Identification of the Conserved and Novel miRNAs in Mulberry by High-Throughput Sequencing

<div><p>miRNAs are a class of non-coding endogenous small RNAs. They play vital roles in plant growth, development, and response to biotic and abiotic stress by negatively regulating genes. Mulberry trees are economically important species with multiple uses. However, to date, little is known about mulberry miRNAs and their target genes. In the present study, three small mulberry RNA libraries were constructed and sequenced using high-throughput sequencing technology. Results showed 85 conserved miRNAs belonging to 31 miRNA families and 262 novel miRNAs at 371 loci. Quantitative real-time PCR (qRT-PCR) analysis confirmed the expression pattern of 9 conserved and 5 novel miRNAs in leaves, bark, and male flowers. A total of 332 potential target genes were predicted to be associated with these 113 novel miRNAs. These results provide a basis for further understanding of mulberry miRNAs and the biological processes in which they are involved.</p></div

The expression profile of 10 target genes belonging to 6 miRNAs in three mulberry tissues (leaf, bark, and male flower) using RT-PCR.

<p>Morus015493 and Morus018032 are the predicted target genes of miR156, so as Morus012124, Morus012122, and Morus012121 for miRn51, Morus008520 for miRn247, Morus019289 for miRn67, Morus011908 for miRn62, Morus002508 and Morus014466 for miRn157. The X axis represents different tissues. L indicates leaf tissue. B indicates bark tissue. MF indicates male flower tissue. The Y axis represents the relative expression level of target genes. MorusXXXXXX represented the target gene ID.</p

Relative expression of 14 miRNAs in three mulberry tissues (leaf, bark, and male flower) using stem-loop RT-PCR.

<p>The X axis represents different tissues. L indicates leaf tissue. B indicates bark tissue. MF indicates male flower tissue. The Y axis represents the relative expression level of miRNAs.</p

Reads per million of conserved miRNAs in mulberry leaves, bark, and male flowers.

<p>A) miRNAs with >1000 reads; B) miRNAs with 30–1000 reads; C) miRNAs with <30 reads. The X axis represents different conserved miRNAs. The Y axis represents the reads per million for different miRNAs. RPM means reads per million.</p

Classification of small RNAs in the mulberry leaves, bark, and male flowers.

<p>Classification of small RNAs in the mulberry leaves, bark, and male flowers.</p

Additional file 1 of Overexpression of a WRKY transcription factor McWRKY57-like from Mentha canadensis L. enhances drought tolerance in transgenic Arabidopsis

Supplementary Material

Sequence length (bp) and percentage GC content (100%) of the ITS region in the genus <i>Morus</i>.

<p>Sequence length (bp) and percentage GC content (100%) of the ITS region in the genus <i>Morus</i>.</p

Additional file 2 of Overexpression of a WRKY transcription factor McWRKY57-like from Mentha canadensis L. enhances drought tolerance in transgenic Arabidopsis

Supplementary Material

Sequence length (bp) and percentage GC content (100%) of the ITS region in the genus <i>Morus</i>.

<p>Sequence length (bp) and percentage GC content (100%) of the ITS region in the genus <i>Morus</i>.</p

Definition of Eight Mulberry Species in the Genus <i>Morus</i> by Internal Transcribed Spacer-Based Phylogeny

<div><p>Mulberry, belonging to the order Rosales, family Moraceae, and genus <i>Morus</i>, has received attention because of both its economic and medicinal value, as well as for its important ecological function. The genus <i>Morus</i> has a worldwide distribution, however, its taxonomy remains complex and disputed. Many studies have attempted to classify <i>Morus</i> species, resulting in varied numbers of designated <i>Morus</i> spp. To address this issue, we used information from internal transcribed spacer (ITS) genetic sequences to study the taxonomy of all the members of generally accepted genus <i>Morus</i>. We found that intraspecific 5.8S rRNA sequences were identical but that interspecific 5.8S sequences were diverse. <i>M</i>. <i>alba</i> and <i>M</i>. <i>notabilis</i> showed the shortest (215 bp) and the longest (233 bp) ITS1 sequence length, respectively. With the completion of the mulberry genome, we could identify single nucleotide polymorphisms within the ITS locus in the <i>M</i>. <i>notabilis</i> genome. From reconstruction of a phylogenetic tree based on the complete ITS data, we propose that the <i>Morus</i> genus should be classified into eight species, including <i>M</i>. <i>alba</i>, <i>M</i>. <i>nigra</i>, <i>M</i>. <i>notabilis</i>, <i>M</i>. <i>serrata</i>, <i>M</i>. <i>celtidifolia</i>, <i>M</i>. <i>insignis</i>, <i>M</i>. <i>rubra</i>, and <i>M</i>. <i>mesozygia</i>. Furthermore, the classification of the ITS sequences of known interspecific hybrid clones into both paternal and maternal clades indicated that ITS variation was sufficient to distinguish interspecific hybrids in the genus <i>Morus</i>.</p></div