Comparative expression analysis of microRNAs and their targets in emerging bio-fuel crop sweet sorghum (Sorghum bicolor L.)

Sweet sorghum (Sorghum bicolor L) is a C4 plant, and it recently has emerged as a model feedstock for biofuel production. MicroRNAs (miRNAs) are known as key regulators in different biological processes in plant including sugar accumulation. Despite bioinformatics analyses for the discovery of miRNAs and prediction of targets in recent years, little information is available on the expression of mature candidate miRNAs and biological validation of their targets in sweet sorghum. This study aims to gain an insight on the conserved and novel miRNAs as well as their potential targets associated with biofuel related traits, with the help of next generation RNA sequencing (RNA-seq) followed by RT-qPCR validation. In order to identify biofuel related miRNAs and their potential targets in sweet sorghum, small RNA libraries were constructed from four varieties, i.e., Achi Turi, Dale, Dasht Local, and Topper 76-6 after being grown under controlled conditions, and sequenced by Illumina Hiseq 2000 platform. Sequencing yield from 12 libraries, 3 from each variety i.e., leaf vegetative stage, leaf reproductive stage and stem reproductive stage, ranged from 19 to 48 million reads. Mapping with Sorghum bicolor ensemble genome while aligned with miRBase-release 21, 276 conserved and novel biofuel relevant miRNAs were predicted in all varieties. Target prediction by employing psRNA Target tool indicated 475 biofuel relevant targets for identified conserved and novel miRNAs. We performed the RT-qPCR analysis on selected 36 candidate mature miRNAs along with their 18 targets. Potential biofuel relevant targets identified in our study included the sucrose phosphate synthase (SPS), sucrose synthase (SUS), sucrose transporters (SUT) and invertase (INV) enzymes, which are believed to play key role in sucrose synthesis, accumulation and transport. Altogether, 19 predicted miRNAs and their 15 target mRNAs showed significant change in expression under the three growth stages of four varieties. Significant down-regulation at reproductive stage was observed with Sbi-miR160, Sbi-miR164, Sbi-miR166 and Sbi-miR168 in most of the varieties while significant upregulation was observed for Sbi-miR156a and Sbi-miR156e during vegetative stages. This study showed a positive as well as negative correlations between levels of miRNA expressions and that of their target mRNAs. These findings, both computational and experimental, provide valuable insights on the role of miRNAs and their targets during sugar accumulation stages. The identification of these significantly expressed miRNAs and their targets will help to explore the molecular mechanisms for sugar accumulation in sweet sorghum.