Dynamic changes in the expression pattern of miRNAs and associated target genes during coconut somatic embryogenesis.

Genome-wide analysis of small RNAs identifies somatic embryogenesis- specific miRNAs and their targets and provides novel insights into the mechanisms governing somatic embryogenesis in coconut, a highly in vitro recalcitrant species. Coconut, a major plantation crop of the tropics is recalcitrant to in vitro culture with a very low rate of somatic embryo turnover. Clonal propagation to enhance the production of high yielding, disease-free planting material in coconut has remained a distant reality. To better understand the molecular basis of this recalcitrance and to throw light on the complex regulatory network involved in the transition of coconut somatic cells to embryogenic calli, genome-wide profiling of small RNAs from embryogenic (EC) and non-embryogenic calli (NEC) was undertaken using Illumina Hiseq 2000 platform. We have identified a total of 110 conserved miRNAs (representing 46 known miRNA families) in both types of calli. In addition, 97 novel miRNAs (48 specific to EC, 21 specific to NEC and 28 common to both the libraries) were also identified. Among the conserved miRNAs, 10 were found to be differentially expressed between NEC and EC libraries with a log2 fold change > 2 following RPM-based normalization. miR156f, miR167c, miR169a, miR319a, miR535a, and miR5179 are upregulated and miR160a, miR166a, miR171a, and miR319b are down-regulated in NEC. To confirm the differential expression pattern and their regulatory role in SE, the expression patterns of miRNAs and their putative targets were analyzed using qRT- PCR and most of the analyzed miRNA-target pairs showed inverse correlation during somatic embryogenesis. Selected targets were further validated by RNA ligase mediated rapid amplification of 5' cDNA ends (5'RLM-RACE). Our data suggest that a few conserved miRNAs and species-specific miRNAs act in concert to regulate the process of somatic embryogenesis in coconut. The results of this study provide the first overview into the regulatory landscape of somatic embryogenesis in coconut and possible strategies for fine-tuning or reprogramming to enhance somatic embryo turn over in coconut.