Elucidation of miRNAs-Mediated Responses to Low Nitrogen Stress by Deep Sequencing of Two Soybean Genotypes

<div><p>Nitrogen (N) is a major limiting factor in crop production, and plant adaptive responses to low N are involved in many post-transcriptional regulation. Recent studies indicate that miRNAs play important roles in adaptive responses. However, miRNAs in soybean adaptive responses to N limitation have been not reported. We constructed sixteen libraries to identify low N-responsive miRNAs on a genome-wide scale using samples from 2 different genotypes (low N sensitive and low N tolerant) subjected to various periods of low nitrogen stress. Using high-throughput sequencing technology (Illumina-Solexa), we identified 362 known miRNAs variants belonging to 158 families and 90 new miRNAs belonging to 55 families. Among these known miRNAs variants, almost 50% were not different from annotated miRNAs in miRBase. Analyses of their expression patterns showed 150 known miRNAs variants as well as 2 novel miRNAs with differential expressions. These differentially expressed miRNAs between the two soybean genotypes were compared and classified into three groups based on their expression patterns. Predicted targets of these miRNAs were involved in various metabolic and regulatory pathways such as protein degradation, carbohydrate metabolism, hormone signaling pathway, and cellular transport. These findings suggest that miRNAs play important roles in soybean response to low N and contribute to the understanding of the genetic basis of differences in adaptive responses to N limitation between the two soybean genotypes. Our study provides basis for expounding the complex gene regulatory network of these miRNAs.</p></div

The length distribution of soybean sRNAs.

<p>(a) Length distribution of total sRNAs reads in eight root libraries. The y-axis indicates percent of total sRNAs, and the x-axis indicates length of total sRNAs (nt). (b) Length distribution of total sRNAs reads in eight shoot libraries: The y-axis indicates percent of total sRNAs, and the x-axis indicates length of total sRNAs (nt). (c) Length distribution of unique sRNAs reads in eight root libraries. The y-axis indicates percent of unique sRNAs, and the x-axis indicates length of unique sRNAs(nt). (d) Length distribution of unique sRNAs reads in eight shoot libraries. The y-axis indicates percent of unique sRNAs, and the x-axis indicates length of unique sRNAs(nt). 116RS, 116-root short-term treatment; 116RL, 116-root long-term treatment; 84RS, 84-70-root short-term treatment; 84RL, 84-70-root long-term treatment; 116SS, 116-shoot short-term treatment; 116SL, 116-shoot long-term treatment; 84SS, 84-70-shoot short-term treatment; 84SL, 84-70-shoot long-term treatment; 116RSC, 116-root short-term control; 116RLC, 116-root long-term control; 84RSC, 84-70-root short-term control; 84RLC, 84-70-root long-term control; 116SSC, 116-shoot short-term control; 116SLC, 116-shoot long-term control; 84SSC, 84-70-shoot short-term control; 84SLC, 84-70-shoot long-term control.</p

Statistics of sequenced reads from all libraries.

<p>Clean reads are those remaining after low-quality reads have been removed from total raw reads. Unique reads are different types of clean reads. The number of the total clean reads and unique reads from the sixteen libraries that matched to the genome sequences are also listed.</p><p>Note: 116RS, 116-root short-term treatment; 116RL, 116-root long-term treatment; 84RS, 84-70-root short-term treatment; 84RL, 84-70-root long-term treatment; 116SS, 116-shoot short-term treatment; 116SL, 116-shoot long-term treatment; 84SS, 84-70-shoot short-term treatment; 84SL, 84-70-shoot long-term treatment; 116RSC, 116-root short-term control; 116RLC, 116-root long-term control; 84RSC, 84-70-root short-term control; 84RLC, 84-70-root long-term control;116SSC, 116-shoot short-term control; 116SLC, 116-shoot long-term control; 84SSC, 84-70-shoot short-term control; 84SLC, 84-70-shoot long-term control.</p

Summary of new miRNAs.

<p>Novel miRNAs are the small sequences which meet the critical criterion of novel miRNA annotation. miRNA* are the sequences which can form miRNA::miRNA* duplexes with novel miRNA. Families are the novel miRNAs with similar sequences.</p

Comparison between qRT-PCR and the deep sequencing in No.116 root exposed to short-term low nitrogen stress.

<p>The y-axis indicate the relative expression levels of twelve selected miRNA in qRT-PCR and in Solexa sequencing analysis. The x-axis indicates twelve selected miRNAs, which are respectively as follows: 1. gma-miR1511-3p; 2. gma-miR390a/c-3p; 3. gma-miR396b/c/d/f/g-5p; 4. gma-miR396b/d/g-3p; 5. gma-miR398a/b-3p; 6. gma-miR4348-5p; 7. gma-miR4413a-5p; 8. gma-miR482a-3p; 9. gma-miR156p-5p; 10. gma-miR171n-3p; 11. gma-miR171o-5p; 12. gma-miR172l-3p. gma-miR1520d-3p and gma-miR156b-5p was chosen as endogenous reference genes.</p

Examples where the most abundant sequences were different from the annotated miRNA.

<p>only sequences with more than 10 reads are shown except the most abundant variants and the annotated miRNA, the sequences that was the most abundant variants on the two arm of pre-miRNAs were shown in red, and the annotated miRNAs were shown in pink.</p

Specific and common response miRNAs in soybean shoots from differential compared libraries.

<p>116SS, 116-shoot short-term treatment; 116SL, 116-shoot long-term treatment; 84SS, 84-70-shoot short-term treatment; 84SL, 84-70-shoot long-term treatment; 116SSC, 116-shoot short-term control; 116SLC, 116-shoot long-term control; 84SSC, 84-70-shoot short-term control; 84SLC, 84-70-shoot long-term control.</p

Specific and common response miRNAs in soybean roots from differential compared libraries.

<p>116RS, 116-root short-term treatment; 116RL, 116-root long-term treatment; 84RS, 84-70-root short-term treatment; 84RL, 84-70-root long-term treatment; 116-root short-term control; 116RLC, 116-root long-term control; 84RSC, 84-70-root short-term control; 84RLC, 84-70-root long-term control.</p

Numbers of up-regulated and downregulated miRNA were summarized in compared libraries.

<p>116RS, 116-root short-term treatment; 116RL, 116-root long-term treatment; 84RS, 84-70-root short-term treatment; 84RL, 84-70-root long-term treatment; 116SS, 116-shoot short-term treatment; 116SL, 116-shoot long-term treatment; 84SS, 84-70-shoot short-term treatment; 84SL, 84-70-shoot long-term treatment; 116RSC, 116-root short-term control; 116RLC, 116-root long-term control; 84RSC, 84-70-root short-term control; 84RLC, 84-70-root long-term control;116SSC, 116-shoot short-term control; 116SLC, 116-shoot long-term control; 84SSC, 84-70-shoot short-term control; 84SLC, g00484-70-shoot long-term control.</p

Some new miRNAs located in the introns or exons of protein-coding genes.

<p>Some new miRNAs located in the introns or exons of protein-coding genes.</p