Genome-Wide Identification of the Rehmannia glutinosa miRNA Family and Exploration of Their Expression Characteristics Caused by the Replant Disease Formation-Related Principal Factor

<b>Background/Objectives:</b> <i>Rehmannia glutinosa</i>, a highly valuable medicinal plant in China, is encountering severe replant disease. Replant disease represents a complex stress driven by multiple principal factors (RDFs), including allelochemicals, microbes, and their interactions. miRNAs are recognized as key regulators of plant response to stresses; however, their specific roles within RDFs are not entirely clear. <b>Methods:</b> This study builds six RDF treatments, comprising <i>R. glutinosa</i> continuously planted (SP), normally planted (NP), and NP treated with ferulic acid (FA), <i>Fusarium oxysporum</i> (FO), and a combination of FA with FO (FAFO). sRNA-seq technology was used to identify crucial miRNAs in response to diverse RDFs. <b>Results:</b> In total, 30 sRNA datasets were generated from the SP, NP, FA, FO, and FAFO samples. A total of 160 known and 41 novel miRNAs (<i>RgmiRNAs</i>) were identified in the <i>R. glutinosa</i> genome based on the sRNA database. Abundance analysis revealed that <i>RgmiRNAs</i> in SP exhibited a distinct expression profile in comparison with others. Of these, 124, 86, 86, and 90 <i>RgmiRNAs</i> were differentially expressed in SP, FA, FO, and FAFO compared with NP. Target analysis indicated that <i>RgmiRNAs</i> downregulated in both SP and RDFs impede the organism growth of <i>R. glutinosa</i>. <i>RgmiRNAs</i> upregulated in SP can disrupt root formation and nutrient metabolism, in which, two <i>RgmiR398</i> were uniquely expressed in SP. It was confirmed to target <i>RgCSD</i> genes. The expression patterns of <i>RgmiR398</i> and <i>RgCSD</i> indicated that replant disease induces the oxidative damage of <i>R. glutinosa</i> through <i>RgmiR398</i>. <b>Conclusions:</b><i>RgmiRNA</i> profiling under RDFs provides a theoretical basis for the further clarification of <i>RgmiRNA</i> function in replant disease.