Identification, expression analysis and target prediction of flax genotroph microRNAs under normal and nutrient stress conditions. flaxgenotrophmirnas

Cultivated flax (Linum usitatissimum L.) is a valuable for industry plant. Some flax lines (called plastic) are able to undergo heritable phenotypic and genotypic (LIS-1 insertion is the most known) changes in response to the nutrient stress. Offspring of plastic line, which stably inherited the changes, were called genotrophs. MicroRNA (miRNA) is a crucial regulatory mechanism of gene expression that previously was assumed to take part in nutrient stress response and therefore can participate in genotroph formation. In this work we performed high-throughput sequencing of small RNAs of flax plants grown under normal, deficient and excess nutrient to identify miRNAs and evaluate their expression. We revealed 96 conserved miRNAs from 21 families that were expressed in flax. Moreover, 475 novel potential miRNAs were identified for the first time, and their targets were predicted. However, none of revealed in our work miRNAs can be transcribed from LIS-1. Expression of 7 miRNAs (miR168, miR169, miR395, miR398, miR399, miR408, and lus-miR-N1) with up- or down-regulation under nutrient stress (on the basis of high-throughput sequencing data) was evaluated on extended sampling using qPCR. Reference gene search identified ETIF3H and ETIF3E genes as the most suitable for this issue. Down-regulation of novel potential lus-miR-N1 (3-fold, p=0.01) and up-regulation of conserved miR399 (3-fold, p<0.01) was revealed under the phosphate deficiency. Besides, the negative correlation of expression of lus-miR-N1 and its predicted target ubiquitin activating enzyme E1 gene as well as miR399 and its predicted target ubiquitin conjugating enzyme E2 gene was shown (rs = -0.32 and -0.64 correspondingly). Thus, in our study, miRNAs expressed in flax plastic lines and genotrophs were identified and their expression and expression of their targets was evaluated using high-throughput sequencing and qPCR for the first time. These data provide a new knowledge on nutrient stress response regulation in plastic flax cultivars.