Genome-Wide Identification of Different Dormant <i>Medicago sativa</i> L. MicroRNAs in Response to Fall Dormancy

<div><p>Background</p><p>MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that regulate gene post-transcriptional expression in plants and animals. High-throughput sequencing technology is capable of identifying small RNAs in plant species. Alfalfa (<i>Medicago sativa</i> L.) is one of the most widely cultivated perennial forage legumes worldwide, and fall dormancy is an adaptive characteristic related to the biomass production and winter survival in alfalfa. Here, we applied high-throughput sRNA sequencing to identify some miRNAs that were responsive to fall dormancy in standard variety (Maverick and CUF101) of alfalfa.</p><p>Results</p><p>Four sRNA libraries were generated and sequenced from alfalfa leaves in two typical varieties at distinct seasons. Through integrative analysis, we identified 51 novel miRNA candidates of 206 families. Additionally, we identified 28 miRNAs associated with fall dormancy in standard variety (Maverick and CUF101), including 20 known miRNAs and eight novel miRNAs. Both high-throughput sequencing and RT-qPCR confirmed that eight known miRNA members were up-regulated and six known miRNA members were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101). Among the 51 novel miRNA candidates, five miRNAs were up-regulated and three miRNAs were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101), and five of them were confirmed by Northern blot analysis.</p><p>Conclusion</p><p>We identified 20 known miRNAs and eight new miRNA candidates that were responsive to fall dormancy in standard variety (Maverick and CUF101) by high-throughput sequencing of small RNAs from <i>Medicago sativa</i>. Our data provide a useful resource for investigating miRNA-mediated regulatory mechanisms of fall dormancy in alfalfa, and these findings are important for our understanding of the roles played by miRNAs in the response of plants to abiotic stress in general and fall dormancy in alfalfa.</p></div

The probes designed for Northern blot.

<p>Note: All the nucleic acid sequences contain T7 promoter.</p><p>The probes designed for Northern blot.</p

Statistics of sRNA sequences for four alfalfa libraries.

<p>Note: 1. Mapping of sequence reads back to transcriptome, using Trinity assembler with the aligner bowtie.</p><p>2. Screening sRNA within the scope of a certain length (15∼40 nt) for subsequent analysis (base on clean reads).</p><p>Statistics of sRNA sequences for four alfalfa libraries.</p

Distribution of sRNAs detected in four alfalfa libraries.

<p>The length distribution of sRNA reads in dormant alfalfa (DM1, and DS3) and non-dormant alfalfa (NM2, and NS4) were detected with Solexa technology.</p

Sequencing and qRT-PCR.

<p>Quantitative RT-PCR validation of differentially expressed genes in non-dormant alfalfa (NM2, and NS4) and dormant alfalfa (DM1, and DS3), including 14 genes. All data were normalized to the expression level of GAPDH. Data represent Log₂ (Fold Change) of relative quantifications for DM1, NM2, and NS4 vs DS3. The error bars represent the range of the fold change as determined by the data assist software.</p

Northern blot analysis.

<p>Sequencing Data represent Log₂ (Fold Change) of quantification of DM1, NM2, DS3, and NS4. Northern blot analysis showed the different expressions of miR-novel_1, miR-novel_3, miR-novel_45, miR-novel_17 and miR-novel_76 in non-dormant alfalfa (NM2, and NS4) and dormant alfalfa (DM1, and DS3). GAPDH was used as a control.</p

Natural height and leaf area of plants selected for sequencing.

<p>Plants heights and leaf areas were measured using Mean±SD (standard deviation).</p><p>Natural height and leaf area of plants selected for sequencing.</p

Predicted targets of fall dormancy miRNAs and their function annotations.

<p>Predicted targets of fall dormancy miRNAs and their function annotations.</p