A detailed gene expression study of the Miscanthus genus reveals changes in the transcriptome associated with the rejuvenation of spring rhizomes

Abstract

Background: The Miscanthus genus of perennial C4 grasses contains promising biofuel crops for temperate climates. However, few genomic resources exist for Miscanthus, which limits understanding of its interesting biology and future genetic improvement. A comprehensive catalog of expressed sequences were generated from a variety of Miscanthus species and tissue types, with an emphasis on characterizing gene expression changes in spring compared to fall rhizomes.

Results: Illumina short read sequencing technology was used to produce transcriptome sequences from different tissues and organs during distinct developmental stages for multiple Miscanthus species, including Miscanthus sinensis, Miscanthus sacchariflorus, and their interspecific hybrid Miscanthus × giganteus. More than fifty billion base-pairs of Miscanthus transcript sequence were produced. Overall, 26,230 Sorghum gene models (i.e., ~ 96% of predicted Sorghum genes) had at least five Miscanthus reads mapped to them, suggesting that a large portion of the Miscanthus transcriptome is represented in this dataset. The Miscanthus × giganteus data was used to identify genes preferentially expressed in a single tissue, such as the spring rhizome, using Sorghum bicolor as a reference. Quantitative real-time PCR was used to verify examples of preferential expression predicted via RNA-Seq. Contiguous consensus transcript sequences were assembled for each species and annotated using InterProScan. Sequences from the assembled transcriptome were used to amplify genomic segments from a doubled haploid Miscanthus sinensis and from Miscanthus × giganteus to further disentangle the allelic and paralogous variations in genes.

Conclusions: This large expressed sequence tag collection creates a valuable resource for the study of Miscanthus biology by providing detailed gene sequence information and tissue preferred expression patterns. We have successfully generated a database of transcriptome assemblies and demonstrated its use in the study of genes of interest. Analysis of gene expression profiles revealed biological pathways that exhibit altered regulation in spring compared to fall rhizomes, which are consistent with their different physiological functions. The expression profiles of the subterranean rhizome provides a better understanding of the biological activities of the underground stem structures that are essentials for perenniality and the storage or remobilization of carbon and nutrient resources.

Figure 1

Sampled Miscanthus × giganteus tissue types and relatedness of EST profiles using Sorghum bicolor gene models as references. Panel A is an image identifying many of the M. × giganteus tissues used in this study. Panel B displays the relatedness of the sequenced tissue types by hierarchical clustering of the expression profiles using Manhattan distance and complete linkage.

Figure 2

Reads from each Miscanthus tissue mapped to Sorghum bicolor. Panel A displays read count matching to S. bicolor gene models for each sequenced M. × giganteus tissue uniquely, non-uniquely (i.e., between two and five matches), or not at all; approximately 53% to 71% of the M. × giganteus reads mapped uniquely to the Sorghum transcripts. Panel B shows the number of Sorghum gene models represented by a minimum of five M. × giganteus reads for each sequenced M. × giganteus tissue. Panels C and D show similarities and differences in the profiles of Sorghum gene models represented with a minimum of five reads for select M. × giganteus tissues. Panel E shows a histogram of the total number of reads mapped per Sorghum gene model for each M. × giganteus library. Panel F shows the distribution of the number of reads mapped per Sorghum gene model in the unique categories of the Venn diagrams in panels C and D.

Figure 3

Verification of differentially expressed genes. Comparison of RPKM data and RT-qPCR results for five separate M. × giganteus tissue types. RPKM values are shown as dashed lines with values on the right y-axis. Relative expression via RT-qPCR is shown as bars with values on the left y-axis.

Figure 4

Basic assembly statistics for the transcriptomes from eight Miscanthus accessions. Panel A compares assembly statistics for each accession. Height of bars indicates number of contigs (left Y-axis) and lines represent length of contigs (right Y-axis). Panel B shows the number of reads from each accession (indicated by letters) which mapped back to either the assembly produced from that specific accession (red letters) or to the more complete assembly derived from all sequenced M. × giganteus libraries (blue letters). The assemblies from individual accessions where a mixture of tissues were combined into one RNA-Seq library are contained within the purple circle, whereas those assemblies derived from only leaf tissue are contained within the green circles. Mapped reads from each accessions are denoted as follows: “Z” M. sinensis ‘Zebrinus’, “A” M. sinensis ‘Amur Silvergrass,’ “W” M. sinensis ‘White Kaskade,’ “O” M. sinensis ‘Goliath,’ “S” M. sacchariflorus ‘Golf Course,’ “U” M. sinensis ‘Undine,’ “G” M. sinensis ‘Grosse Fontaine,’ and “M” is M. × giganteus.

Figure 5

Evolutionary relationships among Miscanthus gene fragments. Maximum likelihood trees were generated for two genes; significant branches are denoted by their bootstrap value. The trees are drawn to scale, with branch lengths measured by the number of substitutions per site. Panel A displays a tree drawn from the alignment of a 691 bp genomic sequence homologous to Sb01g001670, which is a single copy gene annotated as a putative membrane component member of the ER protein translocation complex. Panel B displays a tree drawn from the alignment of a 1,097 bp exonic segment of Sb03g010280, similar to Cycling DOF Factor 1 (CDF1). The Miscanthus EST contigs (M × g TContig35100 and GO TContig29030) are also included in the tree. Abbreviations for accession names: M. sinensis ‘IGR-2011-001’ (DH1), M. sinensis ‘IGR-2011-002’ (DH2), M. sinensis ‘IGR-2011-003’ (DH1P), M. sinensis ‘IGR-2011-004’ (DH2P), M. sacchariflorus ‘Hercules’ (HK), M. sacchariflorus ‘Golf Course’ (GC), M. sinensis ‘Goliath’ (GO), M. sinensis ‘Silbertum’ (ST), M. sinensis ‘White Kaskade’ (WK), and Miscanthus × giganteus (M × g).

Figure 6

Comparison of the assembled Miscanthus transcripts to gene models and ESTs of other grasses. In Panel A, Miscanthus × giganteus EST contigs were compared to Sugarcane transcripts (purple), and gene models of Sorghum bicolor (orange), Zea mays (brown), Oryza sativa (red) and Brachypodium (black). The graph shows the number of contigs that match each grass transcript dataset with a given percent nucleotide identity. Panel B represents the clustering of Miscanthus contigs with Sorghum bicolor gene models and contigs from the Sugarcane assembled EST database (SOGI). In total, 449 clusters contain at least one Miscanthus contig with no match in Sorghum bicolor or in the SOGI database at 90% identity over 90% of its length.

Figure 7

Miscanthus assemblies aligned to the Sorghum genome. Miscanthus transcriptome assemblies aligned to the Sorghum bicolor genome in Phytozome. M. sacchariflorus contigs are shown in green, M. × giganteus contigs are in blue and M. sinensis contigs are brown. The Sorghum coding region is shown in orange and the UTRs in dark grey. The two transcripts shown in Panels A (homologous to Sb01g005150) and B (homolgous to Sb07g004190) are rhizome-preferred transcripts shown in Figure‘3. Panel C shows transcript homologous to Sb01g001670, which is expressed in all tissues.