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Identification and Characterization of Maize MicroRNAs Involved in the Very Early Stage of Seed Germination

ABSTRACT: In this study, a small RNA library from maize seed 24 hours after imbibition was sequenced by the Solexa technology. A total of 11,338,273 reads were obtained. 1,047,447 total reads representing 431 unique sRNAs matched to known maize miRNAs. Further analysis confirmed the authenticity of 115 known miRNAs belonging to 24 miRNA families and the discovery of 167 novel miRNAs in maize. Both the known and the novel miRNAs were confirmed by sequencing of a second small RNA library constructed the same way as the one used in the first sequencing. We also found 10 miRNAs that had not been reported in maize, but had been reported in other plant species. All novel sequences had not been earlier described in other plant species. In addition, seven miRNA* sequences were also obtained. Putative targets for 106 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in maize imbibed seed. This study led to the confirmation of the authenticity of 115 known miRNAs and the discovery of 167 novel miRNAs in maize. Identification of novel miRNAs resulted in significant enrichment of the repertoire of maize miRNAs and provided insights into miRNA regulation of genes expressed in imbibed seed.

ORGANISM(S): Zea mays

PROVIDER: GSE27664 | GEO | 2014/03/03

SECONDARY ACCESSION(S): PRJNA138475

REPOSITORIES: GEO

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Project description:In plants, MicroRNAs (miRNAs) are a new class of endogenous small RNAs that play essential regulatory roles in plant growth, development and stress response. Extensive studies of miRNAs have been performed in model plants such as rice, Arabidopsis thaliana and other plants. However, the number of miRNAs discovered in maize is relatively low and little is known about miRNAs involved in the four stages during maize ear development. Here, we use deep-sequencing, miRNA microarray assays and computational methods to identify, proM-oM-,M-^Ale, and describe conserved and non-conserved miRNAs at four developmental stages. A total of 27 conserved miRNA families were identiM-oM-,M-^Aed in all four stages, In addition to known miRNAs, we also found 23 new maize-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. We have found that many known and new miRNAs showed temporally expression. Finally, a total of 251 transcripts (140 genes) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.This study led to the confirmation of the authenticity of 27 conserved miRNA families and the discovery of 23 novel miRNAs in maize. In addition, we have identified 130 targets of known and new miRNAs and ta-siRNA using recently developed tools for the global identification of miRNA targets. Identification and characterization of this important class of regulatory genes in maize may improve our understanding of molecular mechanism controlling flower development. The seeds of maize inbred line B73 were first sterilized and germinated in an incubator, then grown in a controlled environment with 28M-BM-0C/21M-BM-0C (day/night) under a 16-h day/8-h night photoperiod with a relative humidity of 70%. Ear development can be divided into four stages: the growth point elongation phase, spikelet differentiation phase, the floret primordium differentiation phase and floret organ differentiation phase. Plant materials (ears) were collected as described previously. In brief, ears were manually collected at the four developmental stages according to the plant features (number of visible leaves, leaf age index, and number of unfolded and folded leaves) combined with microscopic observation.

Project description:Background: MicroRNAs regulate various biological processes in plants. Considerable data are available on miRNAs involved in the development of rice, maize and barley. In contrast, little is known about miRNAs and their functions in the development of wheat. In this study, five small RNA (sRNA) libraries from wheat seedlings, flag leaves, and developing seeds were developed and sequenced to identify miRNAs and understand their functions in wheat development. Results: Twenty-four known miRNAs belonging to 15 miRNA families were identified from 18 MIRNA loci in wheat in the present study, including 15 (9 MIRNA loci) first identified in wheat, 13 miRNA families (16 MIRNA loci) being highly conserved and 2 (2 MIRNAs loci) moderately conserved. In addition, fifty-five novel miRNAs were also identified. The potential target genes for 15 known miRNAs and 37 novel miRNAs were predicted using strict criteria, and these target genes are involved in a wide range of biological functions. Four of the 15 known miRNA families and 22 of the 55 novel miRNAs were preferentially expressed in the developing seeds with logarithm of the fold change of 1.0～7.6, and half of them were seed-specific, suggesting that they participate in regulating wheat seed development and metabolism. From 5 days post-anthesis to 20 days post-anthesis, miR164 and miR160 increased in abundance in developing seeds, whereas miR169 decreased, suggesting their coordinating functions in the different developmental stages of wheat seed. Moreover, eight known miRNA families and 28 novel miRNAs exhibited tissue-biased expression in wheat flag leaves, with the logarithm of the fold changes of 0.5～5.2. The putative targets of these tissue-preferential miRNAs were involved in various metabolism and biological processes, suggesting complexity of the regulatory networks in different tissues. Our data also suggested that wheat flag leaves have more complicated regulatory networks of miRNAs than developing seeds. Conclusions: Our work identified and characterised wheat miRNAs, their targets and expression patterns. This study is the first to elucidate the regulatory networks of miRNAs involved in wheat flag leaves and developing seeds, and provided a foundation for future studies on specific functions of these miRNAs.

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Identification and Characterization of Maize MicroRNAs Involved in the Very Early Stage of Seed Germination

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