Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development. The transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat. This research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species.

Sheepgrass (Leymus chinensis) is an important native forage grass, and it is widely grown in north China. Differential dormancy exists in sheepgrass germplasms with different seed colors. To decipher and find potential genes underlying this phenomenon, we compared the transcript profiles of yellow seeds with weak dormancy and brown seeds with strong dormancy. We identified a transcription factor gene LcbHLH92 which is negatively correlated with those of anthocyanin/proanthocyanidin-specific pathway genes, anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). LcbHLH92 had two transcripts, LcbHLH92a and LcbHLH92b, and their expression could be induced by abscisic acid, cold, and NaCl. Overexpression of LcbHLH92a or LcbHLH92b in Arabidopsis significantly inhibited the transcript levels of dihydroflavonol reductase (DFR) and ANS genes in leaves and seeds, which resulted in a decrease in anthocyanins and proanthocyanidins, respectively. Importantly, transgenic Arabidopsis seeds with a yellow color showed a higher germination rate than did the wild-type with a brown seed color. Moreover, LcbHLH92a and LcbHLH92b repressed the transcription of Transparent Testa8, ANS, DFR, and ANR, possibly by elevating the transcript levels of jasmonate-ZIM domain proteins through binding to their promoters. Together, our results demonstrate that LcbHLH92a and LcbHLH92b are negative regulators of the anthocyanin/proanthocyanidin pathway and influence seed dormancy.

Water is a critical environmental factor that restricts the geographic distribution of plants. Sheepgrass (Leymus chinensis, (Trin.) Tzvel) is an important forage grass in the Eurasia Steppe and a close germplasm for wheat and barley. This native grass adapts well to adverse environments such as cold, salinity, alkalinity and drought, and it can survive when the soil moisture may be less than 6% in dry seasons. However, little is known about how sheepgrass tolerates water stress at the molecular level. Here, drought stress experiment and RNA-sequencing (RNA-seq) was performed in three pools of RNA samples (control, drought stress, and rewatering). We found that sheepgrass seedlings could still survive when the soil water content (SWC) was reduced to 14.09%. Differentially expressed genes (DEGs) analysis showed that 7320 genes exhibited significant responses to drought stress. Of these DEGs, 2671 presented opposite expression trends before and after rewatering. Furthermore, approximately 680 putative sheepgrass-specific water responsive genes were revealed that can be studied deeply. Gene ontology (GO) annotation revealed that stress-associated genes were activated extensively by drought treatment. Interestingly, cold stress-related genes were up-regulated greatly after drought stress. The DEGs of MAPK and calcium signal pathways, plant hormone ABA, jasmonate, ethylene, brassinosteroid signal pathways, cold response CBF pathway participated coordinatively in sheepgrass drought stress response. In addition, we identified 288 putative transcription factors (TFs) involved in drought response, among them, the WRKY, NAC, AP2/ERF, bHLH, bZIP and MYB families were enriched, and might play crucial and significant roles in drought stress response of sheepgrass. Our research provided new and valuable information for understanding the mechanism of drought tolerance in sheepgrass. Moreover, the identification of genes involved in drought response can facilitate the genetic improvement of crops by molecular breeding.