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Comparison of miRNAs and Their Targets in Seed Development between Two Maize Inbred Lines by High-Throughput Sequencing and Degradome Analysis.

ABSTRACT: MicroRNAs (miRNAs) play an important role in plant growth, development, and response to environment. For identifying and comparing miRNAs and their targets in seed development between two maize inbred lines (i.e. PH6WC and PH4CV), two sRNAs and two degradome libraries were constructed. Through high-throughput sequencing and miRNA identification, 55 conserved and 24 novel unique miRNA sequences were identified in two sRNA libraries; moreover, through degradome sequencing and analysis, 137 target transcripts corresponding to 38 unique miRNA sequences were identified in two degradome libraries. Subsequently, 16 significantly differentially expressed miRNA sequences were verified by qRT-PCR, in which 9 verified sequences obviously target 30 transcripts mainly involved with regulation in flowering and development in embryo. Therefore, the results suggested that some miRNAs (e.g. miR156, miR171, miR396 and miR444) related reproductive development might differentially express in seed development between the PH6WC and PH4CV maize inbred lines in this present study.

SUBMITTER: Wu FY

PROVIDER: S-EPMC4962988 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMicroRNAs (miRNAs), a class of non-coding small RNAs (sRNAs), regulate various biological processes. Although miRNAs have been identified and characterized in several plant species, miRNAs in Asparagus officinalis have not been reported. As a dioecious plant with homomorphic sex chromosomes, asparagus is regarded as an important model system for studying mechanisms of plant sex determination.ResultsTwo independent sRNA libraries from male and female asparagus plants were sequenced with Illumina sequencing, thereby generating 4.13 and 5.88 million final clean reads, respectively. Both libraries predominantly contained 24-nt sRNAs, followed by 21-nt sRNAs. Further analysis identified 154 conserved miRNAs, which belong to 26 families, and 39 novel miRNA candidates seemed to be specific to asparagus. Comparative profiling revealed that 63 miRNAs exhibited significant differential expression between male and female plants, which was confirmed by real-time quantitative PCR analysis. Among them, 37 miRNAs were significantly up-regulated in the female library, whereas the others were preferentially expressed in the male library. Furthermore, 40 target mRNAs representing 44 conserved and seven novel miRNAs were identified in asparagus through high-throughput degradome sequencing. Functional annotation showed that these target mRNAs were involved in a wide range of developmental and metabolic processes.ConclusionsWe identified a large set of conserved and specific miRNAs and compared their expression levels between male and female asparagus plants. Several asparagus miRNAs, which belong to the miR159, miR167, and miR172 families involved in reproductive organ development, were differentially expressed between male and female plants, as well as during flower development. Consistently, several predicted targets of asparagus miRNAs were associated with floral organ development. These findings suggest the potential roles of miRNAs in sex determination and reproductive developmental processes in asparagus.

Project description:BACKGROUND: MicroRNAs (miRNAs) regulate the expression of target genes by mediating gene silencing in both plants and animals. The miRNA targets have been extensively investigated in Arabidopsis and rice using computational prediction, experimental validation by overexpression in transgenic plants, and by degradome or PARE (parallel analysis of RNA ends) sequencing. However, miRNA targets mostly remain unknown in soybean (Glycine max). More specifically miRNA mediated gene regulation at different seed developmental stages in soybean is largely unexplored. In order to dissect miRNA guided gene regulation in soybean developing seeds, we performed a transcriptome-wide experimental method using degradome sequencing to directly detect cleaved miRNA targets. RESULTS: In this study, degradome libraries were separately prepared from immature soybean cotyledons representing three stages of development and from seed coats of two stages. Sequencing and analysis of 10 to 40 million reads from each library resulted in identification of 183 different targets for 53 known soybean miRNAs. Among these, some were found only in the cotyledons representing cleavage by 25 miRNAs and others were found only in the seed coats reflecting cleavage by 12 miRNAs. A large number of targets for 16 miRNAs families were identified in both tissues irrespective of the stage. Interestingly, we identified more miRNA targets in the desiccating cotyledons of late seed maturation than in immature seed. We validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5' rapid amplification of cDNA ends (RLM-5'RACE). Gene Ontology (GO) analysis indicated the involvement of miRNA target genes in various cellular processes during seed development. CONCLUSIONS: The miRNA targets in both the cotyledons and seed coats of several stages of soybean seed development have been elucidated by experimental evidence from comprehensive, high throughput sequencing of the enriched fragments resulting from miRNA-guided cleavage of messenger RNAs. Nearly 50% of the miRNA targets were transcription factors in pathways that are likely important in setting or maintaining the developmental program leading to high quality soybean seeds that are one of the dominant sources of protein and oil in world markets.

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Comparison of miRNAs and Their Targets in Seed Development between Two Maize Inbred Lines by High-Throughput Sequencing and Degradome Analysis.

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