Identification of responsive miRNAs involved in combination stresses of phosphate starvation and salt stress in soybean root

Salt stress and phosphate (Pi) starvation severely affect plants growth and development. MicroRNAs involved in single stress of Pi starvation or salt stress have been reported in many plant species. However, the combination stresses of Pi starvation and salt stress responsive microRNAs and their target genes have not been reported. In the study, we generated 7 small RNA libraries under 4 different treatments with three independent biological replicates: CK, salt stress (SR1, SR5), Pi starvation (LPR1, LPR5), and the double stresses of salt stress/Pi starvation (DR1, DR5) in soybean root by using Illumina HiSeqTM2000 sequencing technique. Total of 5285 miRNAs including 3616 conserved and 1669 novel miRNAs were identified. Among them, 21 miRNAs including gma-miR169l-3p, gma-miR5036, gma-miR862a, and gma-398a/b, were detected to respond to the single stress and the double stresses of Pi starvation and salt stress. The target genes for the identified miRNAs such as ethylene-responsive transcription factor 4, protein phosphatase 2C, and superoxide dismutase [Cu-Zn], were classified into different biological processes including metabolic process, signaling transduction. The expression patterns of several identified miRNAs were validated by RT-qPCR and the results of RT-qPCR were consistent with the result of IlluminaHiSeq. The Na⁺, K⁺, and PO₄ ³⁻ contents under different treatments were detected in soybean roots and leaves. Our findings identified the differentially expressed miRNAs in response to the combination stresses of Pi starvation and salt stress, which providing the essential information for enhancing the resistance to salt stress and Pi starvation in soybean.