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Selenium (Se), an essential element, plays important roles in human health as well as environmental sustainability. Se hyperaccumulating plants are thought as an alternative selenium resource, recently. Astragalus species are known as hyperaccumulator of Se by converting it to nonaminoacid compounds. However, Se-metabolism-related hyperaccumulation is not elucidated in plants yet. Micro RNAs (mi RNAs) are key molecules in many biological and metabolic processes via targeting mRNAs, which may also play an important role in Se accumulation in plants. In this study, we identified 418 known mi RNAs, belonging to 380 families, and 151 novel mi RNAs induced by Se exposure in Astragalus chyrsochlorus callus. Among known mi RNAs, the expression of 287 families was common in both libraries, besides 71 families were expressed only in Se-treated sample, whereas 60 conserved families were expressed in control tissue. miR1507a, miR1869 and miR2867-3p were mostly up-regulated, whereas miR1507-5p and miR8781b were significantly down-regulated by Se exposure. Computational analysis shows that the targets of mi RNAs are involved in different types of biological mechanisms including 47 types of cellular component, 103 types of molecular function and 144 types of biological process. Degradome analysis shows that 1256 mRNAs were targeted by 499 mi RNAs. We conclude that some known and novel mi RNAs such as miR167a, miR319, miR1507a, miR4346, miR7767-3p, miR7800, miR9748 and miR-n93 target transcription factors, disease resistance proteins and some specific genes like cysteine synthase and might be related to plant hormone signal transduction, plant-pathogen interaction and sulphur metabolism pathways. [ABSTRACT FROM AUTHOR]