Using UPLC-LTQ-Orbitrap-MS and HPLC-CAD to Identify Impurities in Cycloastragenol, Which Is a Pre-Clinical Candidate for COPD.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent disease that has become the third leading cause of death worldwide. Cycloastragenol (CAG), which is the genuine sapogenin of the main active triterpene saponins in Astragali radix, is a bioavailable pre-clinical candidate for chronic obstructive pulmonary disease (COPD), and it was investigated in our previous study. In order to progress medical research, it was first efficiently produced on a 2.5-kg scale via Smith degradation from astragaloside IV (AS-IV). Simultaneously, since the impurity profiling of a drug is critical for performing CMC documentation in pre-clinical development, a study on impurities was carried out. As these structures do not contain chromophores and possess weak UV absorption characteristics, HPLC-CAD and UPLC-LTQ-Orbitrap-MS were employed to carry out the quality control of the impurities. Then, column chromatography (CC), preparative thin-layer chromatography (PTLC), and crystallization led to the identification of 15 impurities from CAG API. Among these impurities, compounds 1, 4, 9, 10, 14, and 15 were elucidated via spectroscopic analysis, and 2-3, 5-8, and 11-13 were putatively identified. Interestingly, the new compounds 9 and 14 were rare 10, 19-secocycloartane triterpenoids that displayed certain anti-inflammatory activities against LPS-induced lymphocyte cells and CSE-induced MLE-12 cells. Additionally, a plausible structural transformation pathway of the degradation compounds from CAG or AS IV was proposed. The information obtained will provide a material basis to carry out the quality control and clinical safety assurance of API and related prescriptions. Reasonable guidance will also be provided regarding the compounds with weak UV absorption characteristics.

Paenibacillus turpanensis sp. nov., isolated from a salt lake of Turpan city in Xinjiang province, north-west China.

Strain YIM B00363T, a Gram-positive, aerobic, non-motile, rod-shaped, spore-forming bacterium, was isolated from saline soil samples collected from a salt lake in Xinjiang province, north-west China, and was characterized using a polyphasic approach. The optimum growth temperature was 37 °C and the optimum pH was 7.5-8.0. The major menaquinone was MK-7; anteiso-C15:0 (53.52%), iso-C15:0 (15.04%) and C16:0 (12.76%) were the predominant cellular fatty acids. The diagnostic diamino acid of the cell wall peptidoglycan was meso-diaminopimelic acid. The phospholipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, unidentified glycolipids and unknown lipids. The DNA G + C content of the type strain was 50.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain YIM B00363T belonged to a cluster comprising species of the genus Paenibacillus. The nearest relatives were P. residui MC-246T and P. senegalensis JC66T, with 93.2% and 92.8% gene sequence similarities, respectively. On the basis of its phenotypic characteristics and phylogenetic distinctivenes, strain YIM B00363T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus turpanensis sp. nov. is proposed. The type strain is YIM B00363T (= CGMCC 1.17507T = KCTC 43184T).

Evolutionary legacy of a forest plantation tree species (Pinus armandii): Implications for widespread afforestation.

Many natural systems are subject to profound and persistent anthropogenic influence. Human-induced gene movement through afforestation and the selective transportation of genotypes might enhance the potential for intraspecific hybridization, which could lead to outbreeding depression. However, the evolutionary legacy of afforestation on the spatial genetic structure of forest tree species has barely been investigated. To do this properly, the effects of anthropogenic and natural processes must be examined simultaneously. A multidisciplinary approach, integrating phylogeography, population genetics, species distribution modeling, and niche divergence would permit evaluation of potential anthropogenic impacts, such as mass planting near-native material. Here, these approaches were applied to Pinus armandii, a Chinese endemic coniferous tree species, that has been mass planted across its native range. Population genetic analyses showed that natural populations of P. armandii comprised three lineages that diverged around the late Miocene, during a period of massive uplifts of the Hengduan Mountains, and intensification of Asian Summer Monsoon. Only limited gene flow was detected between lineages, indicating that each largely maintained is genetic integrity. Moreover, most or all planted populations were found to have been sourced within the same region, minimizing disruption of large-scale spatial genetic structure within P. armandii. This might be because each of the three lineages had a distinct climatic niche, according to ecological niche modeling and niche divergence tests. The current study provides empirical genetic and ecological evidence for the site-species matching principle in forestry and will be useful to manage restoration efforts by identifying suitable areas and climates for introducing and planting new forests. Our results also highlight the urgent need to evaluate the genetic impacts of large-scale afforestation in other native tree species.

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.

Spatial Genetic Structure and Demographic History of the Dominant Forest Oak Quercus fabri Hance in Subtropical China.

Oak trees (Quercus L.) are important models for estimating abiotic impacts on the population structure and demography of long life span tree species. In this study, we generated genetic data for 17 nuclear microsatellite loci in 29 natural populations of Quercus fabri to estimate the population genetic structure. We also integrated approximate Bayesian computation (ABC) and ecological niche analysis to infer the population differentiation processes and demographic history of this oak species. The genetic analyses indicated two genetic clusters across the 29 populations collected, where most approximately corresponded to the intraspecific differentiation among populations from western and eastern China, whereas admixed populations were mainly found in central mountains of China. The best model obtained from hierarchical ABC simulations suggested that the initial intraspecific divergence of Q. fabri potentially occurred during the late Pliocene (ca. 3.99 Ma) to form the two genetic clusters, and the admixed population group might have been generated by genetic admixture of the two differentiated groups at ca. 53.76 ka. Ecological analyses demonstrated clear differentiation among the Q. fabri population structures, and association estimations also indicated significant correlations between geography and climate with the genetic variation in this oak species. Our results suggest abiotic influences, including past climatic changes and ecological factors, might have affected the genetic differentiation and demographic history of Q. fabri in subtropical China.

Evaluating Population Genetic Structure and Demographic History of Quercus spinosa (Fagaceae) Based on Specific Length Amplified Fragment Sequencing.

Effectively identifying the genetic structure and related factors of a species can facilitate understanding the evolutionary history of the species. Phylogeographic patterns and genetic data are essential in investigating the species historical processes and diversification that response to environmental, climatic and geological influences. In this study, Specific Length Amplified Fragment Sequencing (SLAF-seq) data and ecological niche models (ENMs) are combined to identify the genetic structure and demographic modeling of Quercus spinosa, and evaluate the impacts of historical range shifts, climatic variation, and landscape factors on this species. The population topology and genetic divergence of the Cenozoic were inferred by a site frequency spectrum based composite-likelihood approach which is a novel strategy for maximizing the utility of linked SLAF markers. The overall genetic structure using model-based and model-free clustering methods was consistently identified as two geographically distinct genetic clusters. A deep divergence between two natural lineages (i.e., a western Himalaya-Hengduan Mountains lineage and an eastern Qin-ling Mountains lineage) was observed. The demographic modeling and Niche reconstruction indicated that the two groups were diverged in the late Miocene and then presented as two distinct genetic lineages. With the Quaternary glacial climate fluctuation, two groups had continuous asymmetrical secondary contact and gene exchange in the Sichuan Basin during the last glacial maximum. Besides, a significant relationship between genetic distance and geography in all individuals was identified by the Mantel test. Overall, this study 1) contributes to a better understanding of the role played by Quaternary climatic fluctuation in the present-day distributions of Q. spinosa; 2) provides a comprehensive view of the genome-wide variation of sclerophyllous forests in ecological adaptive evolution; 3) indicates that dispersal limitation and ecological divergence contribute to the genome-wide differentiation of Q. spinosa, which supports a hypothesis that complex geography and climatic changes strongly influence the evolutionary origin and history of the species.

Species delimitation and interspecific relationships of the endangered herb genus Notopterygium inferred from multilocus variations.

Species identification and discrimination is the basis of biodiversity research. In general, it is considered that numerous nucleotide variations (e.g., whole chloroplast genomes) can identify species with higher resolution than a few loci, e.g., partial chloroplast or nuclear gene fragments. In this study, we tested this hypothesis by sampling population genetics samples of the endangered herb genus Notopterygium. We sequenced the complete plastomes, five nuclear gene regions, three chloroplast DNA fragments, and a nuclear internal transcribed spacer (nrITS) region for 18 populations sampled throughout most of the geographic ranges of all six Notopterygium species. Species identification analysis showed that four DNA barcodes (matK, rbcL, trnS-trnG, and nrITS) and/or combinations of these markers achieved Notopterygium species discrimination at higher resolution than the general plastomes and nuclear gene sequences. In particular, nrITS had the highest discriminatory power among all of the individual markers. Molecular data sets and morphological evidence indicated that all six Notopterygium species could be reclassified unambiguously to four putative species clades. N. oviforme and N. franchetii had the closest relationship. Molecular dating showed that the origin and divergence of Notopterygium species was significantly associated with geological and climatic fluctuations during the middle of the Pliocene. In conclusion, our results suggest that a few nucleotide variations can achieve species discrimination with higher resolution than numerous plastomes and general nuclear gene fragments when discerning related Notopterygium species.

Effects of Geological and Environmental Events on the Diversity and Genetic Divergence of Four Closely Related Pines: Pinus koraiensis, P. armandii, P. griffithii, and P. pumila.

The effects of mountain uplift and environmental oscillations on nucleotide variability and species divergence remain largely unknown in East Asia. In this study, based on multiple nuclear DNA markers, we investigated the levels and patterns of nucleotide diversity and interspecific divergence in four closely related pines in China, i.e., Pinus koraiensis, P. armandii, P. griffithii, and P. pumila. The four pine taxa shared low levels of nucleotide polymorphisms at the species level. P. pumila had the highest silent nucleotide diversity (πsil = 0.00661) whereas P. griffithii had the lowest (πsil = 0.00175), while the levels of genetic polymorphism in P. armandii (πsil = 0.00508) and P. koraiensis (πsil = 0.00652) were intermediate between the other two species. Population genetic structure analysis showed that variations primarily existed within populations of the four pine species, presumably due to habitat fragmentation or the island-like distributions of Pinus species. Population divergence (FST) analysis showed that the genetic divergence between P. griffithii and P. koraiensis was much greater than that between P. koraiensis and the other two pines species. Isolation-with-migration analysis suggested that asymmetric gene flow had occurred between any two pairs of pine species. Phylogenetic analyses indicated that the four allied species split into two groups about 1.37 million years ago, where P. armandii and P. pumila were closer and clustered as sister species, whereas P. koraiensis and P. griffithii were clustered on another branch. Our results and those obtained in previous studies suggest that mountain uplift and geological climate oscillations may have led to the patterns of genetic divergence and nucleotide variations in these four pine species.

Functional Genetic Diversity and Culturability of Petroleum-Degrading Bacteria Isolated From Oil-Contaminated Soils.

In this study, we compared the culturability of aerobic bacteria isolated from long-term oil-contaminated soils via enrichment and direct-plating methods; bacteria were cultured at 30°C or ambient temperatures. Two soil samples were collected from two sites in the Shengli oilfield located in Dongying, China. One sample (S0) was close to the outlet of an oil-production water treatment plant, and the other sample (S1) was located 500 m downstream of the outlet. In total, 595 bacterial isolates belonging to 56 genera were isolated, distributed in Actinobacteria, Firmicutes, Bacterioidetes, and Proteobacteria. It was interesting that Actinobacteria and Firmicutes were not detected from the 16S rRNA gene clone library. The results suggested the activation of rare species during culture. Using the enrichment method, 239 isolates (31 genera) and 96 (22 genera) isolates were obtained at ambient temperatures and 30°C, respectively, from S0 soil. Using the direct-plating method, 97 isolates (15 genera) and 163 isolates (20 genera) were obtained at ambient temperatures and 30°C, respectively, from two soils. Of the 595 isolates, 244 isolates (41.7% of total isolates) could degrade n-hexadecane. A greater number of alkane-degraders was isolated at ambient temperatures using the enrichment method, suggesting that this method could significantly improve bacterial culturability. Interestingly, the proportion of alkane degrading isolates was lower in the isolates obtained using enrichment method than that obtained using direct-plating methods. Considering the greater species diversity of isolates obtained via the enrichment method, this technique could be used to increase the diversity of the microbial consortia. Furthermore, phenol hydroxylase genes (pheN), medium-chain alkane monooxygenases genes (alkB and CYP153A), and long-chain alkane monooxygenase gene (almA) were detected in 60 isolates (11 genotypes), 91 isolates (27 genotypes) and 93 isolates (24 genotypes), and 34 isolates (14 genotypes), respectively. This study could provide new insights into microbial resources from oil fields or other environments, and this information will be beneficial for bioremediation of petroleum contamination and for other industrial applications.

Effects of the Tanaka Line on the genetic structure of Bombax ceiba (Malvaceae) in dry-hot valley areas of southwest China.

Southwest China is an important biodiversity hotspot. The interactions among the complex topography, climate change, and ecological factors in the dry-hot valley areas in southwest China may have profoundly affected the genetic structure of plant species in this region. In this study, we determined the effects of the Tanaka Line on genetic variation in the wild Bombax ceiba tree in southwest China. We sampled 224 individuals from 17 populations throughout the dry-hot valley regions. Six polymorphic expressed sequence tag-simple sequence repeat primers were employed to sequence the PCR products using the first-generation Sanger technique. The analysis based on population genetics suggested that B. ceiba exhibited a high level of gene diversity (HE: 0.2377-0.4775; I: 0.3997-0.7848). The 17 populations were divided into two groups by cluster analysis, which corresponded to geographic characters on each side of the Tanaka Line. In addition, a Mantel test indicated that the phylogeographic structure among the populations could be fitted to the isolation-by-distance model (r2 = .2553, p < .001). A barrier test indicated that there were obstacles among populations and between the two groups due to complex terrain isolation and geographic heterogeneity. We inferred that the Tanaka Line might have promoted the intraspecific phylogeographic subdivision and divergence of B. ceiba. These results provide new insights into the effects of the Tanaka Line on genetic isolation and population differentiation of plant species in southwest China.

Population genetics, phylogenomics and hybrid speciation of Juglans in China determined from whole chloroplast genomes, transcriptomes, and genotyping-by-sequencing (GBS).

Genomic data are a powerful tool for elucidating the processes involved in the evolution and divergence of species. The speciation and phylogenetic relationships among Chinese Juglans remain unclear. Here, we used results from phylogenomic and population genetic analyses, transcriptomics, Genotyping-By-Sequencing (GBS), and whole chloroplast genomes (Cp genome) data to infer processes of lineage formation among the five native Chinese species of the walnut genus (Juglans, Juglandaceae), a widespread, economically important group. We found that the processes of isolation generated diversity during glaciations, but that the recent range expansion of J. regia, probably from multiple refugia, led to hybrid formation both within and between sections of the genus. In southern China, human dispersal of J. regia brought it into contact with J. sigillata, which we determined to be an ecotype of J. regia that is now maintained as a landrace. In northern China, walnut hybridized with a distinct lineage of J. mandshurica to form J. hopeiensis, a controversial taxon (considered threatened) that our data indicate is a horticultural variety. Comparisons among whole chloroplast genomes and nuclear transcriptome analyses provided conflicting evidence for the timing of the divergence of Chinese Juglans taxa. J. cathayensis and J. mandshurica are poorly differentiated based our genomic data. Reconstruction of Juglans evolutionary history indicate that episodes of climatic variation over the past 4.5 to 33.80 million years, associated with glacial advances and retreats and population isolation, have shaped Chinese walnut demography and evolution, even in the presence of gene flow and introgression.

Comparative Transcriptome Analysis Reveals Adaptive Evolution of Notopterygium incisum and Notopterygium franchetii, Two High-Alpine Herbal Species Endemic to China.

The extreme conditions (e.g., cold, low oxygen, and strong ultraviolet radiation) of the high mountains provide an ideal natural laboratory for studies on speciation and the adaptive evolution of organisms. Up to now, few genome/transcriptome-based studies have been carried out on how plants adapt to conditions at extremely high altitudes. Notopterygium incisum and Notopterygium franchetii (Notopterygium, Apiaceae) are two endangered high-alpine herbal plants endemic to China. To explore the molecular genetic mechanisms of adaptation to high altitudes, we performed high-throughput RNA sequencing (RNA-seq) to characterize the transcriptomes of the two species. In total, more than 130 million sequence reads, 81,446 and 63,153 unigenes with total lengths of 86,924,837 and 62,615,693 bp, were generated for the two herbal species, respectively. OrthoMCL analysis identified 6375 single-copy orthologous genes between N. incisum and N. franchetii. In total, 381 positively-selected candidate genes were identified for both plants by using estimations of the non-synonymous to synonymous substitution rate. At least 18 of these genes potentially participate in RNA splicing, DNA repair, glutathione metabolism and the plant-pathogen interaction pathway, which were further enriched in various functional gene categories possibly responsible for environment adaptation in high mountains. Meanwhile, we detected various transcription factors that regulated the material and energy metabolism in N. incisum and N. franchetii, which probably play vital roles in the tolerance to stress in surroundings. In addition, 60 primer pairs based on orthologous microsatellite-containing sequences between the both Notopterygium species were determined. Finally, 17 polymorphic microsatellite markers (SSR) were successfully characterized for the two endangered species. Based on these candidate orthologous and SSR markers, we detected that the adaptive evolution and species divergence of N. incisum and N. franchetii were significantly associated with the extremely heterogeneous environments and climatic oscillations in high-altitude areas. This work provides important insights into the molecular mechanisms of adaptation to high-altitudes in alpine herbal plants.

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.

Efficient Identification of the Forest Tree Species in Aceraceae Using DNA Barcodes.

Aceraceae is a large forest tree family that comprises many economically and ecologically important species. However, because interspecific and/or intraspecific morphological variations result from frequent interspecific hybridization and introgression, it is challenging for non-taxonomists to accurately recognize and identify the tree species in Aceraceae based on a traditional approach. DNA barcoding is a powerful tool that has been proposed to accurately distinguish between species. In this study, we assessed the effectiveness of three core standard markers (matK, rbcL and ITS) plus the chloroplast locus trnS-trnG as Aceraceae barcodes. A total of 231 sequences representing 85 species in this forest family were collected. Of these four barcode markers, the discrimination power was highest for the ITS (I) region (50%) and was progressively reduced in the other three chloroplast barcodes matK (M), trnS-trnG (T) and rbcL (R); the discrimination efficiency of the ITS marker was also greater than any two-locus combination of chloroplast barcodes. However, the combinations of ITS plus single or combined chloroplast barcodes could improve species resolution significantly; T+I (90.5% resolution) and R+M+T+I (90.5% resolution) differentiated the highest portion of species in Aceraceae. Our current results show that the nuclear ITS fragment represents a more promising DNA barcode marker than the maternally inherited chloroplast barcodes. The most efficient and economical method to identify tree species in Aceraceae among single or combined DNA barcodes is the combination of T+I (90.5% resolution).

Genetic Structure and Evolutionary History of Three Alpine Sclerophyllous Oaks in East Himalaya-Hengduan Mountains and Adjacent Regions.

The East Himalaya-Hengduan Mountains (EH-HM) region has a high biodiversity and harbors numerous endemic alpine plants. This is probably the result of combined orographic and climate oscillations occurring since late Tertiary. Here, we determined the genetic structure and evolutionary history of alpine oak species (including Quercus spinosa, Quercus aquifolioides, and Quercus rehderiana) using both cytoplasmic-nuclear markers and ecological niche models (ENMs), and elucidated the impacts of climate oscillations and environmental heterogeneity on their population demography. Our results indicate there were mixed genetic structure and asymmetric contemporary gene flow within them. The ENMs revealed a similar demographic history for the three species expanded their ranges from the last interglacial (LIG) to the last glacial maximum (LGM), which was consistent with effective population sizes changes. Effects of genetic drift and fragmentation of habitats were responsible for the high differentiation and the lack of phylogeographic structure. Our results support that geological and climatic factors since Miocene triggered the differentiation, evolutionary origin and range shifts of the three oak species in the studied area and also emphasize that a multidisciplinary approach combining molecular markers, ENMs and population genetics can yield deep insights into diversification and evolutionary dynamics of species.

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.

De novo assembly and characterization of the leaf, bud, and fruit transcriptome from the vulnerable tree Juglans mandshurica for the development of 20 new microsatellite markers using Illumina sequencing.

Manchurian walnut (Juglans mandshurica Maxim.) is a vulnerable, temperate deciduous tree valued for its wood and nut, but transcriptomic and genomic data for the species are very limited. Next generation sequencing (NGS) has made it possible to develop molecular markers for this species rapidly and efficiently. Our goal is to use transcriptome information from RNA-Seq to understand development in J. mandshurica and develop polymorphic simple sequence repeats (SSRs, microsatellites) to understand the species' population genetics. In this study, more than 47.7 million clean reads were generated using Illumina sequencing technology. De novo assembly yielded 99,869 unigenes with an average length of 747 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.32 %) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) identified 15,903 (16.9 %) unigenes. Further, we identified and characterized 63 new transcriptome-derived microsatellite markers. By testing the markers on 4 to 14 individuals from four populations, we found that 20 were polymorphic and easily amplified. The number of alleles per locus ranged from 2 to 8. The observed and expected heterozygosity per locus ranged from 0.209 to 0.813 and 0.335 to 0.842, respectively. These twenty microsatellite markers will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species. Moreover, the information uncovered in this research will also serve as a useful genetic resource for understanding the transcriptome and development of J. mandshurica and other Juglans species.

The complete mitochondrial genome of Heliconius pachinus (Insecta: Lepidoptera: Nymphalidae).

The complete mitochondrial genome of Heliconius pachinus has been reconstructed from the whole-genome Illumina sequencing data. The circular genome is 15,369 bp in length, and comprises the typical components: 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and 1 D-loop region. PCGs are mostly initiated with either the ATN codons (COII, COIII, Cytb, ND2, ND3, ND4, ND4L, ND5, ND6, ATP6 & ATP8) or the TTG codon (ND1); the COI gene may use the unusual CGA as its initiation codon, as suggested for other lepidopteran species. Some PCGs harbor TAG (ND3) or incomplete termination codon T (COI, COII & ND4), while the others use TAA as their termination codons. The nucleotide composition is highly asymmetric (39.2% A, 42.0% T, 7.7% G, 11.1% C) with an overall GC content of 18.8%.

The complete chloroplast genome of Armand pine Pinus armandii, an endemic conifer tree species to China.

The complete chloroplast genome (cpDNA) sequence of an endemic conifer species, Armand pine Pinus armandii Franch., is determined in this study. The cpDNA was 117,265 bp in length, containing a pair of 475 bp inverted repeat (IR) regions those distinguished in large and small single copy (LSC and SSC) regions of 64,548 and 51,767 bp in length, respectively. The cpDNA contained 114 genes, including 74 protein-coding genes (74 PCG species), 4 ribosomal RNA genes (four rRNA species) and 36 transfer RNA genes (33 tRNA species). Out of these genes, 12 harbor a single intron and most of the genes occurred in a single copy. The overall AT content of the Armand pine cpDNA was 61.2%, while the corresponding values of the LSC, SSC and IR regions were 62.0%, 60.2% and 62.7%, respectively. A phylogenetic analysis revealed that P. armandii chloroplast genome is closely related to that of the P. koraiensis within the genus Pinus.

The complete plastid genome of Bunge's pine Pinus bungeana (Pinaceae).

The complete nucleotide sequence of Bunge's pine Pinus bungeana Zucc. ex Endl. chloroplast genome (cp DNA) was determined in this study. The cpDNA was 117 861 bp in length, containing a pair of 475 bp inverted repeat regions (IRa and IRb), which were separated by large and small single copy regions (LSC and SSC) of 65 373 and 51 538 bp, respectively. The cpDNA contained 111 genes, including 71 protein-coding genes (71 PCG species), 4 ribosomal RNA genes (4 rRNA species) and 36 tRNA genes (32 tRNA species). In these genes, 13 harbored a single intron and 1 (ycf3) contained a couple of introns. The overall AT content of Bunge's pine cpDNA is 61.2%, while the corresponding values of the LSC, SSC and IR regions are 61.9%, 60.2% and 62.5%, respectively. A phylogenetic reconstruction based on the maximum parsimony analysis suggested that all the sampled Pinus species clustered a monophyletic clade and have a high bootstrap support, and the cpDNA of P. bungeana is closely related to that of congeneric P. gerardiana.