Investigation of genetic relationships within three Miscanthus species using SNP markers identified with SLAF-seq.

BACKGROUND: Miscanthus, which is a leading dedicated-energy grass in Europe and in parts of Asia, is expected to play a key role in the development of the future bioeconomy. However, due to its complex genetic background, it is difficult to investigate phylogenetic relationships in this genus. Here, we investigated 50 Miscanthus germplasms: 1 female parent (M. lutarioriparius), 30 candidate male parents (M. lutarioriparius, M. sinensis, and M. sacchariflorus), and 19 offspring. We used high-throughput Specific-Locus Amplified Fragment sequencing (SLAF-seq) to identify informative single nucleotide polymorphisms (SNPs) in all germplasms. RESULTS: We identified 257,889 SLAF tags, of which 87,162 were polymorphic. Each tag was 264-364 bp long. The obtained 724,773 population SNPs were used to investigate genetic relationships within three species of Miscanthus. We constructed a phylogenetic tree of the 50 germplasms using the obtained SNPs and grouped them into two clades: one clade comprised of M. sinensis alone and the other one included the offspring, M. lutarioriparius, and M. sacchariflorus. Genetic cluster analysis had revealed that M. lutarioriparius germplasm C3 was the most likely male parent of the offspring. CONCLUSIONS: As a high-throughput sequencing method, SLAF-seq can be used to identify informative SNPs in Miscanthus germplasms and to rapidly characterize genetic relationships within this genus. Our results will support the development of breeding programs with the focus on utilizing Miscanthus cultivars with elite biomass- or fiber-production potential for the developing bioeconomy.

The trait and state negative affect can be separately predicted by stable and variable resting-state functional connectivity.

BACKGROUND: Many emotional experiences such as anxiety and depression are influenced by negative affect (NA). NA has both trait and state features, which play different roles in physiological and mental health. Attending to NA common to various emotional experiences and their trait-state features might help deepen the understanding of the shared foundation of related emotional disorders. METHODS: The principal component of five measures was calculated to indicate individuals' NA level. Applying the connectivity-based correlation analysis, we first identified resting-state functional connectives (FCs) relating to NA in sample 1 (n = 367), which were validated through an independent sample (n = 232; sample 2). Next, based on the variability of FCs across large timescale, we further divided the NA-related FCs into high- and low-variability groups. Finally, FCs in different variability groups were separately applied to predict individuals' neuroticism level (which is assumed to be the core trait-related factor underlying NA), and the change of NA level (which represents the state-related fluctuation of NA). RESULTS: The low-variability FCs were primarily within the default mode network (DMN) and between the DMN and dorsal attention network/sensory system and significantly predicted trait rather than state NA. The high-variability FCs were primarily between the DMN and ventral attention network, the fronto-parietal network and DMN/sensory system, and significantly predicted the change of NA level. CONCLUSIONS: The trait and state NA can be separately predicted by stable and variable spontaneous FCs with different attentional processes and emotion regulatory mechanisms, which could deepen our understanding of NA.

TGF-ß-induced α-SMA expression is mediated by C/EBPß acetylation in human alveolar epithelial cells.

BACKGROUND: Although the morbidity and mortality rates associated with idiopathic pulmonary fibrosis (IPF) are high, there is still lack of powerful and precise therapeutic options for IPF. OBJECT: Through in vitro model, this study sought to determine whether binding of acetylated CCAAT/enhancer binding protein ß (C/EBPß) to alpha-smooth muscle actin (α-SMA) promoter could affect the activity of the latter as well as assess if it is essential for epithelial-to-mesenchymal transition (EMT) and extracellular matrix deposition in IPF. METHODS: The expression of EMT and C/EBPß in A549 cells treated with transforming growth factor-beta (TGF-ß) as pulmonary fibrotic model was detected by western blotting and qPCR. Collagen-I expression using ELISA was performed. The luciferase activity was used to examine the activity of C/EBPß. Knockdown of C/EBPß was performed by siRNA. We also investigated the effect of deacetylation of C/EBPß on EMT using sirtuin 1 (SIRT1). The binding ability of C/EBPß with α-SMA promoter was affirmed via chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assay (EMSA). The relationship between α-SMA and acetylated C/EBPß was determined with co-immunoprecipitation (Co-IP). SiRNA-mediated knockdown of C/EBPß in A549 cells attenuated TGF-ß1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPß and α-SMA promoter was dynamically monitored. RESULTS: It was confirmed that deacetylation of C/EBPß in A549 cells successfully ameliorated TGF-ß1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. CONCLUSION: The EMT and fibrotic effect of TGF-ß1 is dependent on acetylated C/EBPß-mediated regulation of α-SMA gene activity. Thus, C/EBPß acetylation may play a central role in pulmonary fibrosis.

Interspecific genetic maps in Miscanthus floridulus and M. sacchariflorus accelerate detection of QTLs associated with plant height and inflorescence.

Miscanthus is recognized as a promising lignocellulosic crop for the production of bioethanol and bioproducts worldwide. To facilitate the identification of agronomical important traits and establish genetics knowledge, two genetic maps were developed from a controlled interspecific cross between M. floridulus and M. sacchariflorus. A total of 650 SSR markers were mapped in M. floridulus, spanning 19 linkage groups and 2053.31 cM with an average interval of 3.25 cM. The map of M. sacchariflorus comprised 495 SSR markers in 19 linkage groups covering 1684.86 cM with an average interval of 3.54 cM. The estimation on genome length indicated that the genome coverage of parental genetic maps were 93.87% and 89.91%, respectively. Eighty-eight bi-parental common markers were allowed to connect the two maps, and six pairs of syntenic linkage groups were recognized. Furthermore, quantitative trait loci (QTL) mapping of three agronomic traits, namely, plant height (PH), heading time (HT), and flowering time (FT), demonstrated that a total of 66 QTLs were identified in four consecutive years using interval mapping and multiple-QTL model. The LOD value of these QTLs ranged from 2.51 to 10.60, and the phenotypic variation explained varied from 9.50 to 37.10%. QTL cluster in syntenic groups MF19/MS7 contained six stable QTLs associated with PH, HT, and FT. In conclusion, we report for the first time the genetic mapping of biomass traits in M. floridulus and M. sacchariflorus. These results will be a valuable genetic resource, facilitating the discovery of essential genes and breeding of Miscanthus.

Intronic regions of plant genes potentially encode RDR (RNA-dependent RNA polymerase)-dependent small RNAs.

Recent research has linked the non-coding intronic regions of plant genes to the production of small RNAs (sRNAs). Certain introns, called 'mirtrons' and 'sirtrons', could serve as the single-stranded RNA precursors for the generation of microRNA and small interfering RNA, respectively. However, whether the intronic regions could serve as the template for double-stranded RNA synthesis and then for sRNA biogenesis through an RDR (RNA-dependent RNA polymerase)-dependent pathway remains unclear. In this study, a genome-wide search was made for the RDR-dependent sRNA loci within the intronic regions of the Arabidopsis genes. Hundreds of intronic regions encoding three or more RDR-dependent sRNAs were found to be covered by dsRNA-seq (double-stranded RNA sequencing) reads, indicating that the intron-derived sRNAs were indeed generated from long double-stranded RNA precursors. More interestingly, phase-distributed sRNAs were discovered on some of the dsRNA-seq read-covered intronic regions, and those sRNAs were largely 24 nt in length. Based on these results, the opinion is put forward that the intronic regions might serve as the genomic origins for the RDR-dependent sRNAs. This opinion might add a novel layer to the current biogenesis model of the intron-derived sRNAs.

Overexpression of a Miscanthus lutarioriparius NAC gene MlNAC5 confers enhanced drought and cold tolerance in Arabidopsis.

KEY MESSAGE: MLNAC5 functions as a stress-responsive NAC transcription factor gene and enhances drought and cold stress tolerance in transgenic Arabidopsis via the ABA-dependent signaling pathway. NAC transcription factors (TFs) play crucial roles in plant responses to abiotic stress. Miscanthus lutarioriparius is one of Miscanthus species native to East Asia. It has attracted much attention as a bioenergy crop because of its superior biomass productivity as well as wide adaptability to different environments. However, the functions of stress-related NAC TFs remain to be elucidated in M. lutarioriparius. In this study, a detailed functional characterization of MlNAC5 was carried out. MlNAC5 was a member of ATAF subfamily and it showed the highest sequence identity to ATAF1. Subcellular localization of MlNAC5-YFP fusion protein in tobacco leaves indicated that MlNAC5 is a nuclear protein. Transactivation assay in yeast cells demonstrated that MlNAC5 functions as a transcription activator and its activation domain is located in the C-terminus. Overexpression of MlNAC5 in Arabidopsis had impacts on plant development including dwarfism, leaf senescence, leaf morphology, and late flowering under normal growth conditions. Furthermore, MlNAC5 overexpression lines in Arabidopsis exhibited hypersensitivity to abscisic acid (ABA) and NaCl. Moreover, overexpression of MlNAC5 in Arabidopsis significantly enhanced drought and cold tolerance by transcriptionally regulating some stress-responsive marker genes. Collectively, our results indicated that MlNAC5 functions as an important regulator during the process of plant development and responses to salinity, drought and cold stresses.

Molecular cloning, characterization and expression analysis of trypsin-like serine protease from triangle-shell pearl mussel (Hyriopsis cumingii).

Trypsin-like serine protease (TLS) is ubiquitous in animals and plays a number of diverse roles, including dietary protein digestion, hemolymph coagulation, antimicrobial activity and immune responses, among others. This study reports the isolation of a 1048 bp full-length cDNA sequence of TLS from triangle-shell pearl mussel (Hyriopsis cumingii), including a 12 bp 5' UTR (untranslated region), a 172 bp 3' UTR, and an open reading frame (ORF) of 864 bp by rapid amplification of cDNA ends (RACE). Bioinformatic analysis shows that the gene belongs to the trypsin-like serine protease superfamily, and contains a 15 residues N-terminal signal peptide and a conserved C-terminal domain. In comparison to other serine proteases, the catalytic triad were identified as His-98, Asp-149, and Ser-240. Quantitative real-time PCR (qPCR) showed a broad expression of the TLS gene in ten tested tissues. Time-course expression analysis demonstrated that the expression level of the TLS mRNA was significantly up-regulated in eight tested tissues (liver, intestine, gill, heart, axe foot, adductor muscle, kidney and gonad), but down-regulated in mantle and stomach after Aeromonas hydrophila injection. This is one of the results indicate that TLS may be involved in innate defense reactions against A. hydrophila in triangle-shell pearl mussel.

Molecular cloning and expression analysis of 13 NAC transcription factors in Miscanthus lutarioriparius.

KEY MESSAGE: The 13 MlNAC genes could respond to various abiotic stresses, suggesting their crucial roles in stress response. Overexpression of MlNAC2 in Arabidopsis led to improved drought tolerance. NAC (NAM, ATAF1/2 and CUC2) proteins are plant-specific transcription factors that play crucial roles in plant development, growth and stress responses. In this study, 13 stress-responsive NAC genes were identified from Miscanthus lutarioriparius. Full-length cDNA sequences were obtained for 11 MlNAC genes, which were phylogenetically classified into six subfamilies. Sequence alignment revealed the highly conserved NAC domain in the N-terminus of these MlNACs, while the C-terminus was highly divergent. We performed quantitative real-time RT-PCR to examine the expression profiles of MlNAC genes in different tissues including root, rhizome, mature stem, young stem, leaf and sheath. The 13 MlNAC genes displayed distinct tissue-specific patterns in six tissues examined. To gain further insight into their roles in response to abiotic stresses, expressions of MlNAC genes were analyzed under different stresses and hormone treatments including salt, drought, cold, wounding, abscisic acid, Methyl jasmonate and salicylic acid. The 13 MlNAC genes could respond to at least five stress treatments, and over 100-fold variations in transcript levels of MlNAC1, MlNAC2, MlNAC4, and MlNAC12 were observed in salt, drought and MeJA treatments, which indicated that MlNACs play crucial roles in stress response. Crosstalk among various abiotic stress and hormone responses was also discussed based on the expression of MlNAC genes. Overexpression of MlNAC2 in Arabidopsis (Col-0) led to improved drought tolerance. The water loss rate was significantly lower, and the recovery rate after a 60-min dehydration stress treatment was significantly higher in the MlNAC2 overexpression lines than the control.

Biostimulant and Arbuscular Mycorrhizae Application on Four Major Biomass Crops as the Base of Phytomanagement Strategies in Metal-Contaminated Soils.

Using contaminated land to grow lignocellulosic crops can deliver biomass and, in the long term, improve soil quality. Biostimulants and microorganisms are nowadays an innovative approach to define appropriate phytomanagement strategies to promote plant growth and metal uptake. This study evaluated biostimulants and mycorrhizae application on biomass production and phytoextraction potential of four lignocellulosic crops grown under two metal-contaminated soils. Two greenhouse pot trials were setup to evaluate two annual species (sorghum, hemp) in Italy and two perennial ones (miscanthus, switchgrass) in China, under mycorrhizae (M), root (B2) and foliar (B1) biostimulants treatments, based on humic substances and protein hydrolysates, respectively, applied both alone and in combination (MB1, MB2). MB2 increased the shoot dry weight (DW) yield in hemp (1.9 times more), sorghum (3.6 times more) and miscanthus (tripled) with additional positive effects on sorghum and miscanthus Zn and Cd accumulation, respectively, but no effects on hemp metal accumulation. No treatment promoted switchgrass shoot DW, but M enhanced Cd and Cr shoot concentrations (+84%, 1.6 times more, respectively) and the phytoextraction efficiency. Root biostimulants and mycorrhizae were demonstrated to be more efficient inputs than foliar biostimulants to enhance plant development and productivity in order to design effective phytomanagement strategies in metal-contaminated soil.

Chromosome-level scaffolding of haplotype-resolved assemblies using Hi-C data without reference genomes.

Scaffolding is crucial for constructing most chromosome-level genomes. The high-throughput chromatin conformation capture (Hi-C) technology has become the primary scaffolding strategy due to its convenience and cost-effectiveness. As sequencing technologies and assembly algorithms advance, constructing haplotype-resolved genomes is increasingly preferred because haplotypes can provide additional genetic information on allelic and non-allelic variations. ALLHiC is a widely used allele-aware scaffolding tool designed for this purpose. However, its dependence on chromosome-level reference genomes and a higher chromosome misassignment rate still impede the unravelling of haplotype-resolved genomes. Here we present HapHiC, a reference-independent allele-aware scaffolding tool with superior performance on chromosome assignment as well as contig ordering and orientation. In addition, we provide new insights into the challenges in allele-aware scaffolding by conducting comprehensive analyses on various adverse factors. Finally, with the help of HapHiC, we constructed the haplotype-resolved allotriploid genome for Miscanthus × giganteus, an important lignocellulosic bioenergy crop.

Salt tolerance evaluation and mini-core collection development in Miscanthus sacchariflorus and M. lutarioriparius.

Marginal lands, such as those with saline soils, have potential as alternative resources for cultivating dedicated biomass crops used in the production of renewable energy and chemicals. Optimum utilization of marginal lands can not only alleviate the competition for arable land use with primary food crops, but also contribute to bioenergy products and soil improvement. Miscanthus sacchariflorus and M. lutarioriparius are prominent perennial plants suitable for sustainable bioenergy production in saline soils. However, their responses to salt stress remain largely unexplored. In this study, we utilized 318 genotypes of M. sacchariflorus and M. lutarioriparius to assess their salt tolerance levels under 150 mM NaCl using 14 traits, and subsequently established a mini-core elite collection for salt tolerance. Our results revealed substantial variation in salt tolerance among the evaluated genotypes. Salt-tolerant genotypes exhibited significantly lower Na+ content, and K+ content was positively correlated with Na+ content. Interestingly, a few genotypes with higher Na+ levels in shoots showed improved shoot growth characteristics. This observation suggests that M. sacchariflorus and M. lutarioriparius adapt to salt stress by regulating ion homeostasis, primarily through enhanced K+ uptake, shoot Na+ exclusion, and Na+ sequestration in shoot vacuoles. To evaluate salt tolerance comprehensively, we developed an assessment value (D value) based on the membership function values of the 14 traits. We identified three highly salt-tolerant, 50 salt-tolerant, 127 moderately salt-tolerant, 117 salt-sensitive, and 21 highly salt-sensitive genotypes at the seedling stage by employing the D value. A mathematical evaluation model for salt tolerance was established for M. sacchariflorus and M. lutarioriparius at the seedling stage. Notably, the mini-core collection containing 64 genotypes developed using the Core Hunter algorithm effectively represented the overall variability of the entire collection. This mini-core collection serves as a valuable gene pool for future in-depth investigations of salt tolerance mechanisms in Miscanthus.

Unlocking the potential of Dongting Lake-grown Miscanthus lutarioriparius biomass: A comprehensive quality analysis and bioproduct application study.

Miscanthus lutarioriparius grown in Dongting Lake has an annual biomass yield potential of 1 million tons. However, with the shutdown of its previous utilization for paper-making, abandoning this huge amount of biomass has caused serious economic, ecological, and social problems. Constructing an industrial cluster to continuously convert biomass into various bioproducts is a win-win measure to address this dilemma. With the increasing confirmation of the importance of biomass quality affecting the conservation process, fully understanding the biomass characteristics of Dongting Lake-grown M. lutarioriparius is crucial for building a scientific industrial cluster. The present work is designed to explore the variation in biomass quality across the entire Dongting Lake area. Results show that the biomass contented with Cd, Mn, Zn, and Cr has significant geographical differences, with a general trend of Southern Dongting Lake-grown biomass having a higher concentration than that from Eastern and Western Dongting Lake areas. Moreover, significant differences are found in terms of biomass ash content, lignin content, and the degree of polymerization of cellulose (DP). The biomass with low ash content is generally from the entire Eastern Dongting Lake area and the northern part of the Western Dongting Lake area. Virtually all Western Dongting Lake-grown biomass has a low lignin content (approximately 18 %). Regarding the spatial variation of DP, Eastern Dongting Lake-grown biomass has a higher DP (average at 585.33) than that in Southern (575.15) and then Western Dongting Lake (529.16). Based on these quality indicators, the biomass production potentials for bioethanol, biochar, and xylo-oligosaccharide were calculated and visualized. Results show that biomass from almost the entire Western and Eastern Dongting Lake area is suitable for bioethanol and xylo-oligosaccharide production, while biomass from the Southern Dongting Lake area for biochar production. These results provide scientific guidance for the future utilization of Dongting Lake-grown M. lutarioriparius biomass.

Development of novel chloroplast microsatellite markers for Miscanthus species (Poaceae).

PREMISE OF THE STUDY: A set of novel chloroplast microsatellite markers (cpSSRs) was developed for the bioenergy crop Miscanthus, and their utility in cross-species amplification was evaluated. METHODS AND RESULTS: Twenty-eight novel primers flanking cpSSR loci were designed from a complete chloroplast genome sequence of Saccharum officinarum, a species closely related to Miscanthus. These primers were then tested on eight Miscanthus species, among which 16 cpSSR loci were found to be polymorphic. The number of alleles per polymorphic locus ranged from two to seven, with an average 3.94 alleles. CONCLUSIONS: These cpSSR markers can be applied to all Miscanthus species and will be useful for studying Miscanthus population structure, diversity, and phylogeography.

The performance of Miscanthus hybrids in saline-alkaline soil.

Cultivating the dedicated biomass crop Miscanthus on marginal land is a sustainable means of avoiding competition with food crops for arable land. A large proportion of global marginal land is saline-alkaline; however, little is known about the performance of Miscanthus in saline-alkaline soil. In this study, Miscanthus × giganteus and ten other Miscanthus hybrids grown in the Yellow River Delta were exposed to low and saline-alkaline soils during the 2016-2018 growing season to evaluate the agronomic traits, biomass quality and the potential productive index of eleven Miscanthus genotypes. Plant biomass, plant height, and tiller number significantly decreased in high saline-alkaline soil. In particular, the average plant biomass of ten Miscanthus hybrids in low saline-alkaline soil in 2017 and 2018 were 0.21 and 2.25 kg per plant, respectively, and in high saline-alkaline soil were 0.13 and 0.65 kg per plant, respectively. Cell wall, cellulose, and nitrogen content of all genotypes significantly decreased in high saline-alkaline soil, while hemicellulose, ash, sodium, potassium, magnesium, and calcium content significantly increased. However, high saline-alkaline soil had no observable impact on lignin content of Miscanthus biomass. The effect of high saline-alkaline on biomass quality parameters could provide important information for the application of Miscanthus biomass in saline-alkaline soil. The selected genotypes (A5) could be considered as breeding materials in saline-alkaline soil.

Development of energy plants from hybrids between Miscanthus sacchariflorus and M. lutarioriparius grown on reclaimed mine land in the Loess Plateau of China.

Miscanthus, a promising bioenergy plant, has a high biomass yield with high cellulose content suitable for biofuel production. However, harsh climatic and poor soil conditions, such as barren lands or abandoned mines, pose a challenge to the survival and yield of Miscanthus feedstock on the marginal land. The selection from the interspecific hybrids of Miscanthus might combine high survival rates and high yield, which benefits energy crop development in multi-stressful environments. A total of 113 F1 hybrids between Miscanthus sacchariflorus and M. lutarioriparius together with the parents were planted and evaluated for multiple morphological and physiological traits on the mine land of the Loess Plateau of China. The majority of hybrids had higher establishment rates than M. sacchariflorus while M. lutarioriparius failed to survive for the first winter. Nearly all hybrid genotypes outperformed M. lutarioriparius for yield-related traits including plant height, tiller number, tiller diameter, and leaf area. The average biomass of the hybrids was 20 times higher than that of surviving parent, M. sacchariflorus. Furthermore, the photosynthetic rates and water use efficiency of the hybrids were both significantly higher than those of the parents, which might be partly responsible for their higher yield. A total of 29 hybrids with outstanding traits related to yield and stress tolerance were identified as candidates. The study investigated for the first time the hybrids between local individuals of M. sacchariflorus and high-biomass M. lutarioriparius, suggesting that this could be an effective approach for high-yield energy crop development on vast of marginal lands.

Transcriptomic Characterization of Miscanthus sacchariflorus × M. lutarioriparius and Its Implications for Energy Crop Development in the Semiarid Mine Area.

Miscanthus interspecific hybrids have been proved to have better adaptability in marginal lands than their parents. Miscanthus sacchariflorus and Miscanthus lutarioriparius were used as the parents to develop hybrids. We performed the transcriptome for 110 F1 hybrids of Miscanthus sacchariflorus × Miscanthus lutarioriparius and their parents that had been established on the Loess Plateau mine area, to estimate the population's genetic expression variation, and illuminate the adaptive mechanism of the F1 population. The result speculated that the F1 population has mainly inherited the stress response metabolic pathway of its female parent (M. sacchariflorus), which may be responsible for its higher environmental adaptability and biomass yield compared with male parents. Based on PopART, we assembled a leaf reference transcriptome for M. sacchariflorus (LRTMS) and obtained 8116 high-quality transcripts. When we analyze the differential expression of genes between F1 population and parent, 39 and 56 differentially expressed genes were screened out in the female parent and male parent, respectively. The enrichment analysis showed that pathways of carbohydrate metabolism, lipid metabolism, biosynthesis of secondary metabolites and circadian rhythm-plant played a key role in resisting the harsh environment. The carbohydrate metabolism and lipid metabolism were also significantly enriched, and the synthesis of these substances facilitated the yield. The results provided an insight into breeding Miscanthus hybrids more suited to the harsh environment of the Loess Plateau.

BSD-GAN: Branched Generative Adversarial Network for Scale-Disentangled Representation Learning and Image Synthesis.

We introduce BSD-GAN, a novel multi-branch and scale-disentangled training method which enables unconditional Generative Adversarial Networks (GANs) to learn image representations at multiple scales, benefiting a wide range of generation and editing tasks. The key feature of BSD-GAN is that it is trained in multiple branches, progressively covering both the breadth and depth of the network, as resolutions of the training images increase to reveal finer-scale features. Specifically, each noise vector, as input to the generator network of BSD-GAN, is deliberately split into several sub-vectors, each corresponding to, and is trained to learn, image representations at a particular scale. During training, we progressively "de-freeze" the sub-vectors, one at a time, as a new set of higher-resolution images is employed for training and more network layers are added. A consequence of such an explicit sub-vector designation is that we can directly manipulate and even combine latent (sub-vector) codes which model different feature scales. Extensive experiments demonstrate the effectiveness of our training method in scale-disentangled learning of image representations and synthesis of novel image contents, without any extra labels and without compromising quality of the synthesized high-resolution images. We further demonstrate several image generation and manipulation applications enabled or improved by BSD-GAN.

Genetic diversity and population structure of Miscanthus lutarioriparius, an endemic plant of China.

Miscanthus lutarioriparius is a native perennial Miscanthus species of China, which is currently used as raw material of papermaking and bioenergy crop. It also has been considered as a promising eco-bioindustrial plant, which can offer raw material and gene for the biomass industry. However, lack of germplasm resources and genetic diversity information of M. lutarioriparius have become the bottleneck that prevents the stable and further development of the biomass industry. In the present study, genetic diversity of 153 M. lutarioriparius individuals nine populations was studied using 27 Start Codon Targeted (SCoT) markers. High polymorphic bands (97.67%), polymorphic information content (0.26) and allele number (1.88) showed SCoT as a reliable marker system for genetic analysis in M. lutarioriparius. At the species, the percentage of polymorphic loci [PPL] was 97.2%, Nei's gene diversity [H] was 0.36, Shannon index [I] was 0.54 and Expected Heterozygosity [He] was 0.56. Genetic variation within populations (84.91%) was higher than among populations (15.09%) based on analysis of molecular variance (AMOVA). Moderate level of genetic differentiation was found in M. lutarioriparius populations (Fst = 0.15), which is further confirmed by STRUCTURE, principal coordinates analysis (PCoA) and an unweighted pair group method with arithmetic mean (UPGMA) analysis that could reveal a clear separation between groups of the north and south of Yangtze River. The gene flow of the populations within the respective south and north of Yangtze River area was higher, but lower between the areas. There was no obvious correlation between genetic distance and geographic distance. The breeding systems, geographical isolation and fragmented habitat of M. lutarioriparius may be due to the high level of genetic diversity, moderate genetic differentiation, and the population, structure. The study further suggests some measure for conservation of genetic resources and provides the genetic basis for improving the efficiency of breeding based on the results of diversity analysis.

Molecular cytogenetic characterization and phylogenetic analysis of four Miscanthus species (Poaceae).

Chromosomes of four Miscanthus (Andersson, 1855) species including M. sinensis (Andersson, 1855), M. floridulus (Schumann & Lauterb, 1901), M. sacchariflorus (Hackel, 1882) and M. lutarioriparius (Chen & Renvoize, 2005) were analyzed using sequentially combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. To elucidate the phylogenetic relationship among the four Miscanthus species, the homology of repetitive sequences among the four species was analyzed by comparative genomic in situ hybridization (cGISH). Subsequently four Miscanthus species were clustered based on the internal transcribed spacer (ITS) of 45S rDNA. Molecular cytogenetic karyotypes of the four Miscanthus species were established for the first time using chromosome measurements, fluorochrome bands and 45S rDNA FISH signals, which will provide a cytogenetic tool for the identification of these four species. All the four have the karyotype formula of Miscanthus species, which is 2n = 2x = 38 = 34m(2SAT) + 4sm, and one pair of 45S rDNA sites. The latter were shown as strong red bands by CPD staining. A non-rDNA CPD band emerged in M. floridulus and some blue DAPI bands appeared in M. sinensis and M. floridulus. The hybridization signals of M. floridulus genomic DNA to the chromosomes of M. sinensis and M. lutarioriparius genomic DNA to the chromosomes of M. sacchariflorus were stronger and more evenly distributed than other combinations. Molecular phylogenetic trees showed that M. sinensis and M. floridulus were closest relatives, and M. sacchariflorus and M. lutarioriparius were also closely related. These findings were consistent with the phylogenetic relationships inferred from the cGISH patterns.