Chromosome-scale genome assembly of oil-tea tree Camellia crapnelliana.

Camellia crapnelliana Tutch., belonging to the Theaceae family, is an excellent landscape tree species with high ornamental values. It is particularly an important woody oil-bearing plant species with high ecological, economic, and medicinal values. Here, we first report the chromosome-scale reference genome of C. crapnelliana with integrated technologies of SMRT, Hi-C and Illumina sequencing platforms. The genome assembly had a total length of ~2.94 Gb with contig N50 of ~67.5 Mb, and ~96.34% of contigs were assigned to 15 chromosomes. In total, we predicted 37,390 protein-coding genes, ~99.00% of which could be functionally annotated. The chromosome-scale genome of C. crapnelliana will become valuable resources for understanding the genetic basis of the fatty acid biosynthesis, and greatly facilitate the exploration and conservation of C. crapnelliana.

Transcutaneous auricular vagus nerve stimulation improves anxiety symptoms and cortical activity during verbal fluency task in Parkinson's disease with anxiety.

OBJECTIVE: To investigate the effect of 20/4Hz transcutaneous auricular vagus nerve stimulation (taVNS) on anxiety symptoms in Parkinson's disease (PD) and the potential neural mechanism. METHODS: In the current randomized, double-blind, sham-controlled trial, 30 PD patients with anxiety (PD-A), 30 PD patients without anxiety (PD-nA), and 30 healthy controls (HCs) were enrolled. PD-A patients were randomly (1:1) allotted to real taVNS stimulation group (RS) or sham stimulation group (SS) to explore the efficacy of a two-week treatment of taVNS to promote anxiety recovery. Simultaneously, all participants were measured activation in the bilateral prefrontal cortex during verbal fluency task (VFT) using functional near-infrared spectroscopy. RESULTS: PD-A patients showed significantly decreased oxyhemoglobin in the left triangle part of the inferior frontal gyrus (IFG) during VFT, which was negatively related to the severity of anxiety symptoms. After two-week treatment of taVNS, the interaction of group and time had significant effect on HAMA scores (F = 18.476, p < 0.001, η2 = 0.398). In RS group, compared with baseline, HAMA scores decreased significantly in the post-treatment and follow-up condition (both p < 0.001). Meanwhile, in RS group, HAMA scores were lower than those in SS group in the post-treatment and follow-up condition (p = 0.006, <0.001, respectively). Furthermore, the 20/4Hz taVNS remarkably ameliorated anxiety symptoms in PD patients, directly correlated with the increased activation of the left triangle part of the IFG during VFT in RS group. CONCLUSION: Our results depicted that taVNS could ameliorate the anxiety symptoms of PD-A patients and regulated the function of the left triangle part of the IFG.

Ancient Duplication and Lineage-Specific Transposition Determine Evolutionary Trajectory of ERF Subfamily across Angiosperms.

AP2/ERF transcription factor family plays an important role in plant development and stress responses. Previous studies have shed light on the evolutionary trajectory of the AP2 and DREB subfamilies. However, knowledge about the evolutionary history of the ERF subfamily in angiosperms still remains limited. In this study, we performed a comprehensive analysis of the ERF subfamily from 107 representative angiosperm species by combining phylogenomic and synteny network approaches. We observed that the expansion of the ERF subfamily was driven not only by whole-genome duplication (WGD) but also by tandem duplication (TD) and transposition duplication events. We also found multiple transposition events in Poaceae, Brassicaceae, Poales, Brassicales, and Commelinids. These events may have had notable impacts on copy number variation and subsequent functional divergence of the ERF subfamily. Moreover, we observed a number of ancient tandem duplications occurred in the ERF subfamily across angiosperms, e.g., in Subgroup IX, IXb originated from ancient tandem duplication events within IXa. These findings together provide novel insights into the evolution of this important transcription factor family.

Comparative Genomic Analysis of Asian Cultivated Rice and Its Wild Progenitor (Oryza rufipogon) Has Revealed Evolutionary Innovation of the Pentatricopeptide Repeat Gene Family through Gene Duplication.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in land plants. However, current knowledge about the evolution of the PPR gene family remains largely limited. In this study, we performed a comparative genomic analysis of the PPR gene family in O. sativa and its wild progenitor, O. rufipogon, and outlined a comprehensive landscape of gene duplications. Our findings suggest that the majority of PPR genes originated from dispersed duplications. Although segmental duplications have only expanded approximately 11.30% and 13.57% of the PPR gene families in the O. sativa and O. rufipogon genomes, we interestingly obtained evidence that segmental duplication promotes the structural diversity of PPR genes through incomplete gene duplications. In the O. sativa and O. rufipogon genomes, 10 (~33.33%) and 22 pairs of gene duplications (~45.83%) had non-PPR paralogous genes through incomplete gene duplication. Segmental duplications leading to incomplete gene duplications might result in the acquisition of domains, thus promoting functional innovation and structural diversification of PPR genes. This study offers a unique perspective on the evolution of PPR gene structures and underscores the potential role of segmental duplications in PPR gene structural diversity.

Genome size variation and polyploidy prevalence in the genus Eragrostis are associated with the global dispersal in arid area.

Background: Biologists have long debated the drivers of the genome size evolution and variation ever since Darwin. Assumptions for the adaptive or maladaptive consequences of the associations between genome sizes and environmental factors have been proposed, but the significance of these hypotheses remains controversial. Eragrostis is a large genus in the grass family and is often used as crop or forage during the dry seasons. The wide range and complex ploidy levels make Eragrostis an excellent model for investigating how the genome size variation and evolution is associated with environmental factors and how these changes can ben interpreted. Methods: We reconstructed the Eragrostis phylogeny and estimated genome sizes through flow cytometric analyses. Phylogenetic comparative analyses were performed to explore how genome size variation and evolution is related to their climatic niches and geographical ranges. The genome size evolution and environmental factors were examined using different models to study the phylogenetic signal, mode and tempo throughout evolutionary history. Results: Our results support the monophyly of Eragrostis. The genome sizes in Eragrostis ranged from ~0.66 pg to ~3.80 pg. We found that a moderate phylogenetic conservatism existed in terms of the genome sizes but was absent from environmental factors. In addition, phylogeny-based associations revealed close correlations between genome sizes and precipitation-related variables, indicating that the genome size variation mainly caused by polyploidization may have evolved as an adaptation to various environments in the genus Eragrostis. Conclusion: This is the first study to take a global perspective on the genome size variation and evolution in the genus Eragrostis. Our results suggest that the adaptation and conservatism are manifested in the genome size variation, allowing the arid species of Eragrostis to spread the xeric area throughout the world.

Genome Size Variation and Evolution Driven by Transposable Elements in the Genus Oryza.

Genome size variation and evolutionary forces behind have been long pursued in flowering plants. The genus Oryza, consisting of approximately 25 wild species and two cultivated rice, harbors eleven extant genome types, six of which are diploid (AA, BB, CC, EE, FF, and GG) and five of which are tetraploid (BBCC, CCDD, HHJJ, HHKK, and KKLL). To obtain the most comprehensive knowledge of genome size variation in the genus Oryza, we performed flow cytometry experiments and estimated genome sizes of 166 accessions belonging to 16 non-AA genome Oryza species. k-mer analyses were followed to verify the experimental results of the two accessions for each species. Our results showed that genome sizes largely varied fourfold in the genus Oryza, ranging from 279 Mb in Oryza brachyantha (FF) to 1,203 Mb in Oryza ridleyi (HHJJ). There was a 2-fold variation (ranging from 570 to 1,203 Mb) in genome size among the tetraploid species, while the diploid species had 3-fold variation, ranging from 279 Mb in Oryza brachyantha (FF) to 905 Mb in Oryza australiensis (EE). The genome sizes of the tetraploid species were not always two times larger than those of the diploid species, and some diploid species even had larger genome sizes than those of tetraploids. Nevertheless, we found that genome sizes of newly formed allotetraploids (BBCC-) were almost equal to totaling genome sizes of their parental progenitors. Our results showed that the species belonging to the same genome types had similar genome sizes, while genome sizes exhibited a gradually decreased trend during the evolutionary process in the clade with AA, BB, CC, and EE genome types. Comparative genomic analyses further showed that the species with different rice genome types may had experienced dissimilar amplification histories of retrotransposons, resulting in remarkably different genome sizes. On the other hand, the closely related rice species may have experienced similar amplification history. We observed that the contents of transposable elements, long terminal repeats (LTR) retrotransposons, and particularly LTR/Gypsy retrotransposons varied largely but were significantly correlated with genome sizes. Therefore, this study demonstrated that LTR retrotransposons act as an active driver of genome size variation in the genus Oryza.

Evolution of Rosaceae Plastomes Highlights Unique Cerasus Diversification and Independent Origins of Fruiting Cherry.

Rosaceae comprises numerous types of economically important fruits, ornamentals, and timber. The lack of plastome characteristics has blocked our understanding of the evolution of plastome and plastid genes of Rosaceae crops. Using comparative genomics and phylogenomics, we analyzed 121 Rosaceae plastomes of 54 taxa from 13 genera, predominantly including Cerasus (true cherry) and its relatives. To our knowledge, we generated the first comprehensive map of genomic variation across Rosaceae plastomes. Contraction/expansion of inverted repeat regions and sequence losses of the two single-copy regions underlie large genomic variations in size among Rosaceae plastomes. Plastid protein-coding genes were characterized with a high proportion (over 50%) of synonymous variants and insertion-deletions with multiple triplets. Five photosynthesis-related genes were specially selected in perennial woody trees. Comparative genomic analyses implied divergent evolutionary patterns between pomaceous and drupaceous trees. Across all examined plastomes, unique and divergent evolution was detected in Cerasus plastomes. Phylogenomic analyses and molecular dating highlighted the relatively distant phylogenetic relationship between Cerasus and relatives (Microcerasus, Amygdalus, Prunus, and Armeniaca), which strongly supported treating the monophyletic true cherry group as a separate genus excluding dwarf cherry. High genetic differentiation and distinct phylogenetic relationships implied independent origins and domestication between fruiting cherries, particularly between Prunus pseudocerasus (Cerasus pseudocerasus) and P. avium (C. avium). Well-resolved maternal phylogeny suggested that cultivated P. pseudocerasus originated from Longmenshan Fault zone, the eastern edge of Himalaya-Hengduan Mountains, where it was subjected to frequent genomic introgression between its presumed wild ancestors and relatives.

Genome-Wide Analysis of CCT Transcript Factors to Identify Genes Contributing to Photoperiodic Flowering in Oryza rufipogon.

Photoperiod sensitivity is a dominant determinant for the phase transition in cereal crops. CCT (CONSTANS, CO-like, and TOC1) transcription factors (TFs) are involved in many physiological functions including the regulation of the photoperiodic flowering. However, the functional roles of CCT TFs have not been elucidated in the wild progenitors of crops. In this study, we identified 41 CCT TFs, including 19 CMF, 17 COL, and five PRR TFs in Oryza rufipogon, the presumed wild ancestor of Asian cultivated rice. There are thirty-eight orthologous CCT genes in Oryza sativa, of which ten pairs of duplicated CCT TFs are shared with O. rufipogon. We investigated daily expression patterns, showing that 36 OrCCT genes exhibited circadian rhythmic expression. A total of thirteen OrCCT genes were identified as putative flowering suppressors in O. rufipogon based on rhythmic and developmental expression patterns and transgenic phenotypes. We propose that OrCCT08, OrCCT24, and OrCCT26 are the strong functional alleles of rice DTH2, Ghd7, and OsPRR37, respectively. The SD treatment at 80 DAG stimulated flowering of the LD-grown O. rufipogon plants. Our results further showed that the nine OrCCT genes were significantly downregulated under the treatment. Our findings would provide valuable information for the construction of photoperiodic flowering regulatory network and functional characterization of the CCT TFs in both O. rufipogon and O. sativa.

Characterization of chloroplast genome of Eleusine coracana, a highly adaptable cereal crop with high nutritional reputation.

Eleusine coracana (L.) Gaertn. is a kind of highly adaptable cereal crop with a high nutritional value with the reputation of 'black pearl'. In this study, we sequenced, assembled and characterized the complete chloroplast genome of the grass species. The circular genome of E. coracana was 135,137 bp in length, which comprised two inverted repeat (IRa and IRb) regions of 20,919 bp in length separated by a large single copy (LSC) region of 80,663 bp and a small single copy (SSC) region of 12,636 bp. The total GC content of the E. coracana chloroplast genome was ∼38.13%. A total of 108 functional genes were predicted, including 76 protein-coding genes, 28 tRNA genes, and four rRNA genes. Our phylogenomic analysis of all protein-coding genes further revealed that E. coracana is closely related to Bouteloua curtipendula and B. gracilis, and they are together positioned in the subfamily Chloridoideae clade of the grass family.

Characterization of the chloroplast genome sequence of Bonia amplexicaulis (L.C.Chia, H.L.Fung & Y.L.Yang) N.H.Xia (Poaceae).

Bonia amplexicaulis (L.C.Chia, H.L.Fung & Y.L.Yang) N.H.Xia is a member of the Bambusoideae subfamily in Poaceae. In this study, we sequenced, assembled and characterized the complete chloroplast genome of B. amplexicaulis. The complete chloroplast genome was 139,935 bp in size, including a large single copy region of 83,453 bp, a small single-copy region of 12,860 bp and a pair of reverse repeats of 21,811 bp in size. The annotation of the B. amplexicaulis chloroplast genome indicates that it contained 83 protein-coding genes, 36 tRNA genes and 8 rRNA genes. Our phylogenetic analysis of all protein-coding genes from the 36 complete chroloplast grass genomes using Cyperus rotundus as outgroup showed that B. amplexicaulis is closely related to Otatea glauca and Pariana campestris to form the Bambusoideae lineage of the grass family.

The complete chloroplast genome sequence of Arundo formosana Hack. (Poaceae).

Arundo formosana Hack. belongs to the Arundionideae subfamily of Poaceae. In this study, we sequenced and assembled the complete chloroplast genome of A. formosana. The complete chloroplast genome was 136,919 bp in size, including a large single copy region of 82,039 bp, a small single-copy region of 12,108 bp and a pair of reverse repeats of 21,386 bp in size. The annotation of A. formosana indicates that it contained 81 protein-coding genes, 47 tRNA and 8 rRNA. Our phylogenetic analysis of the 36 grass complete chroloplast genomes of protein-coding genes using Cyperus rotundus as outgroup showed that A. formosana is closely related to Crinipes species to form the Arundionideae lineage of the grass family.

The complete chloroplast genome sequence of Bromus catharticus Vahl. (Poaceae).

Bromus catharticus Vahl. belongs to the Pooideae subfamily of Poaceae. In this study, we sequenced and assembled the complete chloroplast genome of B. catharticus. The complete chloroplast genome was 134,718 bp in size, including a large single-copy region of 80,540 bp, a small single-copy region of 11,806 bp and a pair of reverse repeats of 21,186 bp in size. The annotation of B. catharticus indicates that it contained 89 protein-coding genes, 47 tRNA genes and eight rRNA genes. Our phylogenetic analysis of all protein-coding genes of the 36 grass complete chroloplast genomes using Cyperus rotundus as outgroup showed that B. catharticus is closely related to the Koeleria and Avena species to form the Pooideae lineage of the grass family.

Gapless indica rice genome reveals synergistic contributions of active transposable elements and segmental duplications to rice genome evolution.

The ultimate goal of genome assembly is a high-accuracy gapless genome. Here, we report a new assembly pipeline that is used to produce a gapless genome for the indica rice cultivar Minghui 63. The resulting 397.71-Mb final assembly is composed of 12 contigs with a contig N50 size of 31.93 Mb. Each chromosome is represented by a single contig and the genomic sequences of all chromosomes are gapless. Quality evaluation of this gapless genome assembly showed that gene regions in our assembly have the highest completeness compared with the other 15 reported high-quality rice genomes. Further comparison with the japonica rice genome revealed that the gapless indica genome assembly contains more transposable elements (TEs) and segmental duplications (SDs), the latter of which produce many duplicated genes that can affect agronomic traits through dose effect or sub-/neo-functionalization. The insertion of TEs can also affect the expression of duplicated genes, which may drive the evolution of these genes. Furthermore, we found the expansion of nucleotide-binding site with leucine-rich repeat disease-resistance genes and cis-zeatin-O-glucosyltransferase growth-related genes in SDs in the gapless indica genome assembly, suggesting that SDs contribute to the adaptive evolution of rice disease resistance and developmental processes. Collectively, our findings suggest that active TEs and SDs synergistically contribute to rice genome evolution.

Draft genome of the herbaceous bamboo Raddia distichophylla.

Bamboos are important nontimber forest plants widely distributed in the tropical and subtropical regions of Asia, Africa, America, and Pacific islands. They comprise the Bambusoideae in the grass family (Poaceae), including approximately 1700 described species in 127 genera. In spite of the widespread uses of bamboo for food, construction, and bioenergy, the gene repertoire of bamboo still remains largely unexplored. Raddia distichophylla (Schrad. ex Nees) Chase, belonging to the tribe Olyreae (Bambusoideae, Poaceae), is a diploid herbaceous bamboo with only slightly lignified stems. In this study, we report a draft genome assembly of the ∼589 Mb whole-genome sequence of R. distichophylla with a contig N50 length of 86.36 Kb. Repeat sequences account for ∼49.08% of the genome assembly, of which LTR retrotransposons occupy ∼35.99% of the whole genome. A total of 30,763 protein-coding genes were annotated in the R. distichophylla genome with an average transcript size of 2887 bp. Access to this herbaceous bamboo genome sequence will provide novel insights into biochemistry, molecular marker-assisted breeding programs, and germplasm conservation for bamboo species worldwide.

Improved hybrid de novo genome assembly and annotation of African wild rice, Oryza longistaminata, from Illumina and PacBio sequencing reads.

African wild rice Oryza longistaminata, one of the eight AA- genome species in the genus Oryza, possesses highly valued traits, such as the rhizomatousness for perennial rice breeding, strong tolerance to biotic and abiotic stresses, and high biomass production on poor soils. To obtain the high-quality reference genome for O. longistaminata we employed a hybrid assembly approach through incorporating Illumina and PacBio sequencing datasets. The final genome assembly comprised only 107 scaffolds and was approximately ∼363.5 Mb, representing ∼92.7% of the estimated African wild rice genome (∼392 Mb). The N50 lengths of the assembled contigs and scaffolds were ∼46.49 Kb and ∼6.83 Mb, indicating ∼3.72-fold and ∼18.8-fold improvement in length compared to the earlier released assembly (∼12.5 Kb and 364 Kb, respectively). Aided with Hi-C data and syntenic relationship with O. sativa, these assembled scaffolds were anchored into 12 pseudo-chromosomes. Genome annotation and comparative genomic analysis reveal that lineage-specific expansion of gene families that respond to biotic- and abiotic stresses are of great potential for mining novel alleles to overcome major diseases and abiotic adaptation in rice breeding programs. This reference genome of African wild rice will greatly enlarge the existing database of rice genome resources and unquestionably form a solid base to understand genomic basis underlying highly valued phenotypic traits and search for novel gene sources in O. longistaminata for the future rice breeding programs.

SMRT- and Illumina-based RNA-seq analyses unveil the ginsinoside biosynthesis and transcriptomic complexity in Panax notoginseng.

Panax notoginseng is one of the most widely used traditional Chinese herbs with particularly valued roots. Triterpenoid saponins are mainly specialized secondary metabolites, which medically act as bioactive components. Knowledge of the ginsenoside biosynthesis in P. notoginseng, which is of great importance in the industrial biosynthesis and genetic breeding program, remains largely undetermined. Here we combined single molecular real time (SMRT) and Second-Generation Sequencing (SGS) technologies to generate a widespread transcriptome atlas of P. notoginseng. We mapped 2,383 full-length non-chimeric (FLNC) reads to adjacently annotated genes, corrected 1,925 mis-annotated genes and merged into 927 new genes. We identified 8,111 novel transcript isoforms that have improved the annotation of the current genome assembly, of which we found 2,664 novel lncRNAs. We characterized more alternative splicing (AS) events from SMRT reads (20,015 AS in 6,324 genes) than Illumina reads (18,498 AS in 9,550 genes), which contained a number of AS events associated with the ginsenoside biosynthesis. The comprehensive transcriptome landscape reveals that the ginsenoside biosynthesis predominantly occurs in flowers compared to leaves and roots, substantiated by levels of gene expression, which is supported by tissue-specific abundance of isoforms in flowers compared to roots and rhizomes. Comparative metabolic analyses further show that a total of 17 characteristic ginsenosides increasingly accumulated, and roots contained the most ginsenosides with variable contents, which are extraordinarily abundant in roots of the three-year old plants. We observed that roots were rich in protopanaxatriol- and protopanaxadiol-type saponins, whereas protopanaxadiol-type saponins predominated in aerial parts (leaves, stems and flowers). The obtained results will greatly enhance our understanding about the ginsenoside biosynthetic machinery in the genus Panax.

Development and characterization of EST-SSR markers for Camellia reticulata.

PREMISE: Camellia reticulata, which is native to southwestern China, is an economically important plant belonging to the family Theaceae. We developed expressed sequence tag-simple sequence repeat (EST-SSR) markers for C. reticulata, which can be used to investigate its genetic diversity, population structure, and evolutionary history. METHODS AND RESULTS: We detected 4780 SSRs in C. reticulata from Camellia RNA-Seq data deposited in the National Center for Biotechnology Information's expressed sequence tags database (dbEST). Primer pairs for 70 SSR loci were designed and used for PCR amplification using 90 individuals from four populations of C. reticulata. Of these loci, 50 microsatellite markers were successfully identified, including 11 polymorphic markers. The allele number per locus ranged from two to seven (mean = 4.182), and the levels of observed and expected heterozygosity ranged from 0.044 to 0.567 and from 0.166 to 0.642, respectively. Eleven primer pairs amplified PCR products in three other species of Camellia (C. saluenensis, C. pitardii, and C. yunnanensis). CONCLUSIONS: The set of microsatellite markers developed here can be used to study the genetic variation and population structure of C. reticulata and related species and thereby help to develop conservation strategies for this species.

Draft genomes of two outcrossing wild rice, Oryza rufipogon and O. longistaminata, reveal genomic features associated with mating-system evolution.

Oryza rufipogon and O. longistaminata are important wild relatives of cultivated rice, harboring a promising source of novel genes for rice breeding programs. Here, we present de novo assembled draft genomes and annotation of O. rufipogon and O. longistaminata. Our analysis reveals a considerable number of lineage-specific gene families associated with the self-incompatibility (SI) and formation of reproductive separation. We show how lineage-specific expansion or contraction of gene families with functional enrichment of the recognition of pollen, thus enlightening their reproductive diversification. We documented a large number of lineage-specific gene families enriched in salt stress, antifungal response, and disease resistance. Our comparative analysis further shows a genome-wide expansion of genes encoding NBS-LRR proteins in these two outcrossing wild species in contrast to six other selfing rice species. Conserved noncoding sequences (CNSs) in the two wild rice genomes rapidly evolve relative to selfing rice species, resulting in the reduction of genomic variation owing to shifts of mating systems. We find that numerous genes related to these rapidly evolving CNSs are enriched in reproductive structure development, flower development, and postembryonic development, which may associate with SI in O. rufipogon and O. longistaminata.

Artemisinin is highly soluble in polyethylene Glycol 4000 and such solution has multiple biological effects.

Artemisinin has a significant role in treatment of malaria, as well as effective anti-inflammatory and anti-cancer activities. However, such problems as poor water solubility and easy recrystallization limit its application. In this study, polyethylene glycol, a solvent which is widely used in pharmaceutics, was introduced to prepare an artemisinin dissolution. Under the action of hydrogen bonding in 12% polyethylene glycol 4000 solvent, the maximum solubility of artemisinin could reach up to 1 mg/mL. Meanwhile, biological functions of such artemisinin solution were evaluated. The obtained artemisinin solution had a significant inhibitory effect on Gram-positive bacteria, Gram-negative bacteria and fungi. As for the anti-inflammatory property, 0.031 mg/mL artemisinin solution had an obvious inhibitory effect on nitric oxide release in inflammatory cells, and the survival rate of cells was greater than 50%. Low concentration of the obtained artemisinin solution (0.031 mg/mL) had no significant cytotoxicity, while it displayed selective inhibition in cancer cells. IC50 for human hepatoma cells BEL-7404, SMMC7721 and Hep G2 is 0.0016 mg/mL, 0.0084 mg/mL and 0.0541 mg/mL, respectively. In conclusion, the 12% PEG4000-assisted artemisinin solution has a good biological effect and it can be further applied in pharmaceutics, biomaterials and medicine.