Genome assemblies of 11 bamboo species highlight diversification induced by dynamic subgenome dominance.

Polyploidy (genome duplication) is a pivotal force in evolution. However, the interactions between parental genomes in a polyploid nucleus, frequently involving subgenome dominance, are poorly understood. Here we showcase analyses of a bamboo system (Poaceae: Bambusoideae) comprising a series of lineages from diploid (herbaceous) to tetraploid and hexaploid (woody), with 11 chromosome-level de novo genome assemblies and 476 transcriptome samples. We find that woody bamboo subgenomes exhibit stunning karyotype stability, with parallel subgenome dominance in the two tetraploid clades and a gradual shift of dominance in the hexaploid clade. Allopolyploidization and subgenome dominance have shaped the evolution of tree-like lignified culms, rapid growth and synchronous flowering characteristic of woody bamboos as large grasses. Our work provides insights into genome dominance in a remarkable polyploid system, including its dependence on genomic context and its ability to switch which subgenomes are dominant over evolutionary time.

Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia.

Fargesia, the largest genus within the temperate bamboo tribe Arundinarieae, has more than 90 species mainly distributed in the mountains of Southwest China. The Fargesia bamboos are important components of the subalpine forest ecosystems that provide food and habitat for many endangered animals, including the giant panda. However, species-level identification of Fargesia is difficult. Moreover, the rapid radiation and slow molecular evolutionary rate of Fargesia pose a significant challenge to using DNA barcoding with standard plant barcodes (rbcL, matK, and ITS) in bamboos. With progress in the sequencing technologies, complete plastid genomes (plastomes) and nuclear ribosomal DNA (nrDNA) sequences have been proposed as organelle barcodes for species identification; however, these have not been tested in bamboos. We collected 196 individuals representing 62 species of Fargesia to comprehensively evaluate the discriminatory power of plastomes and nrDNA sequences compared to standard barcodes. Our analysis indicates that complete plastomes have substantially higher discriminatory power (28.6%) than standard barcodes (5.7%), whereas nrDNA sequences show a moderate improvement (65.4%) compared to ITS (47.2%). We also found that nuclear markers performed better than plastid markers, and ITS alone had higher discriminatory power than complete plastomes. The study also demonstrated that plastomes and nrDNA sequences can contribute to intrageneric phylogenetic resolution in Fargesia. However, neither of these sequences were able to discriminate all the sampled species, and therefore, more nuclear markers need to be identified.

The complete chloroplast genome and phylogenetic position of Thamnocalamus unispiculatus (Poaceae: Bambusoideae: Arundinarieae).

Thamnocalamus unispiculatus T.P.Yi & J.Y.Shi 2007 is an important bamboo species with significant ecological and economic value. This study presents the complete chloroplast genome sequence of T. unispiculatus. The sequence was 139,726 bp in length and exhibited a typical quadripartite structure, containing four regions: large single copy regions (LSC, 83,283 bp), small single copy regions (SSC, 12,851 bp) and a pair of inverted repeats (IRs, 21,726 bp). A total of 130 genes were annotated, including 86 protein-coding genes, 36 transfer RNA genes, and eight ribosomal RNA genes. Phylogenetic analysis indicated that T. unispiculatus and T. spathiflorus are sister species, supporting the conclusion that Thamnocalamus is a monophyletic group. The chloroplast genome of T. unispiculatus promotes the protection and exploration of biodiversity, phylogenetic relationships, and genetic research in Bambusoideae.

Extracellular vesicles from the inflammatory microenvironment regulate the osteogenic and odontogenic differentiation of periodontal ligament stem cells by miR-758-5p/LMBR1/BMP2/4 axis.

BACKGROUND: Extracellular vesicles (EVs) play a key role in constructing a microenvironment that favors the differentiation of stem cells. The present work aimed to determine the molecular mechanisms by which EV derived from inflammatory dental pulp stem cell (iDPSC-EV) influence periodontal ligament stem cells (PDLSCs) and provide a potential strategy for bone and dental pulp regeneration. METHODS: The osteogenic and odontogenic differentiation was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, alkaline phosphatase (ALP) activity assay, ALP staining, alizarin red S (ARS) staining, and immunofluorescence staining. To detect proliferation, the Cell Counting Kit-8 (CCK-8) assay, and flow cytometry analysis were used. EVs were isolated by the Exoperfect kit and ultrafiltration and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. The expression profile of miRNAs in EVs was studied using miRNA sequence and bioinformatics, and one of the upregulated miRNAs was evaluated on PDLSCs. RESULTS: The inflammatory microenvironment stimulated osteogenic and odontogenic differentiation of DPSCs and iDPSC-EV behaved alike on PDLSCs. MiR-758-5p was upregulated in iDPSC-EV and was demonstrated to play a significant role in the osteogenic and odontogenic commitment of PDLSCs. A dual-luciferase reporter assay confirmed the binding site between miR-758-5p and limb development membrane protein 1 (LMBR1). The knockdown of LMBR1 also enhanced the above potential. Mechanically, bone morphogenetic protein (BMP) signaling was activated. CONCLUSIONS: EVs from the inflammatory microenvironment enhanced the osteogenic and odontogenic differentiation of PDLSCs partly by shuttering LMBR1-targeting miR-758-5p via BMP signaling.

Inflorescences of Fargesiaangustissima T.P. Yi and Yushaniapauciramificans T.P. Yi (Poaceae, Bambusoideae) shed light on the taxonomy of the Sino-Himalayan alpine bamboos.

The taxonomy of the Sino-Himalayan alpine bamboos is controversial due to their complex evolutionary history and further complicated by the scarcity of inflorescence. Here, we supplement the description of the inflorescence of Fargesiaangustissima T.P. Yi and Yushaniapauciramificans T.P. Yi, which shed light on the taxonomy of Fargesia Franchet, Borinda Stapleton and Yushania Keng. F.angustissima has compressed inflorescence unilateral stretching out from reduced spathe, showing a transitional state between species with condensed inflorescence embraced by spathe-like bracts and species with open inflorescence without bracts. Considering that extensive gene flow existed between several clades of Fargesia found in recent studies, a broadly-defined Fargesia s. l. should be adopted. Meanwhile, the inflorescence of Y.pauciramificans has typical characteristics of Yushania, such as axilla with tuberculate glands, rachilla internodes ciliate and cylindrical florets, supporting the delimitation of Yushania.

MicroRNA Hsa-Let-7b Regulates the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting CTHRC1.

Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells' (MSCs') biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1.

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

BACKGROUND: Long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs). METHODS: Healthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolated from healthy/inflamed periodontal ligament tissues, respectively. The proliferation abilities were determined by CCK-8, EdU assay, and flow cytometry (FCM). The methods of Western blot assay (WB), quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity detection, and immunofluorescence staining were described to determine the biological influences of lncRNA ANRIL on iPDLSCs. Senescence-associated (SA)-ß-galactosidase (gal) staining, Western blot analysis, and qRT-PCR were performed to determine cell senescence. Dual-luciferase reporter assays were conducted to confirm the binding of lncRNA ANRIL and miR-7-5-p, as well as miR-7-5p and insulin-like growth factor receptor (IGF-1R). RESULTS: HPDLSCs and iPDLSCs were isolated and cultured successfully. LncRNA ANRIL and IGF-1R were declined, while miR-7-5p was upregulated in iPDLSCs compared with hPDLSCs. Overexpression of ANRIL enhanced the osteogenic protein expressions of OSX, RUNX2, ALP, and knocked down the aging protein expressions of p16, p21, p53. LncRNA ANRIL could promote the committed differentiation of iPDLSCs by sponging miR-7-5p. Upregulating miR-7-5p inhibited the osteogenic differentiation of iPDLSCs. Further analysis identified IGF-1R as a direct target of miR-7-5p. The direct binding of lncRNA ANRIL and miR-7-5p, miR-7-5p and the 3'-UTR of IGF-1R were verified by dual-luciferase reporter assay. Besides, rescue experiments showed that knockdown of miR-7-5p reversed the inhibitory effect of lncRNA ANRIL deficiency on osteogenesis of iPDLSCs. CONCLUSION: This study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.

Two new species of Yushania (Poaceae: Bambusoideae) from South China, with a taxonomic revision of related species.

Two new species of Yushania (Poaceae, Bambusoideae, Arundinarieae) are described and illustrated from Hunan, China. Yushania longshanensis D.Z. Li & X.Y. Ye is distinguished from related species (Y. confusa, Y. angustifolia and Y. pachyclada) by having a thinner culm (0.2-0.3 cm in diameter), glabrous sheath scar, no oral setae, a large glabrous leaf blade (10-20 × 0.9-1.3 cm) and 3-4 pairs of secondary veins. Yushania stoloniforma D.Z. Li & X.Y. Ye has a distinctive scrambling habit, which differs from its putative close allies. Both of these two new species have a solitary branch at the basal nodes and can be assigned to Yushania sect. Yushania based on morphological features. Additionally, we treated Yushania gigantea T.P. Yi & L. Yang as a new synonym of Y. elevata T.P. Yi and renamed Y. microphylla T.P. Yi & L. Yang as Y. weiningensis D.Z. Li & X.Y. Ye.

Parallel ddRAD and Genome Skimming Analyses Reveal a Radiative and Reticulate Evolutionary History of the Temperate Bamboos.

Rapid evolutionary radiations are among the most challenging phylogenetic problems, wherein different types of data (e.g., morphology and molecular) or genetic markers (e.g., nuclear and organelle) often yield inconsistent results. The tribe Arundinarieae, that is, the temperate bamboos, is a clade of tetraploid originated 22 Ma and subsequently radiated in East Asia. Previous studies of Arundinarieae have found conflicting relationships and/or low support. Here, we obtain nuclear markers from ddRAD data for 213 Arundinarieae taxa and parallel sampling of chloroplast genomes from genome skimming for 147 taxa. We first assess the feasibility of using ddRAD-seq data for phylogenetic estimates of paleopolyploid and rapidly radiated lineages, optimize clustering thresholds, and analysis workflow for orthology identification. Reference-based ddRAD data assembly approaches perform well and yield strongly supported relationships that are generally concordant with morphology-based taxonomy. We recover five major lineages, two of which are notable (the pachymorph and leptomorph lineages), in that they correspond with distinct rhizome morphologies. By contrast, the phylogeny from chloroplast genomes differed significantly. Based on multiple lines of evidence, the ddRAD tree is favored as the best species tree estimation for temperate bamboos. Using a time-calibrated ddRAD tree, we find that Arundinarieae diversified rapidly around the mid-Miocene corresponding with intensification of the East Asian monsoon and the evolution of key innovations including the leptomorph rhizomes. Our results provide a highly resolved phylogeny of Arundinarieae, shed new light on the radiation and reticulate evolutionary history of this tribe, and provide an empirical example for the study of recalcitrant plant radiations. [Arundinarieae; ddRAD; paleopolyploid; genome skimming; rapid diversification; incongruence.].

Two new species of Fargesia (Poaceae, Bambusoideae) from southwestern China.

Two new species of Fargesia, one from Xizang (Tibet) and one from Yunnan, China, are described and illustrated. Fargesia viridis D.Z. Li & X.Y. Ye is characterized by its densely white powder, nearly solid internodes, yellow setose sheath scar and culm sheaths, and 4-6 leaves of large size. Fargesia purpurea D.Z. Li & X.Y. Ye has thinner culms (0.5-1.4 cm in diameter), a ring of 4-5 mm tall brown setae below nodes, fewer branches, glabrous sheath scar and culm sheaths, differentiated from the related species.

Yushania tongpeii (Poaceae, Bambusoideae), a new bamboo species from north-eastern Yunnan, China.

Yushania tongpeii D.Z.Li, Y.X.Zhang & E.D.Liu, a new species of the temperate bamboo tribe Arundinarieae (Poaceae: Bambusoideae), is described and illustrated from north-eastern Yunnan, China. Yushania tongpeii is characterised by taller branching from nodes 1-2 m above the ground, usually three branches at the node, sparse purple spots and thin white powder on the internode, densely purple-spotted culm sheaths, glabrous margins of culm sheaths and tomentose leaf ligules. Based on the morphological features, this new species is assigned to section Yushania.

Genome Sequences Provide Insights into the Reticulate Origin and Unique Traits of Woody Bamboos.

Polyploidization is a major driver of speciation and its importance to plant evolution has been well recognized. Bamboos comprise one diploid herbaceous and three polyploid woody lineages, and are members of the only major subfamily in grasses that diversified in forests, with the woody members having a tree-like lignified culm. In this study, we generated four draft genome assemblies of major bamboo lineages with three different ploidy levels (diploid, tetraploid, and hexaploid). We also constructed a high-density genetic linkage map for a hexaploid species of bamboo, and used a linkage-map-based strategy for genome assembly and identification of subgenomes in polyploids. Further phylogenomic analyses using a large dataset of syntenic genes with expected copies based on ploidy levels revealed that woody bamboos originated subsequent to the divergence of the herbaceous bamboo lineage, and experienced complex reticulate evolution through three independent allopolyploid events involving four extinct diploid ancestors. A shared but distinct subgenome was identified in all polyploid forms, and the progenitor of this subgenome could have been critical in ancient polyploidizations and the origin of woody bamboos. Important genetic clues to the unique flowering behavior and woody trait in bamboos were also found. Taken together, our study provides significant insights into ancient reticulate evolution at the subgenome level in the absence of extant donor species, and offers a potential model scenario for broad-scale study of angiosperm origination by allopolyploidization.

Genome-wide RAD sequencing data provide unprecedented resolution of the phylogeny of temperate bamboos (Poaceae: Bambusoideae).

The temperate bamboos (tribe Arundinarieae, Poaceae) are strongly supported as monophyly in recent molecular studies, but taxonomic delineation and phylogenetic relationships within the tribe lack resolution. Here, we sampled 39 species (36 temperate bamboos and 3 outgroups) for restriction-site associated DNA sequencing (RAD-seq) with an emphasis on Phyllostachys clade and related clades. Using the largest data matrix for the bamboos to date, we were able to infer phylogenetic relationships with unparalleled resolution. The Phyllostachys, Shibataea, and Arundinaria clades defined from plastid phylogeny, were not supported as monophyletic group. However, the RAD-seq phylogeny largely agreed with the morphology-based taxonomy, with two clades having leptomorph rhizomes strongly supported as monophyletic group. We also explored two approaches, BWA-GATK (a mapping system) and Stacks (a grouping system), for differences in SNP calling and phylogeny inference. For the same level of missing data, the BWA-GATK pipeline produced much more SNPs in comparison with Stacks. Phylogenetic analyses of the largest data matrices from both pipelines, using concatenation and coalescent methods provided similar tree topologies, despite the presence of missing data. Our study demonstrates the utility of RAD-seq data for elucidating phylogenetic relationships between genera and higher taxonomic levels in this important but phylogenetically challenging group.

New distribution records of two bamboo species in Yunnan, China with description of the inflorescence for Melocalamus yunnanensis (Poaceae, Bambusoideae).

Ampelocalamus actinotrichus (Merrill & Chun) S. L. Chen, T. H. Wen & G. Y. Sheng and Neomicrocalamus prainii (Gamble) P. C. Keng are reported with new distribution records in southern and southeastern Yunnan, China, respectively. Ampelocalamus actinotrichus was previously recorded to be endemic to Hainan, China, and Neomicrocalamus prainii to be distributed in southern Tibet and western Yunnan in China, northeastern India, and Burma. The identities of individuals collected in southern and southeastern Yunnan of these two species are confirmed by molecular evidence. The new distribution record of Ampelocalamus actinotrichus provides a case at the species level for confirming floristic affinities of southern Yunnan and Hainan Island in south China. The disjunct distribution of Neomicrocalamus prainii in Yunnan is concordant with the ecogeographical diagonal line from northwestern Yunnan to southeastern Yunnan and this may imply a tropical origin of this species. In addition, the inflorescence of Melocalamus yunnanensis (T. H. Wen) T. P. Yi is described.

Combined blood/tissue analysis for cancer biomarker discovery: application to renal cell carcinoma.

A method that relies on subtractive tissue-directed shot-gun proteomics to identify tumor proteins in the blood of a patient newly diagnosed with cancer is described. To avoid analytical and statistical biases caused by physiologic variability of protein expression in the human population, this method was applied on clinical specimens obtained from a single patient diagnosed with nonmetastatic renal cell carcinoma (RCC). The proteomes extracted from tumor, normal adjacent tissue and preoperative plasma were analyzed using 2D-liquid chromatography-mass spectrometry (LC-MS). The lists of identified proteins were filtered to discover proteins that (i) were found in the tumor but not normal tissue, (ii) were identified in matching plasma, and (iii) whose spectral count was higher in tumor tissue than plasma. These filtering criteria resulted in identification of eight tumor proteins in the blood. Subsequent Western-blot analysis confirmed the presence of cadherin-5, cadherin-11, DEAD-box protein-23, and pyruvate kinase in the blood of the patient in the study as well as in the blood of four other patients diagnosed with RCC. These results demonstrate the utility of a combined blood/tissue analysis strategy that permits the detection of tumor proteins in the blood of a patient diagnosed with RCC.