Identification Of Salt-Responsive MiRNAs And The MiRNA Mediated Regulatory Network In Wild Emmer Wheat (Triticum Dicoccoides L.)

Micro RNA(miRNA)is one class of endogenous non-coding single strand RNAs,which acts as the most important negative regulators to play the crucial role in regulating stress tolerance in plant.Wild emmer wheat is A and B genomes donor of common wheat.Due to its excellent disease resistance,abiotic stress tolerance and wide adaptability,it is considered as the indispensable genetic resource and gene pool for wheat genetic improvement.Therefore,it is of great significance to identify and explore the salt-responsive miRNAs for elucidating the mechanism of salt tolerance and breeding in wild emmer wheat.In this study,high-throughput sequencing technology was used to analyze the miRNA expression profile of wild emmer wheat to identify the salt-responsive miRNAs.Intergration of degradome sequencing and transcriptome sequencing,the regulatory network mediated by miRNA related to salt tolerance was constructed.The main results are listed as follows:(1)The salt tolerant genotype A5 and salt susceptible genotype C2 were used as materials,and samples from 0.5h,3h,8h and 27 after salt stress(150mM NaCl)treatment and control group were collected for high-throughput sequencing to obtain the small RNA expression profiles.A total of 780 miRNAs were identified in wild emmer wheat,including 363 known miRNAs belonging to 78 miRNA families and 417 novel miRNAs.In A5 genotype,62 salt-responsive miRNAs were obtained and 45 saltresponsive miRNAs were identified in C2 genotype,of which 15 were shared by them.Expression patterns of these salt-resonsive miRNAs showed that they displayed upregulation in tolerant genotype A5 under salt treatment,while showed down-regulation in susceptible genotype C2.Additionally,most of them showed time point specificity in both genotypes.(2)The target genes of these miRNAs were predicted by bioinformatics and degradome sequencing.A total of 13,643 genes were found to be targeted by 457 miRNAs(232 known and 225 novel miRNA).Furthermore,336 genes associated with abiotic stress response and 94 genes associated with osmotic stress response were found to be targeted by salt-responsive miRNAs.In addition,among the target genes of salt stress-related miRNAs,522 were transcription factor.The most targeted transcription factor family was NAC family(8.6%),followed by b HLH family(6.5%)and WRKY family(6.5%),which suggested that the miRNAs responding to salt stress in wild emmer might further regulate gene expression by regulating key transcription factors.(3)Based on the expression patterns of miRNAs(miRNA-seq)and their target genes(RNA-seq)in response to salt stress,the correlation between miRNAs and their target m RNA expression profiles was analyzed by person correlation coefficient,and miRNA-m RNA interaction pairs were found.In plants,miRNA usually plays a negative regulatory role,so we further analyzed the negative correlation of interaction.A total of 562 pairs of negative regulated DEmiRNA-DEG were found in the comparison group of A5 strain and 77.4% of them were up-regulated by miRNA and 22.6% were down regulated by miRNA.A total of 518 pairs of DEmiRNA-DEG were found in C2 strain under salt stress and 48.8% of them were up-regulated by miRNA and 51.2%were down regulated by miRNA.The interaction pairs of up-regulated miRNA and downregulated genes in A5 strain(435 pairs)were significantly more than those in C2strain(254 pairs).In A5,td-mi R9653b-p5＿5 has the largest number of differentially regulated genes,indicating that td-mi R9653b-p5＿5 may play an important role in the regulation of salt stress.(4)Among the candidate miRNAs,The expression patterns of td-mi R5048-5p,tdmi R9676-5p,td-mi R9653b-p5＿5 and PC-3p-72304757 were opposite to the expression patterns of target genes,and td-mi R5048-5p、td-mi R9676-5p、td-MIR9653b-p5＿5 and pc-3p-72304＿757 cleaved 7 genes in degradome A library and degradome C library.We take these seven genes as the hub genes of regulatory networks,and extract the genes with high connectivity in the co-expression network to construct miRNA mediated regulatory networks.td-MIR9653b-p5＿5 control three sub networks centered on TRIDC3AG052690,TRIDC4AG038750 and TRIDC6BG020020.Novel miRNA PC-3p-72304＿757 mediates two sub networks centered on TRIDC2AG064330 and TRIDC2BG056880.From the functional enrichment of each subnetwork,it can be inferred that td-mi R9653b-p5＿5 may resist salt stress by regulating phenylalanine ammonia lyase and may also respond to salt stress by regulating the light absorption process of photosynthesis.PC-3p-72304＿757 may respond to salt stress by affecting the composition or structure of chloroplast thylakoid membrane.In this study,we used high-throughput sequencing technology to systematically analyze the miRNA expression profiles of wild emmer wheat at different time points under salt stress,identifing salt stress related miRNA and combined with degradomesequencing and transcriptome sequencing to construct the regulatory network of salt stress related miRNA mediated.The purpose of this study is to reveal the mechanism of miRNA response to salt stress in wild emmer wheat,provide important reference for clarifying the salt tolerance mechanism of wild emmer wheat and provide a new way to improve the salt resistance of wild emmer wheat and wheat by miRNA.