Dissecting The Roles Of MicroRNA In Barley Salinity Response And Grain Development By Integrating Multiple 'omics' Analyses

MicroRNA(miRNA)is a class of small non-coding RNA,which served as the key players in the gene regulation networks by transcript degradation or translational repression.Recently studies showed that miRNAs played important roles in plant growth,development,and stress response.Barley(H.vulgare)is the fourth most cultivated crop worldwide and widely grown in arid and semiarid regions.As one of the most salt-tolerant cereal crops,it is widely used for animal feed,beer,and human food.The advent and development of next generation sequencing technology and bioinformatics provided a new opportunity for dissecting the roles of miRNA in barley development and stress response.In this study,we integrated multiple ‘omics' analyses to dissect the role of miRNA in barley.Firstly,we comparatively analyzed the critical miRNAs involved in barley salinity response and grain development.Secondly,we carried out a large-scale investigation of small RNAs in wild barley and compared with cultivar barley.This study could increase miRNA information in barley and also provide important information in dissecting the roles of mi RNAs in barley stress responsive,development and evolution.The main research contents and results as follows:(1)Global identification of microRNAs and their targets in barley under salinity stressThe barley cultivar Morex,a relative salt tolerant genotype was used in this study.At the three-leaf stage,plants were exposed to salinity by adding NaCl to the final concentration of 100 mmol/L.Whole treated plants and counterpart controls were harvested at the 3h,8h and 27 h after reaching the final concentration of 100 mM/L NaCl for downstream experiments.A pool of two replicates was used for small RNA and degradome sequencing.A total of 152 mi RNAs belonging to 126 families were identified,of which 44(39 miRNA familes)were found to be salinity responsive.14 out of the conserved miRNA families were also found to be salinity responsive miRNAs in other plant species,while 4 and 11 salinity responsive mi RNA families were only found in grass and barley,respectively.Compared with control,the majority of the salinity responsive miRNAs were up-regulated at the 8h time point,while down-regulated at the 3h and 27 h time points.Then,the targets of these miRNAs were further detected by degradome sequencing coupled with bioinformatics prediction.86 and 37 target gene families were found to involve in metabolic process and response to stimulus respectively.Under salinity stress,these genes mainly involved in attenuated plant growth and decreased metabolicrate.Finally,partial miRNAs and their target genes which were verified by the degradome sequencing were selected to further confirm by qRT-PCR.Overall,there were at least 39 barley mi RNA families and 123 corresponding target gene families response to salinity,which may consist to a miRNA regulatory network in barley.These results could facilitate future research to understand the molecular mechanism of salinity response in barley and related cereals to improve their salinity tolerance.(2)Characterization of microRNAs and their targets in wild barley(Hordeum vulgare subsp.spontaneum)using deep sequencingWild barley,the progenitor of cultivar barley,is one of the most important gene pool for barley genetic improvement.In the current study,the wild barley accession 3-25,collected from Mt.Gilboa(Israel),was used as the material.Leaves,stems,and roots at four different stages(seedling,jointing,heading,and filling),and spikes at heading and filling stages were collected to construct the small RNA sequencing library.A total of 9885815 clean reads were obtained,leading to the identification of 55 known miRNAs,14 novel variants and 18 novel mi RNAs.Sequence analysis revealed that all of the miRNAs identified in wild barley contained the highly conserved hairpin sequences found in cultivars,especially in the region of mature mi RNA.Then,a set of 32 miRNAs(14 novel variants and 18 novel miRNAs)obtained in our study were used as database to search against a small RNA sequencing dataset from cultivated barley.The results indicated that 21 out of those miRNAs(65.63%,including 7 novel variants and 14 novel miRNAs)could be detected in cultivated barley.Furthermore,the majority of those miRNAs(95.24%,20/21)displayed significantly different expression levels between those two materials.Considering that most of the miRNAs could detect their precursors in different barley genomes,the role of miRNA contributing to phenotype variation between wild barley and cultivar barley might,in most cases,come from their different expression patterns rather than genomic constitution.MiRNA target predictions showed that 12 out of 52 miRNA families were predicted to target transcription factors,including 8 highly conserved miRNA families in plants and 4 wheat–barley conserved miRNA families.In addition to transcription factors,other predicted target genes were involved in diverse physiological and metabolic processes and stress defense.Our study for the first time reported the large-scale investigation of small RNAs in wild barley,which will provide essential information for understanding the regulatory role of miRNAs in wild barley and also shed light on future practical utilization of miRNAs for barley improvement.(3)Integrated analysis of mRNA and small RNAs profiles dissecting an atlas of barley grain developmentBarley is a well-studied model of cereal grain development and maturation.In this study,we performed a comparative study of transcriptome and small RNAs dynamics between four developmental phases of this process: early pre-storage phase(0-5 DPA),late pre-storage or transition phase(6-10 DPA),early storage phase(11-15 DPA)and levels off stages(16-20 DPA).Transcriptome profiling revealed pronounced shifts in the abundance of transcripts of both primary and secondary metabolism during development.Furthermore,ABA and sugar signaling interaction networks played critical roles in the control of transition from pre-storage to storage phase.The transcripts variations were closely coordinated with the physiological changes that a general decrease in transcripts activity were observed during maturation and the largest systematic divergence occurred between the transitions from pre-storage phase to storage phase.Besides,genes related to photosynthesis showed peak expression in the early and middle stages of barley grain development,which were closely related with lipid metabolism and nucleotide metabolism rather than major CHO synthesis.When combined with the small RNAs data,a high proportion of miRNAs showed peak expression at the most inactive and active phase of gene expression,while majority of siRNAs were preferentially expressed at the inactive phases of gene expression.These results indicated that the presence of miRNAs might act as a ‘regulator' participating in several metabolic activities by the regulation of gene expression,whereas siRNAs might act as a ‘silencer' involved in suppressing gene activity until quiescence during barley grain development.In addition,a total of 8908 genes(298 genes for stage-specific expressed and 8610 for significantly differential expression)were considered to be potentially involved in the regulation of barley grain development,of which 131 and 1695 were regulated by mi RNAs and siRNAs,respectively.Our data provide insights into small RNA-mediated dynamic gene regulation mechanisms underlying grain development in barley.