Identification of Drought-Responsive MicroRNAs from Roots and Leaves of Alfalfa by High-Throughput Sequencing

Abstract

Alfalfa, an important forage legume, is an ideal crop for sustainable agriculture and a potential crop for bioenergy resources. Drought, one of the most common environmental stresses, substantially affects plant growth, development, and productivity. MicroRNAs (miRNAs) are newly discovered gene expression regulators that have been linked to several plant stress responses. To elucidate the role of miRNAs in drought stress regulation of alfalfa, a high-throughput sequencing approach was used to analyze 12 small RNA libraries comprising of four samples, each with three biological replicates. From the 12 libraries, we identified 348 known miRNAs belonging to 80 miRNA families, and 281 novel miRNAs, using Mireap software. Eighteen known miRNAs in roots and 12 known miRNAs in leaves were screened as drought-responsive miRNAs. With the exception of miR319d and miR157a which were upregulated under drought stress, the expression pattern of drought-responsive miRNAs was different between roots and leaves in alfalfa. This is the first study that has identified miR3512, miR3630, miR5213, miR5294, miR5368 and miR6173 as drought-responsive miRNAs. Target transcripts of drought-responsive miRNAs were computationally predicted. All 447 target genes for the known miRNAs were predicted using an online tool. This study provides a significant insight on understanding drought-responsive mechanisms of alfalfa.

Legume genetic resources and transcriptome dynamics under abiotic stress conditions

“…NGS technologies have become essential for genome analysis and SNP research, as well as gene discovery, comparative genomics, and studies on the regulation of gene expression (Abdelrahman, El‐Sayed, Sato, et al, ; Celesti, Celesti, Fazio, Bramanti, & Villari, ; Considine et al, ). The NGS‐based, that is, RNA sequencing‐based, transcriptome analysis is considered to be a superior approach for understanding gene function and the molecular basis of many cellular responses in plants exposed to abiotic and biotic stressors, compared with microarrays‐based one (Abdelrahman, Suzumura, et al, ; Abdelrahman, El‐Sayed, Sato, et al, ; Ha et al, ; Li, Cao, et al, ; Li, Wan, et al, ; O'Rourke et al, ). For example, some plant species have similar genomes but have very different gene expression profiles, reflecting a wide range of biochemical, physical, and developmental differences.…”

“…Consequently, interest has increased in the roles miRNAs play in legumes under abiotic stress. Studies using NGS‐based technologies have shown that both novel and conserved miRNAs regulate species‐specific cellular processes in legumes (Bustos‐Sanmamed, Bazin, Hartmann, Crespi, & Lelandais‐Brière, ; Kohli et al, ; Li, Wan, et al, ; Mantri, Basker, Ford, Pang, & Pardeshi, ; Zheng et al, ). One thousand three hundred seventy‐nine miRNA sequences belonging to 285 miRNA families have been identified in legumes, including Acacia auriculiformis , Acacia mangium , G .…”

“…truncatula , common bean, cowpea, and peanut (http://www.mirbase.org/cgi-bin/browse.pl?org=ath). Recently, high‐throughput Illumina Hiseq 2500 sequencing was used to identify drought‐responsive miRNAs from alfalfa roots and leaves, revealing 348 known miRNAs belonging to 80 miRNA families, and 281 novel, predicted miRNAs (Li, Wan, et al, ). The authors suggested that drought induces miRNA‐related pathways with higher proportions of known miRNA families (10.1–12.5%), including the miR166, miR159 , miR482 , and miR2118 families, relative to control plants, and that miRNAs have important regulatory roles in legumes under drought (Li, Wan, et al, ).…”

“…Recently, high‐throughput Illumina Hiseq 2500 sequencing was used to identify drought‐responsive miRNAs from alfalfa roots and leaves, revealing 348 known miRNAs belonging to 80 miRNA families, and 281 novel, predicted miRNAs (Li, Wan, et al, ). The authors suggested that drought induces miRNA‐related pathways with higher proportions of known miRNA families (10.1–12.5%), including the miR166, miR159 , miR482 , and miR2118 families, relative to control plants, and that miRNAs have important regulatory roles in legumes under drought (Li, Wan, et al, ). Although novel miRNA candidates were predicted by computational methods, the expression levels of these predicted miRNAs were lower than that of the known conserved miRNAs, indicating that the conserved miRNAs regulate key genes that are involved in many important biological processes in legume plants under stress (Li, Wan, et al, ).…”

“…The authors suggested that drought induces miRNA‐related pathways with higher proportions of known miRNA families (10.1–12.5%), including the miR166, miR159 , miR482 , and miR2118 families, relative to control plants, and that miRNAs have important regulatory roles in legumes under drought (Li, Wan, et al, ). Although novel miRNA candidates were predicted by computational methods, the expression levels of these predicted miRNAs were lower than that of the known conserved miRNAs, indicating that the conserved miRNAs regulate key genes that are involved in many important biological processes in legume plants under stress (Li, Wan, et al, ). Another recent study by Sosa‐Valencia et al () showed a significant up‐regulation in Pvu‐miR1514a expression in the drought‐tolerant common bean cultivar “Pinto Saltillo,” relative to the drought‐sensitive cultivar “Bayo Madero,” in response to drought.…”