Transcriptome analysis in sheepgrass (Leymus chinensis): a dominant perennial grass of the Eurasian Steppe

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1. Length distribution of reads and contigs.

(A) Reads length distribution. (B) Length distribution of contigs bigger than 100 bp.

Figure 2. Histogram of the average read-depth coverage for assembled contigs.

Figure 3. Scatter plot representing the number of reads per contig for each contig length.

Figure 4. Histogram presentation of Gene Ontology classification.

Venn diagram of the distribution of plant GO terms associated with sheepgrass unigenes represented in biological process, cellular component and molecular function categories.

Figure 5. KOG function classification.

All unigenes were aligned to the KOG database to predict and categorize possible functions. A total of 25,319 unigenes were assigned to 25 classifications.

Figure 6. Histogram presentation of KEGG classification.

The y-axis indicates the number of unigenes assigned to a specific pathway. The x-axis indicates the KEGG pathway.

Figure 7. Top 25 transcription factor families.

The y-axis indicates the number of unigenes assigned to a specific TF family. The x-axis indicates the top 25 TF families.

Figure 8. Strategy for the identification of Poaceae-specific genes and putatively novel sheepgrass genes.

Transcripts that showed significant hits with non-Poaceae plant species are in dotted boxes. ‘Yes’ represents a significant hit and ‘No’ represents no significant hit in BLAST searches for the given criteria (E ≤1e-5 for BLASTX and TBLASTX). The sheepgrass unigenes identified as putatively novel sheepgrass genes and core Poaceae-specific genes are highlighted in red and blue boxes, respectively.

Figure 9. Classification of KOG function for sheepgrass freezing stress-responsive unigenes.

Figure 10. Similarity and phylogenetic analysis of sheepgrass with other Poaceae species.

(A) Similarity analysis of sheepgrass with other Poaceae species. (B) Relationship of sheepgrass to other grasses. Rooted phylogenetic tree based on the combined partial nucleotide sequences of 29 highly expressed genes.