MicroRNAs (miRNAs) are endogenous small RNAs that play a crucial role in plant growth, development, and environmental stress responses. Reaumuria soongorica is a typical halophyte that is widely distributed in saline–alkali desert regions. Under salt stress, R. soongorica can complete germination, a critical biological process in the life cycle of seed plants. To identify miRNAs and predict target mRNAs involved in seed germination during salt stress, nine small-RNA libraries were constructed and analyzed from R. soongorica seeds treated with various concentrations of NaCl. We also obtained transcriptome data under the same treatment conditions. Further analysis identified 88 conserved miRNAs representing 25 defined families and discovered 13 novel miRNAs from nine libraries. A co-expression analysis was performed on the same samples to identify putative miRNA–mRNA interactions that were responsive to salt stress. A comparative analysis of expression during germination under 273 (threshold) and 43 mM (optimal) NaCl treatments identified 13 differentially expressed miRNAs and 23 corresponding target mRNAs, while a comparison between 43 mM NaCl and non-salt-stress conditions uncovered one differentially expressed miRNA and one corresponding target mRNA. These results provide basic data for further study of molecular mechanisms involved in the germination of salt-stressed R. soongorica seeds, and also provide a reference for the improvement of salt tolerance during plant germination.

Background: Nitraria sibirica Pall. is a halophytic shrub with strong environmental adaptability that can survive in extremely saline-alkali and drought-impacted environments. Gene expression analysis aids in the exploration of the molecular mechanisms of plant responses to abiotic stresses. RT–qPCR is the most common technique for studying gene expression. Stable reference genes are a prerequisite for obtaining accurate target gene expression results in RT–qPCR analysis. Results: In this study, a total of 10 candidate reference genes were selected from the transcriptome of N. sibirica, and their expression stability in leaves and roots under different treatment conditions (salt, alkali, drought, cold, heat and ABA) was evaluated with the geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the tissue and experimental conditions tested. ACT7 combined with R3H, GAPDH, TUB or His were the most stable reference genes in the salt- or alkali-treated leaves, salt-treated roots and drought-treated roots, respectively; R3H and GAPDH were the most suitable combination for drought-treated leaves, heat-treated root samples and ABA-treated leaves; DIM1 and His maintained stable expression in roots under alkali stress; and TUB combined with R3H was stable in ABA-treated roots. TBCB and GAPDH exhibited stable expression in heat-treated leaves; TBCB, R3H, and ERF3A were stable in cold-treated leaves; and the three most stable reference genes for cold-treated roots were TBCB, ACT11 and DIM1. The reliability of the selected reference genes was further confirmed by evaluating the expression patterns of the NsP5CS gene under the six treatment conditions. Conclusion: This study provides a theoretical reference for N. sibirica gene expression standardization and quantification under various abiotic stress conditions and will help to reveal the molecular mechanisms that confer stress tolerance to N. sibirica.

Background: Nitraria sibirica Pall. is one of the pioneer tree species in saline–alkali areas due to its extreme salt tolerance. However, the lack of information on its genome limits the further exploration of the molecular mechanisms in N. sibirica under salt stress. Methods: In this study, we used single-molecule real-time (SMRT) technology based on the PacBio Iso-Seq platform to obtain transcriptome data from N. sibirica under salt treatment for the first time, which is helpful for our in-depth analysis of the salt tolerance and molecular characteristics of N. sibirica. Results: Our results suggested that a total of 234,508 circular consensus sequences (CCSs) with a mean read length of 2121 bp were obtained from the 19.26 Gb raw data. Furthermore, based on transcript cluster analysis, 93,713 consensus isoforms were obtained, including 92,116 high-quality isoforms. After removing redundant sequences, 49,240 non-redundant transcripts were obtained from high-quality isoforms. A total of 37,261 SSRs, 1816 LncRNAs and 47,314 CDSs, of which 40,160 carried complete ORFs, were obtained. Based on our transcriptome data, we also analyzed the coding genes of H⁺-PPase, and the results of both bioinformatics and functional analyses indicated that the gene prediction via full-length transcripts obtained by SMRT technology is reliable and effective. In summary, our research data obtained by SMRT technology provides more reliable and accurate information for the further analysis of the regulatory network and molecular mechanism of N. sibirica under salt stress.