MITOGENOME ANNOUNCEMENTSCharacterization of the complete chloroplast genome of the Chinese endangered species Cymbidium iridioides D. Don.

Cymbidium iridioides D. Don 1852 is a Class I endangered species in China having important ornamental and breeding values. This study used Illumina high-throughput sequencing technology to sequence the complete chloroplast genome of C. iridioides. The genome features of C. iridioides and its phylogenetic relationships were determined. The complete chloroplast genome is 156,599 bp, containing a pair of 26,736 bp inverse duplication regions, a large 85,242 bp single-copy region, and a small 17,885 bp single-copy region. The entire genome contains 76 protein-coding genes, 37 tRNA genes, and 4 rRNA genes. A phylogenetic tree of 25 Orchidaceae species revealed that C. iridioides was grouped within Sect. Iridorchis and comprised a clade with Cymbidium tracyanum.

Characterization of the complete chloroplast genome of a hybrid species Cymbidium tracyanum var 'huanghua' × Cymbidium faberi cv 'huanghui'.

Cymbidium tracyanumvar 'huanghua' × Cymbidium fabericv 'huanghui' is a hybrid species with important ornamental value. In the research, Illumina high-throughput sequencing technologies was used to sequence the chloroplastic genome of Cymbidium tracyanumvar 'huanghua' × C. fabericv 'huanghui'. The genome features of Cymbidium tracyanumvar 'huanghua' × C. fabericv 'huanghui' and the phylogenetic relationships were reported and established. The total length of the complete chloroplast genome is 154,741 bp, consisting of a pair of inverse duplication regions 26,968 bp, a large single-copy region 84,410 bp and a small single-copy region 16,395 bp. The complete genome contains 73 protein-coding genes (PCGs), The entire genome contains 73 genes that encode proteins, 30 tRNA genes and 4 rRNA genes. The phylogenetic tree revealed that Cymbidium tracyanumvar 'huanghua' × C. fabericv 'huanghui' is more closely related to Cymbidium erythraeum.

Characterization of the complete chloroplast genome of a Chinese endangered species Cymbidium mannii.

Cymbidium mannii is an endangered species belonging to the first ranking in protection category in China with important ornamental value and breeding value. This study used Illumina high-throughput sequencing technologies to sequence and analyze the complete chloroplast genome of C. mannii. The genome features of C. mannii and the phylogenetic relationships among Orchidaceae species were reported and established. The complete chloroplast genome is 152,544 bp in length, consisting of a pair of inverse duplication regions 25,357 bp, a large single-copy region 86,014 bp and a small single-copy region 15,816 bp. The entire genome contains 74 mRNA genes, 30 tRNA genes and 4 rRNA genes. The phylogenetic tree of 23 Orchidaceae species revealed C. mannii is more closely related to Cymbidium aloifolium.

Characterization of the complete chloroplast genome of the endangered Chinese species Cymbidium lowianum (Rchb.f.) Rchb.f.

Cymbidium lowianum (Rchb.f.) Rchb.f. is a Class I endangered species in China with important ornamental, economic, and breeding value, especially wild C. lowianum. This study used Illumina high-throughput sequencing technologies to sequence and analyze the complete chloroplast genome of C. lowianum. The genome features of C. lowianum and the phylogenetic relationships among Orchidaceae species were reported and established. The complete chloroplast genome is 155,447 bp long, consisting of a pair of inverse duplication regions that are 26,710 bp long, a large single-copy region of 84,184 bp, and a small single-copy region of 17,843 bp. The entire genome contains 74 mRNA genes, 30 tRNA genes, and 4 rRNA genes. The phylogenetic tree of 24 Orchidaceae species revealed Cymbidium lowianum is more closely related to Cymbidium erythraeum.

Enantioselective Olfactory Effects of the Neonicotinoid Dinotefuran on Honey Bees (Apis mellifera L.).

Sublethal exposure to neonicotinoids affects honey bee olfaction, but few studies have investigated the sublethal effects of the enantioselective neonicotinoid dinotefuran on honey bee olfaction. This study assessed the sublethal olfactory toxicity of dinotefuran enantiomers to honey bees. Compared to R-dinotefuran, S-dinotefuran had higher acute oral toxicity, sucrose sensitivity effects, octopamine concentrations, lower learning ability, and memory effects on honey bees. High-throughput circular RNA sequencing of the honey bee brain revealed that R-dinotefuran caused more gene regulatory changes than S-dinotefuran. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that the SERCA, Kca, and Maxik genes may be related to the enantioselective effects of dinotefuran isomers on honey bee olfaction. These results indicated that the current ecotoxicological safety knowledge about chiral dinotefuran effects on honey bees should be amended.

Combined toxicity of chlorantraniliprole, lambda-cyhalothrin, and imidacloprid to the silkworm Bombyx mori (Lepidoptera: Bombycidae).

Insecticides with different modes of action may act in combination, in ways such as drifting, spray equipment residual, or utilizing concurrently in mulberry orchards or nearby agricultural fields. Silkworms may suffer from a diverse impact on the survival. In this study, the toxicity of chlorantraniliprole, lambda-cyhalothrin, and imidacloprid and their combinations to the second instar of silkworms (Bombyx mori (L.)(Lepidoptera: Bombycidae)) were evaluated after 48 and 72 h treatment by the leaf-dipping method and the combination index (CI)-isobologram equation. After 48 h treatment, results indicated that (1) the increasing order of toxicity was imidacloprid < chlorantraniliprole < lambda-cyhalothrin, and that (2) synergism was predominated in most combinations excepted for the lambda-cyhalothrin + imidacloprid combination which displayed an additive effect at fa value 0.5. Then, after 72 h treatment, results exhibited that (1) the increasing order of toxicity was imidacloprid < lambda-cyhalothrin < chlorantraniliprole, and that (2) only the chlorantraniliprole + imidacloprid mixture yielded antagonism at fa value 0.5; the other combinations performed an additive effect at least. Consequently, combined toxicity of mixtures may pose a worse effect on silkworm than single toxicity of insecticides. Therefore, we suggest that insecticide mixtures should be added into ecotoxicological risk assessment.

Simultaneous determination of neonicotinoid insecticides and insect growth regulators residues in honey using LC-MS/MS with anion exchanger-disposable pipette extraction.

In this study, we developed an anion exchanger-disposable pipette extraction (DPX) method to detect the residual concentrations of eight neonicotinoid insecticides (dinotefuran, acetamiprid, clothianidin, thiacloprid, imidachloprid, imidaclothiz, nitenpyram, and thiamethoxam) and eight insect growth regulators (IGRs; triflumuron, cyromazine, buprofezin, methoxyfenozide, tebufenozide, chromafenozide, fenoxycarb, and RH 5849) in Chinese honey samples collected from different floral sources and different geographical regions using liquid chromatography tandem mass spectrometry (LC-MS/MS). QAE Sephadex A-25 was used as the anion exchanger in the DPX column for the purification and cleanup of honey samples. Analytes were eluted with a mixture of acetonitrile and 0.1 M HCl, and the elution was subjected to LC analysis. This method was thoroughly validated for its reproducibility, linearity, trueness, and recovery. Satisfactory recovery of pesticides was obtained ranging from 72% to 111% with intraday RSDs (n = 5) of 1%-10%. High linearity (R2 ≥ 0.9987) was observed for all 16 pesticides. Limits of detection and quantification for all 16 compounds ranged from 0.3 to 3 µg/kg and from 1 to 10 µg/kg, respectively. Pesticide residues (9-113 µg/kg) were found in Chinese honey samples. The anion exchanger-DPX method was effective for removing sugars and retaining target analytes. Moreover, this method was highly reliable and sensitive for detecting neonicotinoids and IGRs in different floral sources of honey and will be applicable to matrixes with high sugar content.

Corrigendum: Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale.

[This corrects the article on p. 2036 in vol. 7, PMID: 28127299.].

Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale.

Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the ß-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D. officinale. These efficient research tools will not only help create novel D. officinale varieties, but will also facilitate the molecular genetic investigation of orchid biology.

Out of the Water: Origin and Diversification of the LBD Gene Family.

LBD (lateral organ boundaries domain) genes are essential to the developmental programs of many fundamental plant organs and function in some of the basic metabolic pathways of plants. However, our historical perspective on the roles of LBD genes during plant evolution has, heretofore, been fragmentary. Here, we show that the LBD gene family underwent an initial radiation that established five gene lineages in the ancestral genome of most charophyte algae and land plants. By inference, the LBD gene family originated after the emergence of the green plants (Viridiplantae), but prior to the diversification of most extant streptophytes. After this initial radiation, we find limited instances of gene family diversification in land plants until successive rounds of expansion in the ancestors of seed plants and flowering plants. The most dynamic phases of LBD gene evolution, therefore, trace to the aquatic ancestors of embryophytes followed by relatively recent lineage-specific expansions on land.

Dechlorination of chlorobenzene in subcritical water with Fe/ZrO2, Ni/ZrO2 and Cu/ZrO2.

To obtain information about dechlorination of organochlorine compounds in subcritical water catalyzed by metals assisted with ZrO(2), dechlorination of chlorobenzene has been investigated in the presence of Fe/ZrO(2), Ni/ZrO(2) and Cu/ZrO(2) catalysts. The dechlorination efficiency was increased with increasing residence time, temperature and pressure. The order of effectiveness of the catalysts was Cu/ZrO(2) < Ni/ZrO(2) < Fe/ZrO(2). The dechlorination of chlorobenze obeyed pseudo-first-order kinetics models. The rate constants in subcritical water were much greater than that in ambient-temperature water; the activation energies were obtained. ZrO(2) in the catalyst had the power to absorb chlorobenzene onto the catalyst surface and promoted the dechlorination ability of the metal. The primary mechanism for dechlorinaton of chlorobenzene involved the reduction of chlorobenzene by reaction with nascent hydrogen. The nascent hydrogen reacted with the chlorobenzene, which adsorbed on the catalyst in non-planar and co-planar form, and formed benzene and chloride ions.

Identification and characterization of differentially expressed genes involved in pharmacological activities of roots of Panax notoginseng during plant growth.

Panax notoginseng is a highly valued Chinese medicinal herb. To understand the molecular mechanism of the much higher pharmacological activities of roots of 3-year-old plants over 1-year-old ones,two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) method. Positive cDNA clones from each of the two libraries were randomly selected for dot-blotting analysis. A total of 110 genes were highly expressed in 3-year-old roots and 80 genes in 1-year-old roots. Of these, 87 cDNA fragments were sequenced, assembled, and compared with sequences in GenBank, and 81 individual cDNAs were identified. These cDNAs were the first expressed sequence tags of P. notoginseng in GenBank. The result of reverse transcription PCR analysis of six genes was consistent with that of the dot-blot analysis. The global gene expression profile showed that there were significant differences between 1- and 3-year-old roots of P. notoginseng plants. Some important structural and regulatory genes which may be involved in isoprenoid biosynthesis were found to be over-expressed in 3-year-old roots, such as genes encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphat-synthase (IspG-protein), multi-copper oxidase type I family protein, NADH flavin oxidoreductase, lipase and aconitase.

Molecular cloning and characterization of the gene encoding squalene epoxidase in Panax notoginseng.

Squalene epoxidase (SE) is one of the rate-limiting enzymes in the triterpene saponins biosynthetic pathway. Panax notoginseng, one of the famous medicinal plants in China, produces bioactive triterpene saponins. Here we report the P. notoginseng SE, which was cloned from the root of P. notoginseng by PCR. The nucleotide sequence of the ORF (GenBank accession no. DQ386734) contains 1611 nucleotides and encodes 537 amino acid residues with molecular weight of 59.14 kDa and pI of 8.81. The gene has 98% identity with P. ginseng but different identities with other SE families. P. notoginseng SE has a FAD function domain, NAD(P)-binding Rossmann-fold domains, hydrophobicity and 4 transmembrane helices. This SE may be a microsomal membrane-associated enzyme. Real time quantitative PCR shows that the cDNA has different expression pattern and is highly expressed in root, especially in 3-year-old root.