Sequencing of transcriptomes from two Miscanthus species reveals functional specificity in rhizomes, and clarifies evolutionary relationships
- Changsoo Kim†1,
- Tae-Ho Lee†1, 4,
- Hui Guo1,
- Sung Jin Chung2,
- Andrew H Paterson1,
- Do-Soon Kim3 and
- Geung-Joo Lee2Email author
© Kim et al.; licensee BioMed Central Ltd. 2014
Received: 5 February 2014
Accepted: 13 May 2014
Published: 18 May 2014
Miscanthus is a promising biomass crop for temperate regions. Despite the increasing interest in this plant, limited sequence information has constrained research into its biology, physiology, and breeding. The whole genome transcriptomes of M. sinensis and M. sacchariflorus presented in this study may provide good resources to understand functional compositions of two important Miscanthus genomes and their evolutionary relationships.
For M. sinensis, a total of 457,891 and 512,950 expressed sequence tags (ESTs) were produced from leaf and rhizome tissues, respectively, which were assembled into 12,166 contigs and 89,648 singletons for leaf, and 13,170 contigs and 112,138 singletons for rhizome. For M. sacchariflorus, a total of 288,806 and 267,952 ESTs from leaf and rhizome tissues, respectively, were assembled into 8,732 contigs and 66,881 singletons for leaf, and 8,104 contigs and 63,212 singletons for rhizome. Based on the distributions of synonymous nucleotide substitution (Ks), sorghum and Miscanthus diverged about 6.2 million years ago (MYA), Saccharum and Miscanthus diverged 4.6 MYA, and M. sinensis and M. sacchariflorus diverged 1.5 MYA. The pairwise alignment of predicted protein sequences from sorghum-Miscanthus and two Miscanthus species found a total of 43,770 and 35,818 nsSNPs, respectively. The impacts of striking mutations found by nsSNPs were much lower between sorghum and Miscanthus than those between the two Miscanthus species, perhaps as a consequence of the much higher level of gene duplication in Miscanthus and resulting ability to buffer essential functions against disturbance.
The ESTs generated in the present study represent a significant addition to Miscanthus functional genomics resources, permitting us to discover some candidate genes associated with enhanced biomass production. Ks distributions based on orthologous ESTs may serve as a guideline for future research into the evolution of Miscanthus species as well as its close relatives sorghum and Saccharum.
KeywordsMiscanthus sinensis Miscanthus sacchariflorus Expressed sequence tags Synonymous substitution Orthologous sequences Nonsynonymous SNPs
Concerns about global warming in combination with increased use of fossil fuel have spurred growing interest in sources of renewable energy such as biofuels. The genus Miscanthus has been considered attractive as a feedstock for cellulosic biofuel production because the plants are adapted to temperate latitudes yet utilize the energy-efficient C4 photosynthetic pathway, producing high yields of biomass but with low-nutrient requirements, adaptation to marginal land due to resistance to abiotic stress, and not competing with use for food [1–3]. At present, 14 Miscanthus species are recognized, most of which are native to eastern Asia, but with a few found in Polynesia, the Himalayas, and southern Africa .
Miscanthus belongs to the Andropogoneae tribe of grasses, including many economically important crops such as maize, sorghum, and sugarcane. In particular, Miscanthus and sugarcane belong to the subtribe Saccharinae that may be characterized by its complex and high ploidy genomes. For example, M. × giganteus (2n = 3× = 57), which is specifically of interest due to its ability to accumulate biomass, is thought to result from crosses between M. sacchariflorus (2n = 4× = 76) and M. sinensis (2n = 2× = 38) . Study of the large and complex Miscanthus genomes is challenging, although the whole genome sequence of closely-related sorghum (2n = 2× = 20)  provides a valuable reference for molecular research in Miscanthus. Genomic study of Miscanthus is currently focused on the construction of genetic maps using AFLP , SSR , RNA-seq , and GBS (genotype by sequencing) .
Expressed sequence tags (ESTs) are frequently useful for plant species that do not have sufficient genomics resources, providing functional profiles of genes as well as a basis of evolutionary study. Owing to massively parallel sequencing technologies, large numbers of ESTs can be easily generated under different growing conditions or tissue types; however, only five Miscanthus ESTs are publicly available in the NCBI’s dbEST as of December, 2013 (http://www.ncbi.nlm.nih.gov/dbEST).
In the current study, we generated transcriptome data sets from M. sacchariflorus and M. sinensis, the suspected progenitors of the M. × giganteus species which is preferred for biomass production. In particular, we chose those two species to scan transcriptomes because they will be served as mapping parents and the collection of gene profile may be necessary in the future work. We determined functional profiles of transcriptome sets from both leaf and rhizome for each Miscanthus species and compared the profiles. M. sinensis is known to have weak (or sometimes no) rhizomes but M. sacchariflorus has vigorous rhizomes, so it was of interest to identify genes showing significantly different expression in rhizomes than other tissues . In addition, using the massive transcriptome data, we estimated the time of divergence among the two Miscanthus species, sorghum and Saccharum.
Results and discussion
454 sequencing and de novo assembly of MiscanthusEST
Sequencing and assembly statistics for Miscanthus sinensis and M. sacchariflorus ESTs
Total read length (bp)
Average read length (bp)
Contig length (bp)
Average length of contig (bp)
Contig range (bp)
> = 1 kb
> = 200 bp
Singleton length (bp)
Functional annotation of ESTs
More than 39.1% of transcripts were specific to M. sinensis or M. sacchariflorus libraries. The leaf libraries of the two Miscanthus species had 5,314 highly similar contigs which was 43.7% in MSIL and 60.9% in MSAL whereas the rhizome libraries had the 4,461 contigs whose proportion in MSIR and MSAR were 33.9% and 55.0%, respectively. The relatively higher proportions of highly similar contigs between M. sacchariflorus and sinensis in M. sacchariflorus than in M. sinensis libraries might be caused by less number of contigs in M. sacchariflorus and that is consistent with the higher proportion of well conserved orthologous genes in M. sacchariflorus library than M. sinensis (Figure 2).
A total of 2,744 transcripts were found to be expressed in all tissue types and species. As expected, GO classification suggested that those transcripts were mostly related to basal plant metabolic processes or structures. Although a large number of transcripts seem to be tissue- or species-specific genes as shown in this work, housekeeping functions appear well conserved in different tissues and species.
Comparative analysis of rhizome-enriched genes of Sorghum spp. to MiscanthusESTs
Putative functions of genes included in the three orthologous groups shown in Figure 4
Serine-threonine kinase receptor-associated protein
Heat shock protein 90
kh domain containing protein
Amino acid expressed
Poly -binding protein
Plasminogen activator inhibitor 1 rna-binding protein
Acyl carrier protein 3
Ras-related protein rab11c
No hits found
40s ribosomal protein s14
No hits found
Reversibly glycosylated polypeptide
60s acidic ribosomal protein p1
nc domain-containing protein
Maize proteinase inhibitor
Ubiquitin-like protein smt3
No hits found
atp synthase gamma
Actin-depolymerizing factor 6
60s ribosomal protein l26-1
40s ribosomal protein s15a
Speciation of Miscanthusspp., sugarcane, and sorghum
The divergence between sugarcane and sorghum has previously been investigated using orthology, with multiple studies estimating that the two species diverged about 7.7 - 9.0 MYA [3, 17, 18]. Since Miscanthus and sugarcane belong to the same subtribe (Saccharinae) and share a common ancestor, the estimation may be consistent with the case which sorghum and Miscanthus are compared; however, our estimation in was about 1.4 - 2.8 million years more recent (6.2 MYA). The discrepancy from previous reports may be due to different sequence data used for the comparison. Kim et al. and Wang et al. estimated the divergence time at 7.7 MYA but Jannoo et al. reported the divergence time at 8.0 - 9.0 MYA. The former two studies used sugarcane ESTs mostly originating from hybrid sugarcane cultivar(s) but the latter used only a single gene, Adh1, to estimate the divergence between the two subtribes. The sugarcane genome is more complex than Miscanthus owing to a higher ploidy number and hybrid sugarcane is still more complex because of its interspecific origin and frequent aneuploidy caused by its breeding history . Therefore, although the common ancestor of Miscanthus and sugarcane diverged from the sorghum lineage at the same time, hybrid sugarcane could be highly polymorphic within single cultivars, and appear more diverged from sorghum than Miscanthus, resulting in the overestimation of Ks values. We also infer that sugarcane and Miscanthus share a common ancestor about 4.6 MYA. Due to lack of nucleotide information in Miscanthus, its divergence from the Saccharum lineage had not been estimated previously . In turn, the estimation of the two genera could be overestimated because of the ESTs from hybrid sugarcane cultivar(s). However, our estimation together with EST sequences may serve as a guideline for future research in the evolution of Miscanthus species until a detail of sugarcane genome is released.
Recently published genetic maps revealed that the Miscanthus lineage experienced whole genome duplication after its divergence from sorghum [8, 10]. Although we tried to find paralogs from the EST datasets to date the duplication, signals were mostly hidden or too weak. Finding recent whole genome duplication events using ESTs is somewhat difficult in that paralogous and redundant sequences are not clearly identifiable in EST datasets and recent whole genome duplication signals are frequently masked by recent single gene duplication signals. If the whole genome duplication occurred in Miscanthus after its divergence from a common ancestor shared with sorghum (ca. 6.2 MYA), two hypotheses are highly likely: (1) the Miscanthus genome had experienced some ‘diploidization’ (loss of one member of duplicated gene pairs) before the two Miscanthus species diverged (ca. 1.5 MYA), and (2) the (diploid) M. sinensis genome experienced appreciable diploidization after the two Miscanthus species diverged. If the former is true, M. sacchariflorus might have had an additional genome-wide duplication after the two species diverged. Considering the recent divergence of the two species (1.5 MYA), the latter may be more plausible. However, this has to be elucidated with additional information.
Functional differentiation of Miscanthusand sorghum genes
Top 10 nsSNPs having large impacts on gene functions
Pleiotropic drug resistance proteins, ABC superfamily
Calcium transporting ATPase
C1-like domain|Cellulose synthase
Glycogen synthase kinase-3
Pleiotropic drug resistance proteins, ABC superfamily
Pleiotropic drug resistance proteins, ABC superfamily
Pleiotropic drug resistance proteins, ABC superfamily
Glycogen synthase kinase-3
Pleiotropic drug resistance proteins, ABC superfamily
The current study provides nearly 227,000 ESTs and 146,000 ESTs from M. sinensis and M. sacchariflorus, respectively, greatly enriching knowledge of the transcriptome of this promising lignocellulosic bioethanol crop. M. sinensis and M. sacchariflorus are of particular interest because they are suspected progenitors of the triploid M. × giganteus which is cultivated as a biomass crop. These ESTs can be utilized in many ways such as seeking molecular markers, gene prediction in genome sequences, and gene expression studies. A core set of (largely housekeeping) genes are expressed in common in the two Miscanthus species and the two tissues studied, with large populations of genes that appear to be tissue specific. Genes showing rhizome-enriched expression in rhizomatous Sorghum species appear to correspond more closely to those in M. sacchariflorus which has aggressive rhizomes, than those in M. sinensis that has weak rhizomes. The analysis of nsSNPs showed that striking mutations are more frequently found between two Miscanthus species than between Miscanthus and sorghum, reflect that Miscanthus seems to be more tolerant of point mutations than sorghum. A plausible explanation of this observation is that genomic redundancy resulting from its recent genome doubling has relaxed selection on duplicated gene copies. We also analyzed ESTs to make preliminary inferences about Miscanthus evolution. As stated in the text, Miscanthus experienced whole genome duplication since its divergence from sorghum. Although ESTs generally provided too little information to clearly identify paralogs which were formed by the whole genome duplication, they were still useful to predict speciation events by providing clear signal of orthologous relationships. The exact timing of the whole-genome duplication event can be evaluated when additional genome sequence and detailed maps are available, and our ESTs will be able to contribute to such detailed evolutionary studies.
Plant materials and growth conditions
M. sinensis and M. sacchariflorus were collected in South Korea, and their ploidy levels and morphological identities were determined as in a previous report . Plants were grown for a year in plastic pots and placed in the greenhouse at Mokpo National University, Mokpo, South Korea. No fertilizer was applied until sampling, but watering was given when the pot soil dried. The greenhouse temperature was controlled from 24 to 28°C during the growth period without supplemental light. Leaves and rhizomes were sampled in the vegetative state before flowering. Newly developed rhizomes (<1 year old) along inner surface of the pot were harvested and pooled after grinding. All samples were sent to the National Instrumentation Center for Environmental Management (NICEM, Seoul, South Korea) for cDNA library construction.
Construction of cDNA library and sequencing of the library
Total RNA was isolated from rhizome and leaf tissues of M. sinensis and M. sacchariflorus using Rneasy Plant Mini Kit (Qiagen, Seoul, Korea) according to the manufacturer’s instructions. Extracted RNA samples were quantified and quality-checked using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Total RNA (6 μg) was reverse transcribed using Super Script II (Life Technologies, Carlsbad, CA). Second strand cDNA was synthesized using Advantage 2 Polymerase Mix (Clontech, Seoul, Korea). cDNA samples were purified using QIAquick PCR purification kit (Qiagen, Seoul, Korea). The purified fragments were then used to create the 454 single-stranded cDNA library using a 454 library preparation kit (Roche). The fragment ends were polished using T4 ligase and T4 polynucleotide kinase and adaptors containing primer sequences and a biotin tag were ligated to the fragment ends (Roche, Brandford, CT). The fragments with properly ligated adaptors were immobilized onto magnetic streptavidin-coated beads (Roche, Brandford, CT). Nicks or gaps between the adaptors and the dscDNA fragments were repaired using the fill-in polymerase. The non-biotinylated strands of the immobilized dscDNA fragments were melted off to generate the single-stranded cDNA library for 454 sequencing.
Assembly of 454 reads and annotation of contigs
Before assembly, extended multiplex identifier (MID) sequences of 3′ and 5′ ends, poly A(T) tail, short sequences (<50 bp) and low complexity were removed. Four EST data sets from leaf and rhizome tissues of two Miscanthus species were individually assembled using GS de novo assembler version 2.6 (Roche Diagnostics Corporation, http://454.com/products/analysis-software/index.asp) with default parameters. From the assembly results, only consensus sequences (contigs) were taken for further analyses due to the short length of singletons. Functional annotation was performed by sequence comparison with public databases. All contigs were blasted against the Phytozome database (http://www.phytozome.org, version 7.0, April 2011) which includes annotated genes with the KOG assignments (e < 10-5).
Comparison to rhizome-enriched genes from S. halepense and S. propinquum
A total of 768 ESTs previously reported as derived from rhizome-enriched genes in S. halepense or S. propinquum were blasted against Miscanthus ESTs to find putative orthologs (e < 10-20). The putative orthologs from five different datasets - rhizome and leaf ESTs from two Miscanthus species and rhizome-enriched genes from Sorghum spp. - were used to construct phylogenetic trees. The five orthologs were multiple-aligned by ClustalW . ClustalW was also implemented for generating phylogenetic trees using the Neighbor Joining algorithm. The phylogenetic trees were grouped based on their topologies by determining symmetric differences among trees with the Treedist function in the PHYLIP package , in order to compare rhizome-enriched genes from the two Sorghum species to ESTs from Miscanthus. The phylogenetic trees were grouped based on their topologies, in order to infer rhizome-enriched ESTs in Miscanthus species. For the candidate rhizome-enriched ESTs in Miscanthus, putative functions were classified using Blast2GO .
Estimation of divergence time in Saccharinae subtribe
To estimate the speciation time among M. sacchariflorus, M. sinensis, sugarcane, and sorghum, Ks values between orthologous ESTs were calculated. Sugarcane ESTs and sorghum gene sequences were obtained from dbEST (http://www.ncbi.nlm.nih.gov/dbEST/) and Phytozome (http://www.phytozome.net/), respectively. To identify putative orthologs among species, each sequence from one species was blasted against all sequences from other species and the best hits were taken. A pair of best hit was defined as putative orthologs when the pair was aligned over 300 bp or more (e < 10-20). Each member of a pair of sequences was subjected to blastx search against predicted sorghum proteins. The best match was considered significant if the alignment length was more than 100 amino acids (e < 10-15). If no significant match was found, the pair of sequences was excluded from further analysis. The cleaned pairs of sequences were translated using the Genewise program, which can take frameshift sites into consideration , with the corresponding best match protein in S. bicolor as reference. For each pair of paralogs, the two translated products were aligned using ClustalW , and the resulting alignment was used as a guide to align the nucleotide sequences. After removing gaps and N-containing codons, the level of synonymous substitution was estimated using the maximum likelihood approach implemented in the program CODEML, which is a part of the PAML package . Batch jobs were performed using in-house python scripts.
Nonsynonymous SNP analysis
Orthologous protein sequences from S. bicolor, M. sacchariflorus and M. sinensis previously defined were used to identify nsSNP. To find nsSNPs between MSA and MSI, pairwise alignment of predicted proteins were conducted. For nsSNPs between sorghum and Miscanthus, the exactly same proteins across the two Miscanthus species were aligned to sorghum proteins. The identified nsSNPs were classified into two groups: One group contains nsSNP that is polymorphic between MSA and MSI. nsSNPs in the other group are between sorghum and the two Miscanthus species. Protein sequences from the 30 published genomes (http://chibba.agtec.uga.edu/duplication/) were clustered and aligned using orthoMCL  and ClustalW . The nsSNPs identified from the three species were aligned to the orthologous gene clusters. The impact of nsSNPs on gene function is evaluated by functional impact score (FIS) using the formula described by Paterson et al..
All the ESTs are downloadable at the NABIC (National Agricultural Biotechnology Information Center; http://nabic.rda.go.kr/) with accession numbers indicated; NN-0642-000001 (MSAL), NN-0643-000001 (MSAR), NN-0639-000001 (MSIL), and NN-0641-000001 (MSIR).
Amplified fragment length polymorphism
Simple sequence repeat
Expressed sequence tag
Million years ago
M. sinensis leaf library
M. sinensis rhizome library
M. sacchariflorus leaf library
M. sacchariflorus rhizome library
Nonsynonymous single nucleotide polymorphism
Synonymous nucleotide substitution.
This work was supported by a grant from the Plant Molecular Breeding Center of the Next-Generation BioGreen 21 Program, RDA (grant no. PJ0081312012); the US Department of Energy-US Department of Agriculture Plant Feedstock Program (project grant number: 112786); the Consortium for Plant Biotechnology Research (CPBR); the National Science Foundation (NSF: DBI 0849896); and resources and technical expertise from the University of Georgia, Georgia Advanced Computing Resource Center, a partnership between the Office of the Vice President for Research and the Office of the Chief Information Officer.
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