Dipterocarpoidae genomics reveal their demography and adaptations to Asian rainforests.

Dipterocarpoideae species form the emergent layer of Asian rainforests. They are the indicator species for Asian rainforest distribution, but they are severely threatened. Here, to understand their adaptation and population decline, we assemble high-quality genomes of seven Dipterocarpoideae species including two autotetraploid species. We estimate the divergence time between Dipterocarpoideae and Malvaceae and within Dipterocarpoideae to be 108.2 (97.8‒118.2) and 88.4 (77.7‒102.9) million years ago, and we identify a whole genome duplication event preceding dipterocarp lineage diversification. We find several genes that showed a signature of selection, likely associated with the adaptation to Asian rainforests. By resequencing of two endangered species, we detect an expansion of effective population size after the last glacial period and a recent sharp decline coinciding with the history of local human activities. Our findings contribute to understanding the diversification and adaptation of dipterocarps and highlight anthropogenic disturbances as a major factor in their endangered status.

A chromosome-scale genome sequence of pitaya (Hylocereus undatus) provides novel insights into the genome evolution and regulation of betalain biosynthesis.

Pitaya (Hylocereus) is the most economically important fleshy-fruited tree of the Cactaceae family that is grown worldwide, and it has attracted significant attention because of its betalain-abundant fruits. Nonetheless, the lack of a pitaya reference genome significantly hinders studies focused on its evolution, as well as the potential for genetic improvement of this crop. Herein, we employed various sequencing approaches, namely, PacBio-SMRT, Illumina HiSeq paired-end, 10× Genomics, and Hi-C (high-throughput chromosome conformation capture) to provide a chromosome-level genomic assembly of 'GHB' pitaya (H. undatus, 2n = 2x = 22 chromosomes). The size of the assembled pitaya genome was 1.41 Gb, with a scaffold N50 of ~127.15 Mb. In total, 27,753 protein-coding genes and 896.31 Mb of repetitive sequences in the H. undatus genome were annotated. Pitaya has undergone a WGT (whole-genome triplication), and a recent WGD (whole-genome duplication) occurred after the gamma event, which is common to the other species in Cactaceae. A total of 29,328 intact LTR-RTs (~696.45 Mb) were obtained in H. undatus, of which two significantly expanded lineages, Ty1/copia and Ty3/gypsy, were the main drivers of the expanded genome. A high-density genetic map of F1 hybrid populations of 'GHB' × 'Dahong' pitayas (H. monacanthus) and their parents were constructed, and a total of 20,872 bin markers were identified (56,380 SNPs) for 11 linkage groups. More importantly, through transcriptomic and WGCNA (weighted gene coexpression network analysis), a global view of the gene regulatory network, including structural genes and the transcription factors involved in pitaya fruit betalain biosynthesis, was presented. Our data present a valuable resource for facilitating molecular breeding programs of pitaya and shed novel light on its genomic evolution, as well as the modulation of betalain biosynthesis in edible fruits.

An all-in-one magnetic SERS nanosensor for ratiometric detection of Escherichia coli in foods.

An all-in-one nanosensor was developed for the magnetic enrichment and ratiometric surface-enhanced Raman scattering (SERS) detection of Escherichia coli (E. coli). The all-in-one nanosensor was constructed through the chemical integration of four components into a single nanoparticle, which include a manganese ferrite nanoparticle serving as the magnetic core, a thin silver shell as the SERS substrate, a self-assembled layer of 4-mercaptobenzoic acid (MBA) molecules as the SERS internal standard, and a MBA-conjugated layer of aptamer sequences as the capture probe of E. coli. In the detection of E. coli in food, the target cells were first captured by the nanosensors and magnetically enriched in a short time of 15 min, and then the ratiometric SERS was performed through the Raman intensity ratio between two specific SERS peaks produced by the captured E. coli and the internal MBA. The pre-concentration and ratiometry enabled the nanosensor to detect E. coli with a detection limit down to 10 CFU/mL. The all-in-one nanosensor was successfully applied for the detection of E. coli in various liquid foods including milk, juice, tea, and coffee, with recoveries ranging from 89 to 110% and relative standard deviation lower than 1.7%. In comparison with the previous sandwich strategy adopted by most SERS sensors, this nanosensor endowed with an easier use and a lower cost is more sensitive and reproducible, leading to a great potential in practical applications.

Wnt/ß-catenin signaling pathway participates in the effect of miR-626 on oral squamous cell carcinoma by targeting RASSF4.

BACKGROUND: The role of miR-626 in oral squamous cell carcinoma (OSCC) was investigated by targeting RASSF4. METHODS: The miR-626 and RASSF4 expression was detected in normal oral mucosa or OSCC tissues and OSCC or normal cells. The methylation status of RASSF4 was analyzed using methylation-specific polymerase chain reaction (PCR). The cytoplasmic/nuclear ratios (C/N ratios) targeted by miR-626 were examined using microarray, followed by a dual-luciferase reporter assay. The subcellular localization of RASSF4 and miR-626 in OSCC cells was determined using RNA fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC), respectively. Ca9-22 and HSC2 cells were divided into mock, inhibitor NC, miR-626 inhibitor, scramble, RASSF4 and miR-626 mimic + RASSF4 groups, and then CCK-8, Annexin V-FITC/PI, wound healing, Transwell, qRT-PCR and western blotting assays were performed. RESULTS: OSCC tissues and cells had increased miR-626 expression and decreased RASSF4 expression. Patients with RASSF4 methylation had lower RASSF4 expression than those without methylation. In addition, a negative correlation between miR-626 and RASSF4 was found in OSCC tissues, both of which were correlated with the pathological grade, pathological stage, lymph node metastasis and patient prognosis. MiR-626 targeted RASSF4 in OSCC cells. Overexpressed RASSF4 inhibited the proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of OSCC cells, promoted cell apoptosis, and blocked the Wnt/ß-Catenin pathway, which was reversed by miR-626 overexpression. CONCLUSIONS: Inhibiting miR-626 can regulate the biological characteristics of OSCC cells, including proliferation, invasion, migration, EMT and apoptosis, by targeting RASSF4, which may be related to the Wnt/ß-Catenin pathway.

The chromosome-level reference genome of Coptis chinensis provides insights into genomic evolution and berberine biosynthesis.

Coptis chinensis Franch, a perennial herb, is mainly distributed in southeastern China. The rhizome of C. chinensis has been used as a traditional medicine for more than 2000 years in China and many other Asian countries. The pharmacological activities of C. chinensis have been validated by research. Here, we present a de novo high-quality genome of C. chinensis with a chromosome-level genome of ~958.20 Mb, a contig N50 of 1.58 Mb, and a scaffold N50 of 4.53 Mb. We found that the relatively large genome size of C. chinensis was caused by the amplification of long terminal repeat (LTR) retrotransposons. In addition, a whole-genome duplication event in ancestral Ranunculales was discovered. Comparative genomic analysis revealed that the tyrosine decarboxylase (TYDC) and (S)-norcoclaurine synthase (NCS) genes were expanded and that the aspartate aminotransferase gene (ASP5) was positively selected in the berberine metabolic pathway. Expression level and HPLC analyses showed that the berberine content was highest in the roots of C. chinensis in the third and fourth years. The chromosome-level reference genome of C. chinensis provides important genomic data for molecular-assisted breeding and active ingredient biosynthesis.

Clinical outcomes and safety of apatinib monotherapy in the treatment of patients with advanced epithelial ovarian carcinoma who progressed after standard regimens and the analysis of the VEGFR2 polymorphism.

The aims of the present study were to investigate the clinical outcomes and safety of apatinib monotherapy in the treatment of patients with advanced epithelial ovarian carcinoma (EOC) who have progressed after standard regimens, and to analyze the vascular endothelial growth factor receptor 2 (VEGFR2) rs2071559 polymorphism. A total of 118 patients with advanced EOC who received apatinib treatment were included in the study. Tumor response was evaluated using progression-free survival (PFS) and overall survival (OS) time, and safety data were documented. Additionally, peripheral blood and peripheral blood mononuclear cell (PBMC) specimens from the patients with EOC were collected to perform the genotyping of genetic polymorphism and assess the mRNA expression of VEGFR2, respectively. The objective response rate across the 118 patients with advanced EOC was 38.98%, the disease control rate was 63.56%, the median PFS time was 4.65 months and the median OS time was 15.10 months. Regarding the polymorphism analysis, the prevalence of rs2071559 in VEGFR2 among the 118 patients with advanced EOC was recorded as the TT genotype in 72 cases (61.02%), TC genotype in 41 cases (34.75%) and CC genotype in 5 cases (4.23%), and the minor allele frequency of rs2071559 was 0.22. The distribution of the three genotypes was in accordance with the Hardy-Weinberg equilibrium (P=0.781). TC and CC genotypes were merged in the subsequent analysis. The prognosis analyses suggested that the median PFS time of patients with the TC/CC genotype and the TT genotype was 3.10 and 5.40 months, respectively (P=0.015). Moreover, the median OS time of the two genotypes was 12.60 and 17.50 months, respectively (P=0.009). However, no association was noted between genotype status of the polymorphism and adverse reactions. Additionally, the mRNA expression analysis indicated that the mRNA expression levels of VEGFR2 in PBMC specimens were significantly different between TT and TC/CC genotypes (P<0.001). The present study suggested that the clinical outcomes of patients with advanced EOC, who progressed after standard regimens and received apatinib treatment, might be influenced by the VEGFR2 rs2071559 polymorphism.

De novo transcriptome assembly of the eight major organs of Sacha Inchi (Plukenetia volubilis) and the identification of genes involved in α-linolenic acid metabolism.

BACKGROUND: Sacha Inchi (Plukenetia volubilis L.), which belongs to the Euphorbiaceae, has been considered a new potential oil crop because of its high content of polyunsaturated fatty acids in its seed oil. The seed oil especially contains high amounts of α-linolenic acid (ALA), which is useful for the prevention of various diseases. However, little is known about the genetic information and genome sequence of Sacha Inchi, which has largely hindered functional genomics and molecular breeding studies. RESULTS: In this study, a de novo transcriptome assembly based on transcripts sequenced in eight major organs, including roots, stems, shoot apexes, mature leaves, male flowers, female flowers, fruits, and seeds of Sacha Inchi was performed, resulting in a set of 124,750 non-redundant putative transcripts having an average length of 851 bp and an N50 value of 1909 bp. Organ-specific unigenes analysis revealed that the most organ-specific transcripts are found in female flowers (2244 unigenes), whereas a relatively small amount of unigenes are detected to be expressed specifically in other organs with the least in stems (24 unigenes). A total of 42,987 simple sequence repeats (SSRs) were detected, which will contribute to the marker assisted selection breeding of Sacha Inchi. We analyzed expression of genes related to the α-linolenic acid metabolism based on the de novo assembly and annotation transcriptome in Sacha Inchi. It appears that Sacha Inchi accumulates high level of ALA in seeds by strong expression of biosynthesis-related genes and weak expression of degradation-related genes. In particular, the up-regulation of FAD3 and FAD7 is consistent with high level of ALA in seeds of Sacha Inchi compared with in other organs. Meanwhile, several transcription factors (ABI3, LEC1 and FUS3) may regulate key genes involved in oil accumulation in seeds of Sacha Inchi. CONCLUSIONS: The transcriptome of major organs of Sacha Inchi has been sequenced and de novo assembled, which will expand the genetic information for functional genomic studies of Sacha Inchi. In addition, the identification of candidate genes involved in ALA metabolism will provide useful resources for the genetic improvement of Sacha Inchi and the metabolic engineering of ALA biosynthesis in other plants.

The complete chloroplast genome sequence of the biofuel plant Sacha Inchi, Plukenetia volubilis.

Sacha Inchi (Plukenetia volubilis) is a potential woody oil seed plant for producing healthy vegetable oil due to high content of α-linolenic acid in its seeds. In this study, we report the structure of the complete chloroplast genome of P. volubilis using high-throughput next-generation sequencing technology. The circular chloroplast genome is 161,733 bp in size, containing a pair of inverted repeat regions (IR) of 27,382 bp each, which were separated by a large single copy region (LSC) of 88,843 bp and a small single copy region (SSC) of 18,126 bp. The chloroplast genome harbors 135 genes, including 92 protein-coding genes, 35 tRNA genes and 8 rRNA genes. Based on the phylogenetic relationships between the chloroplast genome of P. volubilis and those of the other species, P. volubilis is most closely related to castor bean (Ricinus communis).

rRNA Pseudogenes in Filamentous Ascomycetes as Revealed by Genome Data.

The nuclear ribosomal DNA (rDNA) is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S) are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS) region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP) mutation was probably responsible for the formation of those rRNA pseudogenes.

Comparison of different sequencing and assembly strategies for a repeat-rich fungal genome, Ophiocordyceps sinensis.

Ophiocordyceps sinensis is one of the most expensive medicinal fungi world-wide, and has been used as a traditional Chinese medicine for centuries. In a recent report, the genome of this fungus was found to be expanded by extensive repetitive elements after assembly of Roche 454 (223Mb) and Illumina HiSeq (10.6Gb) sequencing data, producing a genome of 87.7Mb with an N50 scaffold length of 12kb and 6972 predicted genes. To test whether the assembly could be improved by deeper sequencing and to assess the amount of data needed for optimal assembly, genomic sequencing was run several times on genomic DNA extractions of a single ascospore isolate (strain 1229) on an Illumina HiSeq platform (25Gb total data). Assemblies were produced using different data types (raw vs. trimmed) and data amounts, and using three freely available assembly programs (ABySS, SOAP and Velvet). In nearly all cases, trimming the data for low quality base calls did not provide assemblies with higher N50 values compared to the non-trimmed data, and increasing the amount of input data (i.e. sequence reads) did not always lead to higher N50 values. Depending on the assembly program and data type, the maximal N50 was reached with between 50% to 90% of the total read data, equivalent to 100× to 200× coverage. The draft genome assembly was improved over the previously published version resulting in a 114Mb assembly, scaffold N50 of 70kb and 9610 predicted genes. Among the predicted genes, 9213 were validated by RNA-Seq analysis in this study, of which 8896 were found to be singletons. Evidence from genome and transcriptome analyses indicated that species assemblies could be improved with defined input material (e.g. haploid mono-ascospore isolate) without the requirement of multiple sequencing technologies, multiple library sizes or data trimming for low quality base calls, and with genome coverages between 100× and 200×.

The complete mitochondrial genome of Celebes wild boar, Sus celebensis (Cetartiodactyla: Suina: Suidae), and comparative mitochondrial genomics of the Sus species.

In this study, the complete mitochondrial (mt) genome sequence of Sus celebensis was firstly determined. The total genome was 16,481 bp in length and its overall base composition was estimated to be 34.9% for A, 25.8% for T, 26.2% for C, 13.1% for G, respectively, indicating an A-T (60.7%)-rich feature in Celebes wild boar mitogenome. It harbored 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a non-coding control region (D-loop region). Comparisons with other publicly available pig mitogenomes revealed abundant nucleotide diversity. This complete mitgenome sequence would accelerate further studies on pig evolution and domestication that will enhance germplasm preservation and breeding programs of the pig gene pool.

The complete mitochondrial genome of eastern lowland gorilla, Gorilla beringei graueri, and comparative mitochondrial genomics of Gorilla species.

In this study, we determined the complete mitochondrial (mt) genome of eastern lowland gorilla, Gorilla beringei graueri for the first time. The total genome was 16,416 bp in length. It contained a total of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region (D-loop region). The base composition was A (30.88%), G (13.10%), C (30.89%) and T (25.13%), indicating that the percentage of A+T (56.01%) was higher than G+C (43.99%). Comparisons with the other publicly available Gorilla mitogenome showed the conservation of gene order and base compositions but a bunch of nucleotide diversity. This complete mitochondrial genome sequence will provide valuable genetic information for further studies on conservation genetics of eastern lowland gorilla.

The complete mitochondrial genome of domestic sheep, Ovis aries.

In this study, we report a complete mitochondrial (mt) genome sequence of the Texel ewe, Ovis aries. The total genome is 16,615 bp in length and its overall base composition was estimated to be 33.68% for A, 27.36% for T, 25.86% for C, and 13.10% for G indicating an AT-rich (61.04%) feature in the O. aries mtgenome. It contains a total of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a control region (D-loop region). Comparisons with other publicly available sheep mitogenomes revealed a bunch of nucleotide diversity. This complete mitgenome sequence would enlarge useful genomic information for further studies on sheep evolution and domestication that will enhance germplasm conservation and breeding programs of O. aries.

The complete mitochondrial genome of European wild boar, Sus scrofa scrofa.

In this study, we report the complete mitochondrial genome sequence of the European wild boar, Sus scrofa scrofa for the first time. The genome is found to be 16,770 bp in length and has a base composition of A (34.63%), G (13.38%), C (26.21%), and T (25.78%), indicating that the percentage of A + T (60.41%) was higher than G + C (39.59%). Similar to other pigs, it contains a typically conserved structure including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). Most of the genes were located on the H-strand except for the ND6 gene and eight tRNA genes. The complete mitochondrial genome sequence provided here would add a new genetic resource and new study on the evolution of the genus Sus.

The complete mitochondrial genome of the central chimpanzee, Pan troglodytes troglodytes.

This study first report the complete mitochondrial genome sequence of the central chimpanzee, Pan troglodytes troglodytes. The genome was a total of 16 556 bp in length and had a base composition of A (31.05%), G (12.95%), C (30.84%), and T (25.16%), indicating that the percentage of A + T (56.21%) is higher than G + C (43.79%). Similar to other primates, it possessed a typically conserved structure, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region (D-loop). Most of these genes were found to locate on the H-strand except for the ND6 gene and 8 tRNA genes. The phylogenetic analysis showed that the P. t. troglodytes mitochondrial genome formed a cluster with the other three Pan troglodytes genomes and that the genus Pan is closely related to the genus Homo. This mitochondrial genome sequence would supply useful genetic resources to help the conservation management of primate germplasm and uncover hominoid evolution.

Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis.

As part of a genome sequencing project for Ophiocordyceps sinensis, strain 1229, a complete mitochondrial (mt) genome was assembled as a single circular dsDNA of 157,510 bp, one of the largest reported for fungi. Conserved genes including the large and small rRNA subunits, 27 tRNA and 15 protein-coding genes, were identified. In addition, 58 non-conserved open reading frames (ncORFs) in the intergenic and intronic regions were also identified. Transcription analyses using RNA-Seq validated the expression of most conserved genes and ncORFs. Fifty-two introns (groups I and II) were found within conserved genes, accounting for 68.5% of the genome. Thirty-two homing endonucleases (HEs) with motif patterns LAGLIDADG (21) and GIY-YIG (11) were identified in group I introns. The ncORFs found in group II introns mostly encoded reverse transcriptases (RTs). As in other hypocrealean fungi, gene contents and order were found to be conserved in the mt genome of O. sinensis, but the genome size was enlarged by longer intergenic regions and numerous introns. Intergenic and intronic regions were composed of abundant repetitive sequences usually associated with mobile elements. It is likely that intronic ncORFs, which encode RTs and HEs, may have contributed to the enlarged mt genome of O. sinensis.