Abstract
BackgroundAlfalfa (Medicago sativa L.) is a forage legume with significant agricultural value worldwide. MicroRNAs (miRNAs) are key components of post-transcriptional gene regulation and essentially regulate many aspects of plant growth and development. Although miRNAs were reported in alfalfa, their expression profiles in different tissues and the discovery of novel miRNAs as well as their targets have not been described in this plant species.ResultsTo identify tissue-specific miRNA profiles in whole plants, shoots and roots of three different alfalfa genotypes (Altet-4, NECS-141and NF08ALF06) were used. Small RNA libraries were generated and sequenced using a high-throughput sequencing platform. Analysis of these libraries enabled identification of100 miRNA families; 21 of them belong to the highly conserved families while the remaining 79 families are conserved at the minimum between M. sativa and the model legume and close relative, M. truncatula. The profiles of the six abundantly expressed miRNA families (miR156, miR159, miR166, miR319, miR396 and miR398) were relatively similar between the whole plants, roots and shoots of these three alfalfa genotypes. In contrast, robust differences between shoots and roots for miR160 and miR408 levels were evident, and their expression was more abundant in the shoots. Additionally, 17 novel miRNAs were identified and the relative abundance of some of these differed between tissue types. Further, the generation and analysis of degradome libraries from the three alfalfa genotypes enabled confirmation of 69 genes as targets for 31 miRNA families in alfalfa.ConclusionsThe miRNA profiles revealed both similarities and differences in the expression profiles between tissues within a genotype as well as between the genotypes. Among the highly conserved miRNA families, miR166 was the most abundantly expressed in almost all tissues from the three genotypes. The identification of conserved and novel miRNAs as well as their targets in different tissues of multiple genotypes increased our understanding of miRNA-mediated gene regulation in alfalfa and could provide valuable insights for practical research and plant improvement applications in alfalfa and related legume species.
Highlights
Alfalfa (Medicago sativa L.) is an important forage legume species with global adaptation, high forage quality and the capacity for harvesting biomass multiple times during the growing season
The quality of the small RNA library generated from the shoots of NF08ALF06 did not meet the threshold criteria, only NECS-141 and Altet-4 were used for the miRNA analyses of shoot tissues
Quantification of miRNA abundances between the genotypes and tissues was preceded by normalizing the expression levels of miRNA families to reads per ten million (RPTM)
Summary
Alfalfa (Medicago sativa L.) is an important forage legume species with global adaptation, high forage quality and the capacity for harvesting biomass multiple times during the growing season. Endogenous non-coding RNAs of approximately 21–22 nucleotides represent plant miRNAs that silence gene expression by binding to complementary sequences of its target mRNA at the post-transcriptional level. Such targeting results in mRNA cleavage and degradation or repression of translation, with the former being more prevalent in plants [4,5,6,7]. MiRNAs were reported in alfalfa, their expression profiles in different tissues and the discovery of novel miRNAs as well as their targets have not been described in this plant species
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