PSMD: An extensive database for pan‐species microsatellite investigation and marker development

Microsatellites are widely distributed throughout nearly all genomes which have been extensively exploited as powerful genetic markers for diverse applications due to their high polymorphisms. Their length variations are involved in gene regulation and implicated in numerous genetic diseases even in cancers. Although much effort has been devoted in microsatellite database construction, the existing microsatellite databases still had some drawbacks, such as limited number of species, unfriendly export format, missing marker development, lack of compound microsatellites and absence of gene annotation, which seriously restricted researchers to perform downstream analysis. In order to overcome the above limitations, we developed PSMD (Pan‐Species Microsatellite Database, http://big.cdu.edu.cn/psmd/ ) as a web‐based database to facilitate researchers to easily identify microsatellites, exploit reliable molecular markers and compare microsatellite distribution pattern on genome‐wide scale. In current release, PSMD comprises 678,106,741 perfect microsatellites and 43,848,943 compound microsatellites from 18,408 organisms, which covered almost all species with available genomic data. In addition to interactive browse interface, PSMD also offers a flexible filter function for users to quickly gain desired microsatellites from large data sets. PSMD allows users to export GFF3 formatted file and CSV formatted statistical file for downstream analysis. We also implemented an online tool for analysing occurrence of microsatellites with user‐defined parameters. Furthermore, Primer3 was embedded to help users to design high‐quality primers with customizable settings. To our knowledge, PSMD is the most extensive resource which is likely to be adopted by scientists engaged in biological, medical, environmental and agricultural research.     

Identification of CR1 retroposons in Arborophila rufipectus and their application to Phasianidae phylogeny

Chicken repeat 1 (CR1), a member of non‐LTR retroposon, is an important phylogenetic marker in avian systematics. In this study, we reported several characteristics of CR1 elements in a draft genome of Arborophila rufipectus (Sichuan partridge). According to the analyses of RepeatMasker, approximately 254 966 CR1 elements were identified in A. rufipectus, covering 6.7% of the genome. Subsequently, we selected eighteen novel CR1 elements by comparing the chicken genome, turkey genome and assembled A. rufipectus scaffolds. Here, a combined data set comprising of 22 CR1 loci, mitochondrial genomes and eight unlinked introns was analysed to infer the evolutionary relationships of twelve Phasianidae species. The applicability of CR1 sequences for inferring avian phylogeny relative to mtDNA and intron sequences was investigated as well. Our results elucidated the position of A. rufipectus in Phasianidae with robust supports that it presented a sister clade to Arborophila ardens/Arborophila brunneopectus, and implied that genus Arborophila was in a basal phylogenetic position within Phasianidae and a phylogenetic affinity between Meleagris gallopavo and Pucrasia macrolopha. Therefore, this work not only resolved some of the confounding relationships among Phasianidae, but also suggested CR1 sequences could provide powerful complementary data for phylogeny reconstruction.     

美洲大蠊胸腺素THY3的表达模式分析

美洲大蠊Periplaneta americana胸腺素基因具有THY1、THY2和THY3三个不同剪接体,其中THY3结构域最多。本研究使用实时荧光定量PCR技术比较分析了THY3在美洲大蠊不同性别、不同发育阶段及不同组织中的表达差异,以及大肠杆菌Escherichia coli诱导对美洲大蠊血淋巴和脂肪体中THY3表达的影响。结果表明:THY3在成虫期的表达量显著高于其他虫期,雌虫表达量显著高于雄虫,脂肪体表达量显著高于血淋巴、头部、肌肉、体壁组织。雌性成虫体腔注射大肠杆菌3 h后血淋巴中THY3表达量显著增高,而在脂肪体中12 h后才检测到THY3表达明显上调,研究结果为进一步研究美洲大蠊胸腺素的功能打下了基础。     

A long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration

A single nicotine exposure increases dopamine levels in the mesolimbic reward system for hours, but nicotine concentrations experienced by smokers desensitize nAChRs on dopamine neurons in seconds to minutes. Here, we show that persistent modulation of both GABAergic and glutamatergic synaptic transmission by nicotine can contribute to the sustained increase in dopamine neuron excitability. Nicotine enhances GABAergic transmission transiently, which is followed by a persistent depression of these inhibitory inputs due to nAChR desensitization. Simultaneously, nicotine enhances glutamatergic transmission through nAChRs that desensitize less than those on GABA neurons. The net effect is a shift toward excitation of the dopamine reward system. These results suggest that spatial and temporal differences in nicotinic receptor activity on both excitatory and inhibitory neurons in reward areas coordinate to reinforce nicotine self-administration.     

The complete mitochondrial genome and phylogenetic analysis of the giant panda (Ailuropoda melanoleuca)

The complete mitochondrial genome sequence of the giant panda, Ailuropoda melanoleuca, was determined by the long and accurate polymerase chain reaction (LA-PCR) with conserved primers and primer walking sequence methods. The complete mitochondrial DNA is 16,805 nucleotides in length and contains two ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and one control region. The total length of the 13 protein-coding genes is longer than the American black bear, brown bear and polar bear by 3 amino acids at the end of ND5 gene. The codon usage also followed the typical vertebrate pattern except for an unusual ATT start codon, which initiates the NADH dehydrogenase subunit 5 (ND5) gene. The molecular phylogenetic analysis was performed on the sequences of 12 concatenated heavy-strand encoded protein-coding genes, and suggested that the giant panda is most closely related to bears.     

Molecular phylogenetic relationships among Asiatic shrewlike moles inferred from the complete mitogenomes

Asiatic shrewlike moles are distributed almost entirely in south‐west China; four of the five species of the genus Uropsilus, Uropsilus aequodonenia, U. andersoni, U. investigator and U. soricipes are endemic to China. Excluding the five species, three cryptic species (U. sp. 1, U. sp. 2 and U. sp. 3) and two putative species, U. nivatus and U. atronates, are recognized. The phylogenetic relationships among the species remain unclear and these preclude investigations of their potential adaptations for living in high altitudes. We sequenced the complete mitochondrial DNA genomes of three species of Asiatic shrewlike moles (U. aequodonenia, U. andersoni and U. nivatus). Phylogenetic analyses of 16 published and our de novo mitogenomes yield single, robust trees with the relationships being (U. soricipes (U. sp. 1 (U. nivatus (U. andersoni, U. aequodonenia)))). Further, the tree verifies the validity of recently described U. aequodonenia. Analyses of selection pressure suggest that the 13 mtDNA‐encoding genes of species in the genus Uropsilus all have experienced strong purifying selection, although ATP8 accumulated a higher ratio of non‐synonymous substitutions than the other loci, which might reflect adaptation of the genus Uropsilus to different environments/elevations.     

Determination of Baylisascaris schroederi Infection in Wild Giant Pandas by an Accurate and Sensitive PCR/CE-SSCP Method

It has been recognized that other than habitat loss, degradation and fragmentation, the infection of the roundworm Baylisascaris schroederi (B. schroederi) is one of the major causes of death in wild giant pandas. However, the prevalence and intensity of the parasite infection has been inconsistently reported through a method that uses sedimentation-floatation followed by a microscope examination. This method fails to accurately determine infection because there are many bamboo residues and/or few B. schroederi eggs in the examined fecal samples. In the present study, we adopted a method that uses PCR and capillary electrophoresis combined with a single-strand conformation polymorphism analysis (PCR/CE-SSCP) to detect B. schroederi infection in wild giant pandas at a nature reserve, and compared it to the traditional microscope approach. The PCR specifically amplified a single band of 279-bp from both fecal samples and positive controls, which was confirmed by sequence analysis to correspond to the mitochondrial COII gene of B. schroederi. Moreover, it was demonstrated that the amount of genomic DNA was linearly correlated with the peak area of the CE-SSCP analysis. Thus, our adopted method can reliably detect the infectious prevalence and intensity of B. schroederi in wild giant pandas. The prevalence of B. schroederi was found to be 54% in the 91 fecal samples examined, and 48% in the fecal samples of 31 identified individual giant pandas. Infectious intensities of the 91 fecal samples were detected to range from 2.8 to 959.2 units/gram, and from 4.8 to 959.2 units/gram in the fecal samples of the 31 identified giant pandas. For comparison, by using the traditional microscope method, the prevalence of B. schroederi was found to be only 33% in the 91 fecal samples, 32% in the fecal samples of the 31 identified giant pandas, and no reliable infectious intensity was observed.     

Identification and characterization of ten polymorphic microsatellite loci in the red panda Ailurus fulgens

Ten polymorphic microsatellite loci were characterized from two genomic DNA-enriched libraries of the red panda (Ailurus fulgens). The number of observed alleles among 35 samples of red pandas ranged from five to 12. Observed and expected heterozygosities were 0.286–0.971 and 0.443–0.894, and the mean polymorphic information content was 0.712. All loci followed Hardy–Weinberg expectations except Aifu-14 and Aifu-16, which may due to the presence of inbreeding or null alleles. Three pairs of loci exhibited significant linkage disequilibrium after Bonferroni correction for multiple comparisons. These microsatellites would be useful to strengthen population management, genetic diversity exploration, and demographic history speculation of this species.     

Applying DNA barcoding to conservation practice: a case study of endangered birds and large mammals in China

Owning to advantages over traditional species identification methods, DNA barcoding is suggested to be a promising tool in conservation research. However, the use of DNA barcoding to accurately identify unknown samples in conservation practices has not been well documented in the literature. To illustrate this issue, we implemented a survey of endangered birds and mammals in China based on mitochondrial Cytochrome c Oxidase subunit I (COI) gene. We included mostly confiscated specimens and non-invasive samples while concealing species information to simulate real-world scenarios of identification. In total, 47 avian and 39 mammalian specimen were re-identified by sequential analyses of online species assignment, genetic distances, phylogenetic reconstruction, and diagnostic nucleotide method. With this multiple analyses approach, 82 individuals were accurately assigned to the species level and four individuals to the genus level. 78.72% of the avian specimen and 87.18% of mammalian specimen identifications were consistent with morphological classification. Among those inconsistent with morphological classification, we identified several potential errors including misidentification based on morphology and mislabelling that may have occurred while combining results from different analytical methods. Our case study not only enriches the barcode database, but also reports a successful application of DNA barcoding identification to conservation practices, which could effectively facilitate species identification of unknown samples in conservation practices in the future.     

Population genetic diversity of Prenant’s schizothoracin, Schizothorax prenanti, inferred from the mitochondrial DNA control region

Wild populations of Prenant’s schizothoracin, Schizothorax prenanti, were collected from the Qingyi, Dadu, and Muli rivers in upper reaches of the Yangtze River. Genetic variation and population structure were examined based on analysis of the mitochondrial DNA control region. Nucleotide sequence analysis defined 30 haplotypes in 41 individuals. The nucleotide and haplotype diversities were very high, and genetic distances between the Dadu and Qingyi populations were much smaller than between either of them and the Muli population. The analysis of molecular variance demonstrated that most variation occurred within samples, and the differentiation between Muli and Dadu or Qingyi was significant. Estimates of gene flow indicated reproductive isolation between Muli and the other populations, and this divergence might be related to geographical location. The results are mostly consistent with the findings from AFLP analysis and also suggested considerable genetic diversity of the populations in the Muli, Dadu, and Qingyi rivers.