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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.
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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...
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Citations
... The development of rhizome of turfgrass is affected by various factors, including carbohydrate and nitrogen levels in plant, environment and phytohormones. Plant carbohydrates and nitrogen status affect plant overwintering, rhizome bud germination, and community reproduction [10,11]. The structural features of rhizomes are similar to those of tillers [12]. ...
Abstract Background Rhizome is vital for carbon and nitrogen metabolism of the whole plant. However, the effect of carbon and nitrogen in the rhizome on rhizome expansion remains unclear. Results Three wild Kentucky bluegrass (Poa pratensis L.) germplasms with different rhizome expansion capacity (strong expansion capacity, ‘YZ’; medium expansion capacity, ‘WY’; and weak expansion capacity, ‘AD’) were planted in the field and the rhizomes number, tiller number, rhizome dry weight, physiological indicators and enzyme activity associated carbon and nitrogen metabolisms were measured. Liquid chromatography coupled to mass spectrometry (LC-MS) was utilized to analyze the metabolomic of the rhizomes. The results showed that the rhizome and tiller numbers of the YZ were 3.26 and 2.69-fold of that of the AD, respectively. The aboveground dry weight of the YZ was the greatest among all three germplasms. Contents of soluble sugar, starch, sucrose, NO3 −-N, and free amino acid were significantly higher in rhizomes of the YZ than those of the WY and AD (P
... Moreover, Miscanthus breeding would benefit from molecular markers to facilitate the selection of varieties with an improved biomass yield and/or composition [6]. Genetic resources for Miscanthus, such as an extensive transcriptome database [7] and a recently completed Miscanthus sinensis draft genome, Miscanthus sinensis v7.1 DOE-JGI, accessible at Phytozome (http://phytozome.jgi.doe.gov/, accessed on 12 November 2021), are now available to explore biological processes with molecular approaches. ...
... In Miscanthus, the NAC TF MsSND1 has been previously characterized as a master regulator orchestrating SCW formation, and MsSCM1, a MYB TF related to MYB20/43/85 [18], has been proposed as specific regulator of lignin biosynthesis [30]. We identified a putative MsMYB103 in a Miscanthus transcriptome [7] by a homology-based approach with AtMYB103 as a query sequence. AtMYB103 has been suggested as a key gene regulating the expression of ferulate-5-hydroxylase (F5H), which directs lignin biosynthesis towards formation of syringyl-rich lignin [23] and was initially suggested to exclusively regulate lignin composition. ...
Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.
... Miscanthus Andersson (Poaceae) is a C4 photosynthetic plant, which have been widely investigated as a potential second-generation bio-energy crop (Barling et al., 2013). The genus Miscanthus includes approximately 20 species, which could be classified into Miscanthus clades and Triarrhena clades (Ge et al., 2017). ...
Miscanthus is not only a perennial fiber biomass crop, but also valuable breeding resource for its low-nutrient requirements, photosynthetic efficiency and strong adaptability to environment. In the present study, the codon usage patterns of five different Miscanthus plants and other two related species were systematically analyzed. The results indicated that the cp genomes of the seven representative species were preference to A/T bases and A/T-ending codons. In addition, 21 common high-frequency codons and 4–11 optimal codons were detected in the seven chloroplast genomes. The results of ENc-plot, PR2-plot and neutrality analysis revealed the codon usage patterns of the seven chloroplast genomes are influenced by multiple factors, in which nature selection is the main influencing factor. Comparative analysis of the codon usage frequencies between the seven representative species and four model organisms suggested that Arabidopsis thaliana , Populus trichocarpa and Saccharomyces cerevisiae could be considered as preferential appropriate exogenous expression receptors. These results might not only provide important reference information for evolutionary analysis, but also shed light on the way to improve the expression efficiency of exogenous gene in transgenic research based on codon optimization.
... Although several Miscanthus species have been considered important feedstocks for biofuels and biorefineries, their genetic resources, including transcriptome data, are currently very limited. Previously, transcriptome profiling has only been reported for three important Miscanthus species, including M. sinensis, M. sacchariflorus, and M. × giganteus, to study their transcriptomic similarity with other grasses, construct genetic maps using NGS based markers, and identify genes probably related to traits like rhizome development and selfincompatibility traits [10][11][12]. Miscanthus sinensis, a genetic diploid (2n = 38), has a genome size of one copy of a single complete genome of 2.4-2.6 Gb [13]. The key limitation to the genetic improvement of Miscanthus sinensis is the complexity of its genome, which makes transcriptome studies of Miscanthus very challenging. ...
... Meanwhile, 102,573 (∼25 %) transcripts remained unknown, so they can be considered as new transcripts putatively unique to Miscanthus (Additional file 2). Of all the hits to the NR proteins from BLASTX, most transcripts (149,618, ∼49 %) were annotated to Sorghum bicolor, followed by Zea mays (59,871; 19.8 %) (Fig. 3b), which is consistent with several previous Miscanthus transcriptome analyses when the sugarcane genome sequences were not yet available [10][11][12]. Thus, it is no surprise that most of our Miscanthus sequences could be mapped to the sugarcane genome but could not be annotated owing to the limited sugarcane genome annotation information in public databases. As an international standardized gene functional classification system, GO offers a dynamic updated controlled vocabulary and a strictly defined concept to describe the properties of genes and their products in any organism [37,38]. ...
Background
Miscanthus sinensis Andersson is a perennial grass that exhibits remarkable lignocellulose characteristics suitable for sustainable bioenergy production. However, knowledge of the genetic resources of this species is relatively limited, which considerably hampers further work on its biology and genetic improvement.
Results
In this study, through analyzing the transcriptome of mixed samples of leaves and stems using the latest PacBio Iso-Seq sequencing technology combined with Illumina HiSeq, we report the first full-length transcriptome dataset of M. sinensis with a total of 58.21 Gb clean data. An average of 15.75 Gb clean reads of each sample were obtained from the PacBio Iso-Seq system, which doubled the data size (6.68 Gb) obtained from the Illumina HiSeq platform. The integrated analyses of PacBio- and Illumina-based transcriptomic data uncovered 408,801 non-redundant transcripts with an average length of 1,685 bp. Of those, 189,406 transcripts were commonly identified by both methods, 169,149 transcripts with an average length of 619 bp were uniquely identified by Illumina HiSeq, and 51,246 transcripts with an average length of 2,535 bp were uniquely identified by PacBio Iso-Seq. Approximately 96 % of the final combined transcripts were mapped back to the Miscanthus genome, reflecting the high quality and coverage of our sequencing results. When comparing our data with genomes of four species of Andropogoneae, M. sinensis showed the closest relationship with sugarcane with up to 93 % mapping ratios, followed by sorghum with up to 80 % mapping ratios, indicating a high conservation of orthologs in these three genomes. Furthermore, 306,228 transcripts were successfully annotated against public databases including cell wall related genes and transcript factor families, thus providing many new insights into gene functions. The PacBio Iso-Seq data also helped identify 3,898 alternative splicing events and 2,963 annotated AS isoforms within 10 function categories.
Conclusions
Taken together, the present study provides a rich data set of full-length transcripts that greatly enriches our understanding of M. sinensis transcriptomic resources, thus facilitating further genetic improvement and molecular studies of the Miscanthus species.
... Raw RNA-seq data of M. × giganteus were collected from NCBI BioProject (PRJNA183625, 17 samples) [57] and our previous study (NCBI SRA accession number: SRR1734721, 1 sample) [48]. After quality filtering and adaptor trimming with fastp (version 0.20.0) ...
Background
Miscanthus × giganteus is widely recognized as a promising lignocellulosic biomass crop due to its advantages of high biomass production, low environmental impacts, and the potential to be cultivated on marginal land. However, the high costs of bioethanol production still limit the current commercialization of lignocellulosic bioethanol. The lignin in the cell wall and its by-products released in the pretreatment step is the main component inhibiting the enzymatic reactions in the saccharification and fermentation processes. Hence, genetic modification of the genes involved in lignin biosynthesis could be a feasible strategy to overcome this barrier by manipulating the lignin content and composition of M. × giganteus. For this purpose, the essential knowledge of these genes and understanding the underlying regulatory mechanisms in M. × giganteus is required.
Results
In this study, MgPAL1, MgPAL5, Mg4CL1, Mg4CL3, MgHCT1, MgHCT2, MgC3′H1, MgCCoAOMT1, MgCCoAOMT3, MgCCR1, MgCCR2, MgF5H, MgCOMT, and MgCAD were identified as the major monolignol biosynthetic genes in M. × giganteus based on genetic and transcriptional evidence. Among them, 12 genes were cloned and sequenced. By combining transcription factor binding site prediction and expression correlation analysis, MYB46, MYB61, MYB63, WRKY24, WRKY35, WRKY12, ERF021, ERF058, and ERF017 were inferred to regulate the expression of these genes directly. On the basis of these results, an integrated model was summarized to depict the monolignol biosynthesis pathway and the underlying regulatory mechanism in M. × giganteus.
Conclusions
This study provides a list of potential gene targets for genetic improvement of lignocellulosic biomass quality of M. × giganteus, and reveals the genetic, transcriptional, and regulatory landscape of the monolignol biosynthesis pathway in M. × giganteus.
... These studies have identified genes specifying rhizomes by comparing gene expression between rhizome tissues, e.g. tips and internodes 7,9,10,15 , between rhizomes and above-ground shoots or leaves [16][17][18] and between rhizomes, roots and above-ground organs 16,19,20 . ...
... Comparisons between rhizomes, roots, leaves and shoots have been reported for clonal grass species www.nature.com/scientificreports/ such as Oryza 16,53 and Miscanthus 19,20 . The reported lists of the genes that were highly expressed in the rhizomes in these studies did not correspond to that of this study, at least for the top twenty genes, probably reflecting distant phylogenetic relationships between monocots and dicots. ...
The rhizome is a plant organ that develops from a shoot apical meristem but penetrates into belowground environments. To characterize the gene expression profile of rhizomes, we compared the rhizome transcriptome with those of the leaves, shoots and roots of a rhizomatous Brassicaceae plant, Cardamine leucantha. Overall, rhizome transcriptomes were characterized by the absence of genes that show rhizome-specific expression and expression profiles intermediate between those of shoots and roots. Our results suggest that both endogenous developmental factors and external environmental factors are important for controlling the rhizome transcriptome. Genes that showed relatively high expression in the rhizome compared to shoots and roots included those related to belowground defense, control of reactive oxygen species and cell elongation under dark conditions. A comparison of transcriptomes further allowed us to identify the presence of an ER body, a defense-related belowground organelle, in epidermal cells of the C. leucantha rhizome, which is the first report of ER bodies in rhizome tissue.
... Although several Miscanthus species have been considered important feedstocks for biofuels and biore neries, their genetic resources, including transcriptome data, are currently very limited. Previously, transcriptome pro ling has only been reported for three important Miscanthus species, including M. sinensis, M. sacchari orus, and M. × giganteus, to study their transcriptomic similarity with other grasses, construct genetic maps using NGS based markers, and identify genes probably related to traits like rhizome development and self-incompatibility traits [10][11][12]. Miscanthus sinensis is a genetic diploid (2n = 38) with a genome size of 1C = 2.4-2.6 Gb [13]. The key limitation to the genetic improvement of Miscanthus sinensis is the complexity of its genome, which makes transcriptome studies of Miscanthus very challenging. ...
... Meanwhile, 102,573 (∼25%) transcripts remained unknown, so they can be considered as new transcripts putatively unique to Miscanthus (Additional le 2). Of all the hits to the NR proteins from BLASTX, most transcripts (149,618, ∼49%) were annotated to Sorghum bicolor, followed by Zea mays (59,871; 19.8%) (Fig. 3b), which is consistent with several previous Miscanthus transcriptome analyses when the sugarcane genome sequences were not yet available [10][11][12]. Thus, it is no surprise that most of our Miscanthus sequences could be mapped to the sugarcane genome but could not be annotated owing to the limited sugarcane genome annotation information in public databases. ...
Background: Miscanthus sinensis Andersson is a perennial grass that exhibits remarkable lignocellulose characteristics suitable for sustainable bioenergy production. However, knowledge of the genetic resources of this species is relatively limited, which considerably hampers further work on its biology and genetic improvement.
Results: In this study, through analyzing the transcriptome of mixed samples of leaves and stems using the latest PacBio Iso-Seq sequencing technology combined with Illumina HiSeq, we report the first full-length transcriptome dataset of M. sinensis with a total of 58.21 Gb clean data. An average of 15.75 Gb clean reads of each sample were obtained from the PacBio Iso-Seq system, which doubled the data size (6.68 Gb) obtained from the Illumina HiSeq platform. The integrated analyses of PacBio- and Illumina-based transcriptomic data uncovered 408,801 non-redundant transcripts with an average length of 1,685 bp. Of those, 189,406 transcripts were commonly identified by both methods, 169,149 transcripts with an average length of 619 bp were uniquely identified by Illumina HiSeq, and 51,246 transcripts with an average length of 2,535 bp were uniquely identified by PacBio Iso-Seq. When comparing our data with genomes of four species of Andropogoneae, M. sinensis showed the closest relationship with sugarcane with up to 93% mapping ratios, followed by sorghum with up to 80% mapping ratios, indicating a high conservation of orthologs in these three genomes. Furthermore, 306,228 transcripts were successfully annotated against public databases including cell wall related genes and transcript factor families, thus providing many new insights into gene functions. The PacBio Iso-Seq data also helped identify 3,898 alternative splicing events and 2,963 annotated AS isoforms within 10 function categories.
Conclusions: Taken together, the present study provides a rich data set of full-length transcripts that greatly enriches our understanding of M. sinensis transcriptomic resources, thus facilitating further genetic improvement and molecular studies of the Miscanthus species.
... Although several Miscanthus species have been considered important feedstocks for biofuels and biore neries, their genetic resources, including transcriptome data, are currently very limited. Previously, transcriptome pro ling has only been reported for three important Miscanthus species, including M. sinensis, M. sacchari orus, and M. × giganteus, to study their transcriptomic similarity with other grasses, construct genetic maps using NGS based markers, and identify genes probably related to traits like rhizome development or self-incompatibility traits [12][13][14]. ...
... Meanwhile, 102,573 (∼25%) transcripts remained unknown, so they can be considered as new transcripts putatively unique to Miscanthus (Additional le 1). Of all the hits to the NR proteins from BLASTX, most transcripts (149,618, ∼49%) were annotated to Sorghum bicolor, and followed by Zea mays (59,871; 19.8%) (Fig. 3b), which is consistent with several previous Miscanthus transcriptome analyses when the sugarcane genome sequences was not yet been sequenced and released [12][13][14]. Thus, it is no surprise that most of our Miscanthus sequences could be mapped to the sugarcane genome but could not be annotated owing to the limited sugarcane genome annotation information in public databases. ...
... With the development of new sequencing techniques, transcriptome analysis has been proven to be a very powerful means of gene discovery, genome annotation, and deep exploration of genes that contribute to phenotypic traits [9][10][11]. A few NGS RNA-Seq transcriptome databases of M. sinensis, M. sacchari orus, and M. lutarioriparius have been reported previously [12,13,38], but these studies were limited by either transcript length and/or the number of transcript isoforms. Recently, the third-generation sequencing technology PacBio Iso-Seq opened up a new era of transcriptome-wide research, which is particularly suitable for the direct generation of comprehensive transcriptomes with accurate alternative splicing isoforms and novel genes in non-model organisms that lack genomic sequences. ...
Background
Miscanthus sinensis Andersson is a perennial grass that exhibits remarkable lignocellulose characteristics for sustainable bioenergy production. However, knowledge of the genetic resources of this species is relatively limited, which significantly hampers further work on its interesting biology and genetic improvement.
Results
In this study, through analyzing the transcriptome of mixed samples of leaves and stems using the latest PacBio Iso-Seq sequencing technology combined with Illumina HiSeq, we report the first full-length transcriptome dataset of M. sinensis with a total of 58.21 Gbp clean data. An average of 15.75 Gbp clean reads of each sample were obtained from PacBio Iso-Seq system, which doubled the data size (6.68 Gbp) obtained from the Illumina HiSeq platform. The integrated analyses of PacBio- and Illumina-based transcriptomic data uncovered 408,801 non-redundant transcripts with an average length of 1,685 bp, representing about 60% of the total number of predicted Miscanthus genes. Of those, 189,406 transcripts were commonly identified by both methods, 169,149 transcripts with an average length of 619 bp were uniquely identified by Illumina HiSeq, and 51,246 transcripts with an average length of 2,535 bp were uniquely identified by PacBio Iso-SEq. When comparing our data with genomes of four Andropogoneae species, M. sinensis showed the closest relationship with sugarcane with up to 93% mapping ratios, followed by sorghum with up to 80% mapping ratios, indicating a high conservation of orthologs in these three genomes. Furthermore, 306,228 transcripts were successfully annotated against public databases including cell wall related genes and transcript factor families, thus providing many new insights into gene functions. The PacBio Iso-Seq data also helped identify 3,898 alternative splicing events and 2,963 annotated AS isoforms within 10 function categories.
Conclusions
Taken together, the present study provides a rich data set of full-length transcripts that greatly enriches our understanding of M. sinensis genomic resources, thus facilitating further genetic improvement and molecular studies of the Miscanthus species.
... Furthermore, a series of widely used reference genes, like Actin 2 (ACTIN), ubiquitin conjugating enzyme (UBC), 18S ribosomal RNA (18S rRNA), 25S ribosomal RNA (25S rRNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were proved to be differentially expressed under different experimental conditions [40][41][42][43]. As no housekeeping genes have been tested or verified for use in M. sinensis, many researches refer to gene expression pattern verification by qRT-PCR often used the common reference genes or control candidates from its genetically close species sorghum, but the results were not all desired [44]. Bustin et al. proposed that the utilization of reference genes must be experimentally validated for different tissues and for a particular experimental condition [45]. ...
Quantitative real-time PCR (qRT-PCR) has been widely used for studying gene expression at the transcript level. Its accuracy usually relies on the reference genes that are utilized for data normalization. Miscanthus sinensis, a perennial C4 grass with high biomass and strong resistance to adversities, is often utilized as a high value energy crop. However, no reliable reference genes have been investigated for normalizing gene expression for this species. In this study, 12 candidate reference genes were selected to identify their stability under five different abiotic stress treatments (drought, salt, cadmium, chromium and arsenic) by using geNorm, NormFinder, BestKeeper and RefFinder softwares. The results showed that 18S rRNA and Unigene33312 were the best reference genes under drought treatments. Unigene33312 and Unigene33024 were found to be the most stably expressed genes under salt stress and Cd stress. Moreover, Unigene33024 and PP2A were the most suitable reference genes under Cr stress and Unigene33024 and Sb09g019750 were deemed more suitable reference genes under As stress. In total, considering all the samples, Unigene33024 and PP2A were the most stable genes while ACTIN and Unigene26576 were the least stable reference genes for internal control. The expression patterns of two target genes (Cu/Zn SOD and CAT) were used to further verify those selected reference genes under different conditions. The results showed that the most and the least stable reference genes had clearly different expression patterns. This work comprehensively estimated the stability of reference genes in M. sinensis which may give insight to the reference genes selection in other tissues as well as other related varieties. These suggested reference genes would assist in further putative gene expression validation in M. sinensis.
... While recent studies in Arabidopsis have identified and characterized a large number of enzymes and transcription factors responsible for these steps, and each can significantly affect lignin content and composition [6], knowledge about lignification in Miscanthus remains thus far limited. However, a recently completed transcriptome analysis, based on developing internodes of Miscanthus lutarioriparius, a Miscanthus transcriptome database [7], and Miscanthus sinensis draft genome (Miscanthus sinensis v7.1 DOE-JGI, http://phytozome.jgi.doe.gov/), revealed many similarities to other species concerning the secondary cell wall biosynthetic machinery [8]. ...
... Using published laccase nucleotide sequences from Arabidopsis, Sorghum, and Brachypodium, we performed tBLASTn analysis of a Miscanthus transcriptome [7]. In total, 95 laccase-like contigs were identified, 28 of them containing complete sequences for putative laccase open reading frames (ORFs). ...
... Compared to dicot species, laccase families in monocots appear to be larger [16,22]. A phylogenetic analysis was performed to classify laccase contigs found in the Miscanthus EST database [7] against Arabidopsis and Brachypodium laccases, respectively ( Fig. 1 and Additional file 1: Figure S1). This analysis identified Mxg_TContig47643 (Fig. 1) as an ORF related to AtLAC17, which is known to be involved in lignification [15]. ...
Background:
Understanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo.
Results:
Using a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4-2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate.
Conclusions:
In summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.
