Analysis of microRNAs and metabolomic profiling of two different populations of Neem (Azadirachta indica A. Juss) from Mexico

Azadirachta indica, common name neem, is an evergreen tree with potential for the treatment of cardiovascular diseases, rheumatism, osteoporosis, and a mosquito repellent; These properties are due to its rich content in phytochemicals. In Mexico, it is established in more than 12 states, but there are no previous reports of phytochemical content for Mexican neem populations. MicroRNAs (miRNAs) are highly conserved, non-coding, short RNA molecules that play important regulatory roles in plant development and metabolism, including the synthesis of secondary metabolites. The aim of the present work was to identify and characterize the miRNAs and their targets involved in the production of secondary metabolites in A. indica, employing a high stringent genome-wide computational-based approach, and following a set of strict filtering norms, followed by experimental validation. As well, to perform the metabolomic profile of two Mexican A. indica samples through gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-QTOF-MS). Thus, a total of 44 potential A. indica miRNAs belonging to 21 families and their corresponding 48 potential target transcripts were identified. Relevant targets included Squamosa promoter binding protein-like proteins, NAC, Scarecrow proteins, Auxin response factor, and F-box proteins. Some of the identified miRNAs showed to be involved in the biosynthesis of phenolic compounds and terpenoids. Gene network analysis was developed to understand the miRNA mediated gene regulation. As well, conservation and phylogenetic studies showed high conservation of miRNA precursors with homologs in other important plant models. Moreover, nine selected miRNAs were experimentally validated by amplification via RT-PCR, and five miRNAs involved in secondary metabolism (ain-miR156l, ain-miR160, ain-miR164, ain-miR171, and ain- vi miR395) were validated by qPCR and their specific differential expression pattern was observed in Queretaro and Chiapas samples. As well, hexane, acetone and methanol extracts were analyzed by GC-MS and 29 compounds were identified, including relevant phenolic compounds, terpenes, vitamins and fatty acids with reported biological activities. Similarly, UHPL-QTOF-MS analysis revealed that methanolic extracts of A. indica from Queretaro and Chiapas presented differential metabolic profiles according to Principal Component Analysis. Among the preliminary compounds identified, flavonoids and triterpenes of pharmacological importance.