies,we speculated that the miRNAs was involved in the mechanism of disease resistance of S.sclerotiorum in B.napus.And we discovered that these miRNAs resist the infection of pathogens through a plant disease resistance control network,such as it negative regulated its target genes to start disease resistance genes,and then activate defense and immune system,regulate plant signal transduction and secondary metabolism and otherways.The strong resistance materials can make quicker response than the weak resistance materials...