The genome of Magnolia hypoleuca provides a new insight into cold tolerance and the evolutionary position of magnoliids.

Magnolia hypoleuca Sieb. & Zucc, a member of the Magnoliaceae of magnoliids, is one of the most economically valuable, phylogenetic and ornamental tree species in Eastern China. Here, the 1.64 Gb chromosome-level assembly covers 96.64% of the genome which is anchored to 19 chromosomes, with a contig N50 value of 1.71 Mb and 33,873 protein-coding genes was predicted. Phylogenetic analyses between M. hypoleuca and other 10 representative angiosperms suggested that magnoliids were placed as a sister group to the eudicots, rather than sister to monocots or both monocots and eudicots. In addition, the relative timing of the whole-genome duplication (WGD) events about 115.32 Mya for magnoliid plants. M. hypoleuca was found to have a common ancestor with M. officinalis approximately 23.4 MYA, and the climate change of OMT (Oligocene-Miocene transition) is the main reason for the divergence of M. hypoleuca and M. officinalis, which was along with the division of Japanese islands. Moreover, the TPS gene expansion observed in M. hypoleuca might contribute to the enhancement of flower fragrance. Tandem and proximal duplicates of younger age that have been preserved have experienced more rapid sequence divergence and a more clustered distribution on chromosomes contributing to fragrance accumulation, especially phenylpropanoid, monoterpenes and sesquiterpenes and cold tolerance. The stronger selective pressure drived the evolution of tandem and proximal duplicates toward plant self-defense and adaptation. The reference M. hypoleuca genome will provide insights into the evolutionary process of M. hypoleuca and the relationships between the magnoliids with monocots and eudicots, and enable us to delve into the fragrance and cold tolerance produced by M. hypoleuca and provide more robust and deep insight of how the Magnoliales evolved and diversified.

The chromosome-scale genome of Magnolia officinalis provides insight into the evolutionary position of magnoliids.

Magnolia officinalis, a representative tall aromatic tree of the Magnoliaceae family, is a medicinal plant that is widely used in diverse industries from medicine to cosmetics. We report a chromosome-scale draft genome of M. officinalis, in which ∼99.66% of the sequences were anchored onto 19 chromosomes with the scaffold N50 of 76.62 Mb. We found that a high proportion of repetitive sequences was a common feature of three Magnoliaceae with known genomic data. Magnoliids were a sister clade to eudicots-monocots, which provided more support for understanding the phylogenetic position among angiosperms. An ancient duplication event occurred in the genome of M. officinalis and was shared with Lauraceae. Based on RNA-seq analysis, we identified several key enzyme-coding gene families associated with the biosynthesis of lignans in the genome. The construction of the M. officinalis genome sequence will serve as a reference for further studies of Magnolia, as well as other Magnoliaceae.

The complete chloroplast genome of Aconitum scaposum.

Aconitum scaposum Franch 1894 belongs to the Genus Aconitum and Subgenus Lycoctonum (Ranunculaceae). It is widely distributed in China and adjacent areas, used as herbal medicine and had highy toxic components. This species has little reasearch information, especially its chloroplast (cp) genome information being unclear. Therefore, with the method of high salt and low pH to extract the cp of A. scaposum, we sequenced and assembled the complete cp genome of A. scaposum using Illumina high-throughput sequencing platform. The results showed the cp genome of A. scaposum was 157 688 bp in length, including a pair of inverted repeated regions (IRa 26 156 bp and IRb 26 232 bp, respectively), large single copy region (LSC 69 309 bp) and small single copy region (SSC 16 917 bp). And cp genome of A. scaposum consisted of 145 unique genes, 8 ribosomal RNA (rRNA) genes and 38 transfer RNA (tRNA) genes, with GC content was 38%. Meanwhile, based on the cp complete genome, we performed the phylogenetic tree of 66 species with maximum likelihood (ML) method, respectively. Among them, we selected one Delphinium species as the outgroup and the bootstrap of each braches were greater than 90%. The results indicated that the phylogenetic relationship of A. scaposum was relatively closely related to A. scaposum var. vaginatum compared to other Aconitum species.

Phylogeography of Schizopygopsis malacanthus Herzenstein (Cypriniformes, Cyprinidae) in relation to the tectonic events and Quaternary climatic oscillations in the Shaluli Mountains Region.

The tectonic events and Quaternary climatic oscillations in the Shaluli Mountains (the margin of the southeastern Tibetan Plateau) had an extensive effect on the genetic structure and distribution patterns of this region's terrestrial fauna and flora. It is not yet clear whether similar mechanisms influence this region's fish fauna. Schizopygopsis malacanthus is limited to high-elevation rivers and lakes, and exhibits distinct adaptations to the mountains of, and near to, the Qinghai-Tibet Plateau (QTP). Therefore, this species is a good candidate for investigating patterns of genetic variation resulting from palaeoenvironmental fluctuations in the Shaluli Mountains (China). Here, we used microsatellite and mitochondrial DNA control region sequences to analyze six populations of S. malacanthus collected from the Jinsha and Yalong River drainages. Genealogical analyses identified four maternal lineages and perhaps even four putative species, of which the Ouqu River lineage played a pivotal role during the course of the species' evolution. Two lineages from the Yalong River drainage did not cluster together, whereas those from different drainages grouped together, suggesting tectonic event impacts that possibly altered regional river drainages have been highly influential in population connectivity and gene flow. Population genetic analysis indicated that the geographic barriers and this species preference for higher elevations both played key roles in the divergence of S. malacanthus populations. Demographic tests suggested large-scale spatial synchrony in population fluctuations of S. malacanthus, accompanying dramatic Pleistocene climatic oscillations. It appears that palaeoenvironmental changes in the Shaluli Mountains influenced the distribution and evolution of studied S. malacanthus populations, which provide important information about the factors that influenced the phylogeographic history of this region's fish fauna. Additionally, our study also has implications for ongoing conservation of this vulnerable fish.

Morphology and molecular identification of the zoological origin of medicinal seahorses in Chinese herbal markets.

Seahorses are a charismatic group of fish that have high economic value for their unique appearance and important medicinal values. They were heavily traded as traditional Chinese medicines. Authenticating the zoological origin of medicinal seahorses is very difficult because of their similar morphology. To study the identification characteristics of dried seahorse, and to provide a scientific basis for seahorse resource conservation and market supervision, 64 dried specimens from China's four major pharmaceutical markets were investigated based on morphology and COI sequences. Sixty-four COI sequences of 662 bp length revealed 43 unique haplotypes, which were divided into 12 main clades in both NJ and UPGMA phylogenetic trees. Eleven species including Hippocampus spinosissimus, H. barbouri, H. kuda, H. comes, H. histrix, H. trimaculatus, H. kelloggi, H. ingens, H. mohnikei, H. erectus and H. jayakari were clustered on different branches and showed respective monophyly. The results were confirmed by morphology and BLAST analysis. Hippocampus capensis and H. fuscus, which were clustered together in the phylogenetic tree, could be distinguished by different morphology. The morphological and molecular determination revealed 13 seahorse species in Chinese herbal markets. The method of DNA sequences analysis combined with morphological characteristics is conducive to accurately identify the zoological origin of commercial seahorses.

Molecular authentication of sika deer (Cervus nippon) based on allele-specific PCR.

Sika deer (Cervus nippon) is listed as a tonic in many ancient Chinese pharmaceutical. Many adulterants of sika deer products have been found in Chinese medicinal materials markets, which led to detrimental impacts in clinical treatment. However, it is lack of the rapid and effective identification method for sika deer. This study amplified 574 bp fragment of mtDNA COI region of 19 samples from seven Cervidae species, and the relevant five sequences from reindeer (Rangifer tarandus) were downloaded from GenBank. It was found that there were two SNP loci for sika deer. Phylogenetic analysis indicated that individuals from sika deer clustered together. Based on SNP locus, one pair specific primer for allele-specific PCR identification of sika deer was designed, which could be used to rapidly and accurately identify sika deer.

Identification of the original species of cubilose based on DNA barcode.

Cubilose, a valuable traditional Chinese medicine, is mainly composed of the saliva by several species of Aerodramus or Collocalia in the Apodidae. Due to rarity, high economic value and huge market demand, its fake or adulteration is frequently found in the market. Therefore, it is urgent to establish a simple and accurate method for authenticating cubilose. DNA barcoding, which is an easy, quick and reliable method, is widely used to trace the origin of traditional Chinese medicine. For identifying the original species of cubilose, cytb gene of 18 cubilose samples including 15 officer cubilose and 3 feather cubilose were amplified and entered into the GenBank database using the BLAST search tool. The genetic distances among 18 cubilose samples were calculated based on the Kimura two parameter (K2P) model. To construct the reference database, 18 cytb sequences of Aerodramus or Collocalia were downloaded from GenBank. The neighbor-joining (NJ) and unweighted pair group method with arithmetic average (UPGMA) trees were constructed based on sequences from GenBank and our dataset. Blast analysis showed that all cubilose samples had the highest similarity with A. fuciphagus, and the sequence similarity reached over 99%. Genetic distance of 18 cubilose samples ranged from 0.000-0.010. Trees constructed by NJ and UPGMA gave similar topology: all cubilose samples clustered together with A. fuciphagus. These result demonstrated that the original species of all 18 cubilose samples were A. fuciphagus, and cytb gene is a good candidate for identifying cubilose.

Identification of marine traditional Chinese medicine dried seahorses in the traditional Chinese medicine market using DNA barcoding.

Seahorse documented in Chinese pharmacopeia possess important medicinal efficacy and are used as an ingredient in traditional Chinese medicines. The growing international trade threatens the species. DNA barcoding holds a great application potentiality in wildlife conservation and might prevent the illegal trade of threatened species. The COI gene was used to identify seahorse, and nine Hippocampus species were found in the three large traditional Chinese medicines markets of China. All inter-specific genetic variations were larger than 2%. Mean genetic distances between species were 17-fold larger than those within the species. Phylogenetic tree showed that each species clustered in the appropriate branch. All results demonstrated that COI-based barcoding technique could be used to identify seahorse species and played a major role in monitoring the seahorse trade.

[Development and evaluation of a rapid PCR detection kit for Ophiocordyceps sinensis].

Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage, expensive price and huge market demand, there are many adulterants of O. sinensis in markets. Therefore, it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the species-specific PCR of O. sinensis, this study developed a detection kit by optimizing the components and evaluated the specificity, detection limit, repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture, it could be detected successfully. Moreover, only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃ for three days, which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions, and in different batch under the same operation conditions gave the same result and accuracy, which showed good repeatability of the kit. It is simple, rapid and accurate to distinguish O. sinensis from its adulterants using the kit, and lays the foundation for commercialization of traditional Chinese medicine fast detection kit.

Assessing genetic diversity of wild and hatchery samples of the Chinese sucker (Myxocyprinus asiaticus) by the mitochondrial DNA control region.

To restore the natural populations of Chinese sucker (Myxocyprinus asiaticus), a hatchery release program has been underway for nearly 10 years. Using DNA sequences of the mitochondrial control region, we assessed the genetic diversity and genetic structure among samples collected from three sites of the wild population as well as from three hatcheries. The haplotype diversity of the wild samples (h = 0.899-0.975) was significantly higher than that of the hatchery ones (h = 0.296-0.666), but the nucleotide diversity was almost identical between them (π = 0.0170-0.0280). Relatively high gene flow was detected between the hatchery and wild samples. Analysis of effective population size indicated that M. asiaticus living in the Yangtze River has been expanding following a bottleneck in the recent past. Our results suggest the hatchery release programs for M. asiaticus have not reduced the genetic diversity, but have influenced the genetic structure of the species in the upper Yangtze River.

Complete mitochondrial genome of Chinese salamander Batrachuperus pinchonii: genome characterization and phylogenetic analysis.

The complete mitogenome sequence of Batrachuperus pinchonii (Caudata: Hynobiidae) has been amplified and sequenced in this study. The overall base composition of B. pinchonii mitogenome is 33.9% for A, 19.7% for C, 13.6% for G, and 32.8% for T and has low GC content of 33.3%. The assembled mitogenome, consisting of 16,381 bp, has 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and 1 D-loop region. All PCGs, except for ND6 gene, were encoded on H-strand. Phylogenetic analysis with the whole mitogenome sequences revealed a close relationship of B. pinchonii with B. tibetanus.

The complete mitochondrial genome of the Statilia maculate. (Mantodea: Mantidae).

The complete mitochondrial genome of the Statilia maculate has been amplified and sequenced in this study. The mitogenome was 15,775bp long with two ribosomal RNA genes, 26 transfer RNA genes, 13 protein-coding genes, and a non-coding control region, with an A + T-rich characteristic (75.8%). Five identical tandem duplication of trnR were found in mitogenome of S. maculate, similar to the other mantis. According to the phylogenetic analysis, S. maculate had a closer genetic relationship with Statilia sp.

The complete mitochondrial DNA genome of the Queensland seahorse Hippocampus queenslandicus Horne, 2001 (Gasterosteiformes: Syngnathidae).

The complete mitochondrial genome of the Queensland seahorse Hippocampus queenslandicus Horne, 2001 (Gasterosteiformes: Syngnathidae) has been amplified and sequenced in this study. The mitogenome was 16,527 bp long with protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and a non-coding control region. The overall base composition of the genomes was 32.2% for A, 30.1% for T, 14.9% for G, and 22.8% for C with an A + T-rich characteristic (62.3%). According to the phylogenetic analysis, H. queenslandicus showed a closer genetic relationship with H. spinosissimus.

Strong population structure of Schizopygopsis chengi and the origin of S. chengi baoxingensis revealed by mtDNA and microsatellite markers.

The Tibetan Plateau underwent dramatic geological and climatic changes, which had important implications for genetic divergence and population dynamics of freshwater fish populations. Fluctuations of the ecogeographical environment and major hydrographic formations might have promoted the formation of new subspecies or species. In order to understand the impact of plateau uplift on freshwater fish evolutionary history, we estimated the genetic diversity and population structure in two subspecies of Schizopygopsis chengi (S. c. chengi and S. c. baoxingensis) in upper Yangtze River in Tibetan Plateau area using mitochondrial DNA control region and eight microsatellite markers, which suggested that there was a close genetic relationship. S. chengi showed some significant genetic structure that did not correlate with geographic distance. Bayesian assignment tests indicated that S. chengi samples in the study could be divided into four populations: upstream population, midstream population, tributary population and S. c. baoxingensis population. S. c. chengi and S. c. baoxingensis showed significant genetic divergence. However, phylogenetic analysis, population structure analysis and historical gene flow estimation suggested that there was close genetic relationship between S. c. baoxingensis and the Dawei population which belongs to populations of S. c. chengi. The time that Dawei population suffered from a bottleneck and S. c. baoxingensis underwent population expansion was congruent with the last glacial period on the Tibetan Plateau. The results confirmed the hypothesis that the Dawei River and Baoxing River were once connected, and the Dawei and Baoxing populations originated from a single population, but were isolated into separate populations because of crustal movements and the Baoxing population evolved as S. c. baoxingensis.

[Identification of original species of Mantidis Oötheca (Sangpiaoxiao) based on DNA barcoding].

Both market research and literature reports both found that the ootheca of mantodea was all used as medicine. However, Chinese Pharmacopoeia only records the ootheca of three mantis species. The clinical use of ootheca unrecorded in Chinese Pharmacopoeia, will pose potential risks to drug safety. It's urgent to identify the origin of Mantidis Oötheca. The current researches about original animal in Mantidis Oötheca are based on morphology and unanimous. DNA barcoding fill gaps of the traditional morphological identification, which is widely used in animal classification studies. This study first use DNA barcoding to analyze genetic distance among different Mantidis Oötheca types, align COI sequences between mantis and Mantidis Oötheca and construct the phylogeny tree. The result confirmed that Tenodera sinensis and Hierodula patellifera were the origin insects of Tuanpiaoxiao and Heipiaoxiao, respectively, and Statilia maculate and Mantis religiosa were the origin insects of Changpiaoxiao.