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Background   Medicago sativa  (alfalfa) is a low-input forage and potential bioenergy crop, and improving its yield and quality has always been a focus of the alfalfa breeding industry. Transgenic alfalfa plants overexpressing a precursor of alfalfa  microRNA156  ( MsmiR156 ) were recently generated by our group. These plants (miR156OE) showed enhanced biomass yield, reduced internodal length, increased shoot branching and trichome density, and a delay in flowering time. Transcripts of three  SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL)  genes ( MsSPL6 ,  MsSPL12 , and  MsSPL13 ) were found to be targeted for cleavage by  MsmiR156  in alfalfa.    Results  To further illustrate the molecular mechanisms underlying the effects of miR156 in alfalfa, two miR156OE genotypes (A11a and A17) were subjected to Next Generation RNA Sequencing with Illumina HiSeq. More than 1.11 billion clean reads were obtained from our available sequenced samples. A total of 160,472 transcripts were generated using Trinity  de novo  assembly and 4,985 significantly differentially expressed genes were detected in miR156OE plants A11a and A17 using the  Medicago truncatula  genome as reference. A total of 17 genes (including upregulated, downregulated, and unchanged) were selected for quantitative real-time PCR (qRT-PCR) validation, which showed that gene expression levels were largely consistent between qRT-PCR and RNA-Seq data. In addition to the established  SPL  genes  MsSPL6 ,  MsSPL12  and  MsSPL13 , four new  SPLs; MsSPL2 ,  MsSPL3 ,  MsSPL4  and  MsSPL9  were also down-regulated significantly in both miR156OE plants. These seven  SPL  genes belong to genes phylogeny clades VI, IV, VIII, V and VII, which have been reported to be targeted by  miR156  in  Arabidopsis thaliana . The gene ontology terms characterized electron transporter, starch synthase activity, sucrose transport, sucrose-phosphate synthase activity, chitin binding, sexual reproduction, flavonoid biosynthesis and lignin catabolism correlate well to the phenotypes of miR156OE alfalfa plants.    Conclusions  This is the first report of changes in global gene expression in response to miR156 overexpression in alfalfa. The discovered miR156-targeted  SPL  genes belonging to different clades indicate miR156 plays fundamental and multifunctional roles in regulating alfalfa plant development.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-016-3014-6) contains supplementary material, which is available to authorized users.

Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa