Water stress seriously constrains plant growth and yield. Long non-coding RNAs (lncRNAs) serve as versatile regulators in various biological regulatory processes. To date, the systematic screening and potential functions of lncRNA have not yet been characterized in Cleistogenes songorica, especially under water stress conditions. In this study, we obtained the root and shoot transcriptomes of young C. songorica plants subjected to different degrees of water stress and recovery treatments by Illumina-based RNA-seq. A total of 3397 lncRNAs were identified through bioinformatics analysis. LncRNA differential expression analysis indicated that the higher response of roots compared to shoots during water stress and recovery. We further identified the 1644 transcription factors, 189 of which were corresponded to 163 lncRNAs in C. songorica. Though comparative analyses with major Poaceae species based on blast, 81 water stress-related orthologues regulated to lncRNAs were identified as a core of evolutionary conserved genes important to regulate water stress responses in the family. Among these target genes, two genes were found to be involved in the abscisic acid (ABA) signalling pathway, and four genes were enriched for starch and sucrose metabolism. Additionally, the 52 lncRNAs were predicted as target mimics for microRNAs (miRNAs) in C. songorica. RT-qPCR results suggested that MSTRG.43964.1 and MSTRG.4400.2 may regulate the expression of miRNA397 and miRNA166, respectively, as target mimics under water stress and during recovery. Finally, a co-expression network was constructed based on the lncRNAs, miRNAs, protein-coding genes (PCgenes) and transcription factors under water stress and during recovery in C. songorica. In C. songorica, lncRNAs, miRNAs, PCgenes and transcription factors constitute a complex transcriptional regulatory network which lncRNAs can regulate PCgenes and miRNAs under water stress and recovery. This study provides fundamental resources to deepen our knowledge on lncRNAs during ubiquitous water stress.

Background: Transcription factors act as important regulators of transcription networks. Basic leucine zipper (bZIP) transcription factors have been shown to be involved in multiple biological processes in plants. However, no information is available for the bZIP family in Cleistogenes songorica, which is an important xerophytic and allotetraploid grass in desert grasslands. Results: In this study, 86 CsbZIPs were identified in the allotetraploid C. songorica genome. For location analysis, CsbZIPs were distributed evenly across two subgenomes of C. songorica. Phylogenetic tree analysis among three species indicated that CsbZIPs were evolutionarily more closely related to OsbZIPs than AtbZIPs. Syntenic and phylogenetic analyses confirmed that the CsbZIPs were mainly expanded by whole-genome duplication events. Furthermore, it was determined that rice and C. songorica might have undergone purified selection during their long evolutionary history by calculating the Ks values and Ka/Ks ratios of orthologous gene pairs. By analysing the expression patterns of CsbZIPs in different tissues and under abiotic stresses, 21 CsbZIP genes were differentially expressed between chasmogamous (CH) and cleistogamous (CL) flowers, including two FLOWERING LOCUS D (FD) genes. In shoots and roots, 79.1 and 87.2% of the CsbZIP genes, respectively, displayed transcript changes under at least one stress treatment, such as heat, cold, drought and salt. Strikingly, 17 common CsbZIP genes showed differential expression under stress response and during CL flowering. Co-expression network, GO annotation and real-time quantitative reverse transcription PCR (qRT-PCR) analyses revealed a close relationship between CL flowering-associated genes and abiotic stress-related genes. Conclusions: BZIP TFs were comprehensively analysed and identified in allotetraploid C. songorica. Our results provide insights into the evolutionary history of the bZIP family in C. songorica and provide abiotic stress-responsive and CL-associated candidate CsbZIP genes for potential applications in the genetic improvement of plants.

Background: As global warming continues, heat stress (HS) is becoming an increasingly significant factor limiting plant growth and reproduction, especially for cool-season grass species. The objective of this study was to determine the transcriptional regulatory network of Cleistogenes songorica under HS via transcriptome profiling, identify of gene families and comparative analysis across major Poaceae species. Results: Physiological analysis revealed significantly decreased leaf relative water content (RWC) but increased proline (Pro) content in C. songorica under 24 h of HS. Transcriptome profiling indicated that 16,028 and 14,645 genes were differentially expressed in the shoots and roots of C. songorica under HS, respectively. Two subgenomes of C. songorica provide equal contribution under HS on the basis of the distribution and expression of differentially expressed genes (DEGs). Furthermore, 216 DEGs were identified as key evolutionarily conserved genes involved in the response to HS in C. songorica via comparative analysis with genes of four Poaceae species; these genes were involved in the ‘response to heat’ and ‘heat acclimation’. Notably, most of the conserved DEGs belonged to the heat-shock protein (HSP) superfamily. Similar results were also obtained from co-expression analysis. Interestingly, hub-genes of co-expression analysis were found to overlap with conserved genes, especially heat-shock protein (HSP). In C. songorica, 84 HSP and 32 heat-shock transcription factor (HSF) genes were identified in the allotetraploid C. songorica genome, and might have undergone purifying selection during evolutionary history based on syntenic and phylogenetic analysis. By analysing the expression patterns of the CsHSPs and CsHSFs, we found that the transcript abundance of 72.7% of the CsHSP genes and of 62.5% of the CsHSF genes changed under heat stress in both the shoots and roots. Finally, a core regulatory network of HS was constructed on the basis of the CsHSP, CsHSF and other responsive genes in C. songorica. Conclusions: Regulatory network and key genes were comprehensively analysed and identified in C. songorica under HS. This study improves our knowledge of thermotolerance mechanisms in native grasses, and also provides candidate genes for potential applications in the genetic improvement of grasses.

Melilotus is an important genus of legumes with industrial and medicinal value, partly due to the production of coumarin. To explore the genetic diversity and population structure of Melilotus, 40 accessions were analyzed using long terminal repeat (LTR) retrotransposon-based markers. A total of 585,894,349 bp of LTR retrotransposon sequences, accounting for 55.28% of the Melilotus genome, were identified using bioinformatics tools. A total of 181,040 LTR retrotransposons were identified and classified as Gypsy, Copia, or another type. A total of 350 pairs of primers were designed for assessing polymorphisms in 15 Melilotus albus accessions. Overall, 47 polymorphic primer pairs were screened for their availability and transferability in 18 Melilotus species. All the primer pairs were transferable, and 292 alleles were detected at 47 LTR retrotransposon loci. The average polymorphism information content (PIC) value was 0.66, which indicated that these markers were highly informative. Based on unweighted pair group method with arithmetic mean (UPGMA) dendrogram cluster analysis, the 18 Melilotus species were classified into three clusters. This study provides important data for future breeding programs and for implementing genetic improvements in the Melilotus genus.