Selection of Reference Genes for Expression Normalization by RT-qPCR in Dracocephalum moldavica L.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes' stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.

Comparative transcriptome analyses of three Gentiana species provides signals for the molecular footprints of selection effects and the phylogenetic relationships.

Species in Gentiana section Cruciata are important alpine plants with a center of diversity and speciation in Qinghai-Tibet Plateau (QTP), and some of these species are sympatrically distributed in northeastern QTP. Studies on genome features and natural selection signatures of sympatric species in section Crucata have been impeded by a lack of genomic resources. Here, we showed transcript characterizations and molecular footprints of selection effects on G. straminea, G. dahurica and G. officinalis based on the comparative transcriptome. A total of 62.97 Gb clean reads were obtained with unigene numbers per species ranging from 141,819 to 236,408 after assembly. We found that these three species had similar distribution of functional categories in different databases, and key enzyme-encoding genes involved in the iridoids biosynthesis were also obtained. The selective pressure analyses indicated that most paired orthologs between these three species were subject to negative selection, and only a low proportion of the orthologs that underwent positive selection were detected. We found that some positive selected genes were involved in "catalytic activity", "metabolic process", "response to stimulus" and "response to stress". Besides, large numbers of SSR primer pairs with transferabilities were successfully designed based on the available transcriptome datasets of three Gentiana species. The phylogenetic relationships reconstructed based on 352 single-copy nuclear genes provided a rough phylogenetic framework for this genus and confirmed the monophyly of section Cruciata. Our study not only provides insights for the natural selection effects on sympatric Gentiana species, but also enhances future genetic breeding or evolutionary studies on Qinjiao species.

Full-length transcriptome analysis and identification of genes involved in asarinin and aristolochic acid biosynthesis in medicinal plant Asarum sieboldii.

Asarum sieboldii, a well-known traditional Chinese medicinal herb, is used for curing inflammation and ache. It contains both the bioactive ingredient asarinin and the toxic compound aristolochic acid. To address further breeding demand, genes involved in the biosynthetic pathways of asarinin and aristolochic acid should be explored. Therefore, the full-length transcriptome of A. sieboldii was sequenced using PacBio Iso-Seq to determine the candidate transcripts that encode the biosynthetic enzymes of asarinin and aristolochic acid. In this study, 63 023 full-length transcripts were generated with an average length of 1371 bp from roots, stems, and leaves, of which 49 593 transcripts (78.69%) were annotated against public databases. Furthermore, 555 alternative splicing (AS) events, 10 869 long noncoding RNAs (lncRNAs) as well as their 11 291 target genes, and 17 909 simple sequence repeats (SSRs) were identified. The data also revealed 97 candidate transcripts related to asarinin metabolism, of which six novel genes that encoded enzymes involved in asarinin biosynthesis were initially reported. In addition, 56 transcripts related to aristolochic acid biosynthesis were also identified, including CYP81B. In summary, these transcriptome data provide a useful resource to study gene function and genetic engineering in A. sieboldii.

Transcriptome Sequencing and Development of Genic SSR Markers of an Endangered Chinese Endemic Genus Dipteronia Oliver (Aceraceae).

Dipteronia Oliver (Aceraceae) is an endangered Chinese endemic genus consisting of two living species, Dipteronia sinensis and Dipteronia dyeriana. However, studies on the population genetics and evolutionary analyses of Dipteronia have been hindered by limited genomic resources and genetic markers. Here, the generation, de novo assembly and annotation of transcriptome datasets, and a large set of microsatellite or simple sequence repeat (SSR) markers derived from Dipteronia have been described. After Illumina pair-end sequencing, approximately 93.2 million reads were generated and assembled to yield a total of 99,358 unigenes. A majority of these unigenes (53%, 52,789) had at least one blast hit against the public protein databases. Further, 12,377 SSR loci were detected and 4179 primer pairs were designed for experimental validation. Of these 4179 primer pairs, 435 primer pairs were randomly selected to test polymorphism. Our results show that products from 132 primer pairs were polymorphic, in which 97 polymorphic SSR markers were further selected to analyze the genetic diversity of 10 natural populations of Dipteronia. The identification of SSR markers during our research will provide the much valuable data for population genetic analyses and evolutionary studies in Dipteronia.

Isolation and characterization of polymorphic microsatellite loci in the endangered plant Dipteronia sinensis (Sapindaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in Dipteronia sinensis to investigate the population genetics of this endangered plant. • METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, 19 microsatellite loci were developed in D. sinensis and evaluated for their variability in 29 samples from a natural population. For the 15 polymorphic loci, the number of alleles ranged from nine to 33, while the observed and expected heterozygosities ranged from 0.3793 to 0.9655 and from 0.6029 to 0.9609, respectively. Their cross-taxa transferability was also investigated in Acer miaotaiense, A. palmatum, and A. pictum subsp. mono, and 10 to 15 loci proved amplifiable in these species. • CONCLUSIONS: These microsatellite markers could be employed to investigate the population genetics of D. sinensis and may potentially be applicable to other related species.

Evaluation of TPGU using entropy - improved TOPSIS - GRA method in China.

China is still one of the countries dominated by thermal power generation. In order to generate more efficient, stable and clean power, it is necessary to evaluate thermal power generation units (TPGU). Firstly, a comprehensive evaluation index system for TPGU with 20 secondary indicators was established from four aspects: reliability indicators, economic indicators, technical supervision indicators, and major operating indicators. Secondly, the entropy weight method can be used to calculate the weight of each second-level index. Mahalanobis Distance improved Technique for Order Preference by Similarity to an Ideal Solution (TOPSIS) method is coupled with the Grey Relational Analysis (GRA), and the comprehensive evaluation values of 5 units (600MW) are respectively 0.4516, 0.5247, 0.3551, 0.5589 and 0.6168 from both vertical and horizontal dimensions. Finally, by comparing and analyzing this method with the above research methods, it is found that the results obtained by this method which re-establishes the coordinate system based on the data set are more accurate. In addition, this method can effectively evaluate the operation of TPGU, which is of great significance for cleaner production while generating electricity. In conclusion, some suggestions on clean production of TPGU are put forward, and the innovation points and limitations of this paper are pointed out.

De novo genome assembly of the medicinal plant Gentiana macrophylla provides insights into the genomic evolution and biosynthesis of iridoids.

Gentiana macrophylla is a perennial herb in the Gentianaceae family, whose dried roots are used in traditional Chinese medicine. Here, we assembled a chromosome-level genome of G. macrophylla using a combination of Nanopore, Illumina, and Hi-C scaffolding approaches. The final genome size was ~1.79 Gb (contig N50 = 720.804 kb), and 98.89% of the genome sequences were anchored on 13 pseudochromosomes (scaffold N50 = 122.73 Mb). The genome contained 55,337 protein-coding genes, and 73.47% of the assemblies were repetitive sequences. Genome evolution analysis indicated that G. macrophylla underwent two rounds of whole-genome duplication after the core eudicot γ genome triplication event. We further identified candidate genes related to the biosynthesis of iridoids, and the corresponding gene families mostly expanded in G. macrophylla. In addition, we found that root-specific genes are enriched in pathways involved in defense responses, which may greatly improve the biological adaptability of G. macrophylla. Phylogenomic analyses showed a sister relationship of asterids and rosids, and all Gentianales species formed a monophyletic group. Our study contributes to the understanding of genome evolution and active component biosynthesis in G. macrophylla and provides important genomic resource for the genetic improvement and breeding of G. macrophylla.

The chromosome-level genome for Toxicodendron vernicifluum provides crucial insights into Anacardiaceae evolution and urushiol biosynthesis.

The lacquer tree (Toxicodendron vernicifluum (Stokes) F.A. Barkley) is an important tree with economic, industrial, and medicinal values. Here, we generated the reference genome of T. vernicifluum at the chromosome level with 491.93 Mb in size, in which 98.26% of the assembled contigs were anchored onto 15 pseudochromosomes with the scaffold N50 of 32.97 Mb. Comparative genomic analysis revealed the gene families related to urushiol biosynthesis were expanded, contributing to the ecological fitness and biological adaptability of the lacquer tree. We combined multi-omics data to identify genes that encode key enzymes in the T. vernicifluum urushiol and lignin biosynthetic pathways. Furthermore, the unique active metabolites, such as butin and fisetin, in cultivar lacquers were identified by metabolism profiling. Our work would provide crucial insights into metabolite synthesis such as urushiol and lignin, meanwhile offer a basis for further exploration of the cultivation and breeding of T. vernicifluum and other Anacardiaceae members.

Research on the coordination of energy in China's economic growth.

This study uses the improved Cobb-Douglas two-factor production function model to explore the potential relationship between economic growth and energy consumption through the multiple co-integration test on the panel data of China from 1985 to 2018. The results show that there is a positive long-term balance between energy consumption and economic growth: economic growth of 1%, total energy consumption growth of 1.53%, which means that economic growth needs higher energy support in the former short term. At the same time, the error correction term will converge energy consumption to a long-term equilibrium state with an adjustment intensity of 134.59%. From the results of variance decomposition, we can also see that as the number of periods increases, the part of real economic growth explained by energy consumption gradually increases.

Framework Phylogeny, Evolution and Complex Diversification of Chinese Oaks.

Oaks (Quercus L.) are ideal models to assess patterns of plant diversity. We integrated the sequence data of five chloroplast and two nuclear loci from 50 Chinese oaks to explore the phylogenetic framework, evolution and diversification patterns of the Chinese oak's lineage. The framework phylogeny strongly supports two subgenera Quercus and Cerris comprising four infrageneric sections Quercus, Cerris, Ilex and Cyclobalanopsis for the Chinese oaks. An evolutionary analysis suggests that the two subgenera probably split during the mid-Eocene, followed by intergroup divergence within the subgenus Cerris around the late Eocene. The initial diversification of sections in the subgenus Cerris was dated between the mid-Oligocene and the Oligocene-Miocene boundary, while a rapid species radiation in section Quercus started in the late Miocene. Diversification simulations indicate a potential evolutionary shift on section Quercus, while several phenotypic shifts likely occur among all sections. We found significant negative correlations between rates of the lineage diversification and phenotypic turnover, suggesting a complex interaction between the species evolution and morphological divergence in Chinese oaks. Our infrageneric phylogeny of Chinese oaks accords with the recently proposed classification of the genus Quercus. The results point to tectonic activity and climatic change during the Tertiary as possible drivers of evolution and diversification in the Chinese oak's lineage.

Characterization of the complete chloroplast genome of Abies chensiensis (Pinaceae), an endemic to China.

Abies chensiensis, an endemic evergreen trees distributed in Qinling Mountains, is listed in China Species Red List as an endangered species. In this study, the complete chloroplast genome (cpDNA) sequence of Abies chensiensis was determined from Illumina pair-end sequencing data. The cpDNA is 121,329 bp in length, contains a large single copy region (LSC) of 72,843 bp and a small single copy region (SSC) of 46,126 bp, which were separated by a pair of inverted repeat (IR) regions of 1180 bp. The genome contains 120 genes, including 81 protein-coding genes, 4 ribosomal RNA genes, and 35 transfer RNA genes. The overall GC content of the whole genome is 38.3%, and the corresponding values of the LSC, SSC, and IR regions are 38.8, 37.1, and 37.5%, respectively. Phylogenetic analysis based on 20 chloroplast genomes indicates that A. chensiensis is closely related to A. sibirica.

Complete chloroplast genome sequence of Fagopyrum dibotrys: genome features, comparative analysis and phylogenetic relationships.

Fagopyrum dibotrys, belongs to Polygonaceae family, is one of national key conserved wild plants of China with important medicinal and economic values. Here, the complete chloroplast (cp) genome sequence of F. dibotrys is reported. The cp genome size is 159,919 bp with a typical quadripartite structure and consisting of a pair of inverted repeat regions (30,738 bp) separated by large single copy region (85,134 bp) and small single copy region (13,309 bp). Sequencing analyses indicated that the cp genome encodes 131 genes, including 80 protein-coding genes, 28 tRNA genes and 4 rRNA genes. The genome structure, gene order and codon usage are typical of angiosperm cp genomes. We also identified 48 simple sequence repeats (SSR) loci, fewer of them are distributed in the protein-coding sequences compared to the noncoding regions. Comparison of F. dibotrys cp genome to other Polygonaceae cp genomes indicated the inverted repeats (IRs) and coding regions were more conserved than single copy and noncoding regions, and several variation hotspots were detected. Coding gene sequence divergence analyses indicated that five genes (ndhK, petL rpoC2, ycf1, ycf2) were subject to positive selection. Phylogenetic analysis among 42 species based on cp genomes and 50 protein-coding genes indicated a close relationship between F. dibotrys and F. tataricum. In summary, the complete cp genome sequence of F. dibotrys reported in this study will provide useful plastid genomic resources for population genetics and pave the way for resolving phylogenetic relationships of order Caryophyllales.

Characterization of the complete chloroplast genome of Fraxinus mandshurica (Oleaceae).

The whole chloroplast (cp) genome sequence of Fraxinus mandshurica has been characterized from Illumina pair-end sequencing. The complete cp genome was 155,530 bp in length, containing a large single copy region (LSC) of 86,415 bp and a small single copy region (SSC) of 19,279 bp, which were separated by a pair of inverted repeat (IR) regions of 25,653 bp. The genome contained 133 genes, including 88 protein-coding genes, 37 tRNA genes and eight ribosomal RNA genes (four rRNA species). Most genes occur as a single copy, while 19 gene species are duplicated. phylogenetic analysis revealed that F. mandshurica is closely related to the species of F. chiisanensis and F. excelsior.

The complete chloroplast genome sequence of Abies chensiensis (Pinaceae: Abietoideae), an endangered species endemic to China.

Chloroplast (cp) genome sequences became a widely used tool for evolutionary and phylogenetic studies in plants. Abies chensiensis (Pinaceae: Abietoideae) is an endangered species endemic to China. To understand its evolutionary characteristics, the complete chloroplast genome of the A. chensiensis has been reconstructed from the whole-genome Illumina sequencing data. The circular genome is 121,784 bp in length and without a typical quadripartite structure due to the loss of IR region. The total GC content of whole genome sequence is 38.3%. The chloroplast genome encodes 109 genes, including 75 protein-coding genes, 30 transfer RNA genes and four ribosomal RNA genes. Among them, 35 genes involved in photosynthesis, while 58 genes involved in self-replication. The Maximum-Likelihood phylogenetic analysis showed a strong sister relationship with A. nephrolepis and A. koreana in Abietoideae. Our findings provide fundamental information for further evolutionary and phylogenetic researches of Abietoideae.

Plastid Genome Comparative and Phylogenetic Analyses of the Key Genera in Fagaceae: Highlighting the Effect of Codon Composition Bias in Phylogenetic Inference.

Fagaceae is one of the largest and economically important taxa within Fagales. Considering the incongruence among inferences from plastid and nuclear genes in the previous Fagaceae phylogeny studies, we assess the performance of plastid phylogenomics in this complex family. We sequenced and assembled four complete plastid genomes (Fagus engleriana, Quercus spinosa, Quercus aquifolioides, and Quercus glauca) using reference-guided assembly approach. All of the other 12 published plastid genomes in Fagaceae were retrieved for genomic analyses (including repeats, sequence divergence and codon usage) and phylogenetic inference. The genomic analyses reveal that plastid genomes in Fagaceae are conserved. Comparing the phylogenetic relationships of the key genera in Fagaceae inferred from different codon positions and gene function datasets, we found that the first two codon sites dataset recovered nearly all relationships and received high support. Thus, the result suggested that codon composition bias had great influence on Fagaceae phylogenetic inference. Our study not only provides basic understanding of Fagaceae plastid genomes, but also illuminates the effectiveness of plastid phylogenomics in resolving relationships of this intractable family.

Clinical Staphylococcus argenteus Develops to Small Colony Variants to Promote Persistent Infection.

Staphylococcus argenteus is a novel staphylococcal species (also considered as a part of Staphylococcus aureus complex) that is infrequently reported on, and clinical S. argenteus infections are largely unstudied. Here, we report a persistent and recurrent hip joint infection case in which a S. argenteus strain and its small colony variants (SCVs) strain were successively isolated. We present features of the two S. argenteus strains and case details of their pathogenicity, explore factors that induce S. argenteus SCVs formation in the course of anti-infection therapy, and reveal potential genetic mechanisms for S. argenteus SCVs formation. S. argenteus strains were identified using phenotypic and genotypic methods. The S. argenteus strain XNO62 and SCV strain XNO106 were characterized using different models. S. argenteus SCVs were induced by the administration of amikacin and by chronic infection course based on the clinical case details. The genomes of both strains were sequenced and aligned in a pair-wise fashion using Mauve. The case details gave us important insights on the characteristics and therapeutic strategies for infections caused by S. argenteus and its SCVs. We found that strain XNO62 and SCV strain XNO106 are genetically-related sequential clones, the SCV strain exhibits reduced virulence but enhanced intracellular persistence compared to strain XNO62, thus promoting persistent infection. The induction experiments for S. argenteus SCVs demonstrated that high concentrations of amikacin greatly induce S. argenteus XNO62 to form SCVs, while a chronic infection of S. argenteus XNO62 slightly induces SCVs formation. Potential genetic mechanisms for S. argenteus SCVs formation were revealed and discussed based on genomic alignments. In conclusion, we report the first case of infection caused by S. argenteus and its SCVs strain. More attention should be paid to infections caused by S. argenteus and its SCVs, as they constitute a challenge to current therapeutic strategies. The problem of S. argenteus SCVs should be noticed, in particular when amikacin is used or in the case of a chronic S. argenteus infection.

Completion of Eight Gynostemma BL. (Cucurbitaceae) Chloroplast Genomes: Characterization, Comparative Analysis, and Phylogenetic Relationships.

Gynostemma BL., belonging to the family Cucurbitaceae, is a genus containing 17 creeping herbaceous species mainly distributed in East Asia. It can be divided into two subgenera based on different fruit morphology. Herein, we report eight complete chloroplast genome sequences of the genus Gynostemma, which were obtained by Illumina paired-end sequencing, assembly, and annotation. The length of the eight complete cp genomes ranged from 157,576 bp (G. pentaphyllum) to 158,273 bp (G. laxiflorum). Each encoded 133 genes, including 87 protein-coding genes, 37 tRNA genes, eight rRNA genes, and one pseudogene. The four types of repeated sequences had been discovered and indicated that the repeated structure for species in the Subgen. Triostellum was greater than that for species in the Subgen. Gynostemma. The percentage of variation of the eight cp genomes in different regions were calculated, which demonstrated that the coding and inverted repeats regions were highly conserved. Phylogenetic analysis based on Bayesian inference and maximum likelihood methods strongly supported the phylogenetic position of the genus Gynostemma as a member of family Cucurbitaceae. The phylogenetic relationships among the eight species were clearly resolved using the complete cp genome sequences in this study. It will also provide potential molecular markers and candidate DNA barcodes for future studies and enrich the valuable complete cp genome resources of Cucurbitaceae.

The complete chloroplast genome of Kolkwitzia amabilis (Caprifoliaceae), an endangered horticultural plant in China.

The complete chloroplast genome of Kolkwitzia amabilis (Caprifoliaceae) is first presented in the current study. The cp genome of K. amabilis was 156 875 bp in length and composed of two short inverted repeat (IRa and IRb) regions of 23 946 bp which were separated by a small single copy (SSC) region of 18 846 bp and a large single copy (LSC) region of 90 137 bp. The genome encoded 130 genes contained 81 coding genes, 39 tRNA genes, and 8 rRNA genes. The overall AT content of K. amabilis is 61.6% and the corresponding values of the SSC, LSC and IR regions are 66.8%, 64.1%, and 57.3%, respectively. The phylogenetic analysis based on the maximum parsimony tree revealed that K. amabilis was closely related to Lonicera japonica.

The complete chloroplast genome of Dipteronia sinensis (Aceraceae), an endangered endemic species to China.

The complete chloroplast sequence of D. sinensis was reported in this study. The total length was 157 080 bp containing a pair of 26 766 bp inverted repeat regions (IRa and IRb), which were separated by a small single copy regions and a large single copy regions (SSC and LSC) of 18 093 and 85 455 bp, respectively. A total of 138 functional genes were annotated, which included 90 protein-coding genes, 40 tRNAs, and eight rRNA genes. The overall GC content of the complete chloroplast genome is 37.8% and in LSC, SSC and IR regions were 35.9%, 32.1%, and 42.7%, respectively. The maximum likelihood (ML) phylogenetic analysis revealed that D. sinensis was closely related to A. buergerianum subsp. Ningpoense in Sapindales order.

Comparative Transcriptome and Chloroplast Genome Analyses of Two Related Dipteronia Species.

Dipteronia (order Sapindales) is an endangered genus endemic to China and has two living species, D.sinensis and D. dyeriana. The plants are closely related to the genus Acer, which is also classified in the order Sapindales. Evolutionary studies on Dipteronia have been hindered by the paucity of information on their genomes and plastids. Here, we used next generation sequencing to characterize the transcriptomes and complete chloroplast genomes of both Dipteronia species. A comparison of the transcriptomes of both species identified a total of 7814 orthologs. Estimation of selection pressures using Ka/Ks ratios showed that only 30 of 5435 orthologous pairs had a ratio significantly >1, i.e., showing positive selection. However, 4041 orthologs had a Ka/Ks < 0.5 (p < 0.05), suggesting that most genes had likely undergone purifying selection. Based on orthologous unigenes, 314 single copy nuclear genes (SCNGs) were identified. Through a combination of de novo and reference guided assembly, plastid genomes were obtained; that of D. sinensis was 157,080 bp and that of D. dyeriana was 157,071 bp. Both plastid genomes encoded 87 protein coding genes, 40 tRNAs, and 8 rRNAs; no significant differences were detected in the size, gene content, and organization of the two plastomes. We used the whole chloroplast genomes to determine the phylogeny of D. sinensis and D. dyeriana and confirmed that the two species were highly divergent. Overall, our study provides comprehensive transcriptomic and chloroplast genomic resources, which will be valuable for future evolutionary studies of Dipteronia.