Identification of sika deer and red deer using partial cytochrome b and 12s ribosomal RNA genes

A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.

Nematode Diversity of Qingdao Coast Inferred from the 18S Ribosomal RNA Gene Sequence Analysis

The 18S ribosomal DNA gene （18S rDNA） sequences （approxtmately 1300 bp in length） were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode specific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin61 restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Although it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve sequences were close to Pontonema vulgate and Adoncholaimus sp., four to Daptonema procerus and two （identical） to Enoplus brews. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.

Taxonomy and phylogeny of the section Chaetoceros(Chaetocerotaceae,Bacillariophyta),with description of two new species

Chaetoceros Ehrenberg is one of the most diverse genera of planktonic diatoms.The species in section Chaetoceros are characterized by cells and setae having numerous chloroplasts and being widely distributed.However,the delimitations of some species are problematic because of limited morphological information in the classical descriptions.Monoclonal strains of the section Chaetoceros were established,morphological features were studied using light and electron microscopy,and the hypervariable D 1-D 3 region of the nuclear ribosomal large subunit gene was sequenced to address phylogenetic relationships.Fifteen species belonging to the section Chaetoceros were recorded,including two new species,C.hainanensis sp.nov.and C.tridiscus sp.nov.Chaetoceros hainanensis was characterized by straight chains,narrowly lanceolate to hexagonal apertures,sibling setae diverging in nearly right angles,stipule-shaped spines on terminal setae and arrowhead-shaped spines on intercalary setae.C.tridiscus had short straight chains,narrowly lanceolate apertures,arrowhead-shaped spines and circular poroids arranged in a grid pattern on terminal and intercalary setae.The phylogenetic analyses revealed six groups formed by 19 species within the section Chaetoceros,which was found to be monophyletic.The subdivision of the section is still not well understood.The morphological characters within each group varied considerably and molecular information on more species are needed to enrich the phylogenetic profiling.

Multi-gene-based investigation on the molecular phylogeny of the hypotrichous family Strongylidiidae(Protista,Ciliophora),with notes on the ontogeny of a new genus and new species

Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups,leading to their systematics remaining unresolved.Phylogenetic relationships within the hypotrich family Strongylidiidae have been ambiguous due to discordance between the morphological and genetic data.In this study,a new strongylidiid genus Heterouroleptus is established,mainly based on the novel mode of origin of the ventral cirral rows:left ventral cirral row(LVR)originates from frontal-ventral-transverse cirral anlagen(FVTA)Ⅲ(anterior portion),IV(middle portion),and V(rear portion);right ventral cirral row comes from the entire FVTA VI.A new species,Hetero-uroleptus weishanensis gen.nov.,sp.nov.,is investigated along with the morphometric and molecular data from a population of Strongylidium wuhanense.Eight new sequences and nuclear gene markers(single-gene and multi-gene)are provided to analyze the phylogenetic relationships of strongylidiids,with the COI gene utilized to uncover further genetic information at species level and below.The results reveal that:(1)Strongylidiidae is monophyletic and has a close relationship with Dorsomarginalia;(2)Heterouroleptus gen.nov.forms a clade that is sister to all the other strongylidiids;(3)Hemiamphisiella Foissner,1988 and Pseudouroleptus Hemberger,1985 should not be synonyms,and both genera should be subdivided due to their variable morphological characteristics;(4)LVR originating from three anlagen is a plesiomorphy of Strongylidiidae.The discovery of the origin of the LVR not only contributes to the establishment of the genus Heterouroleptus,but also helps to improve the diagnosis of the family Strongylidiidae.

A single degenerated primer significantly improves COX1 barcoding performance in soil nematode community profiling

A new COX1 primer for soil nematode metabarcoding was designed,and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products.•The lack of reference database is the main reason that led to the low species recovery in COX1 metabarcoding.•We expanded current NCBI database by adding 51 newly generated COX1 reference sequences.Microscopic nematodes play important roles in soil ecosystems and often serve as bioindicators of soil health.The identification of soil nematodes is often difficult due to their limited diagnostic characters and high phenotypic plasticity.DNA barcoding and metabarcoding techniques are promising but lack universal primers,especially for mitochondrial COX1 gene.In this study a degenerated COX1 forward primer COIFGED was developed.The primer pair(COIFGED/JB5GED)outperforms other four commonly used COX1 primer pairs in species recovery and quantity of polymerase chain reaction(PCR)products.In metabarcoding analysis,the reads obtained from the new primer pair had the highest sequencing saturation threshold and amplicon sequence variant(ASV)diversity in comparison to other COX1 as well as 18S rRNA primers.The annotation of ASVs suggested the new primer pair initially recovered 9 and 6 out of 25 genera from mock communities,respectively,outperformed other COX1 primers,but underperformed the widely used 18S NF1/18Sr2b primers(16 out of 25 genera).By supplementing the COX1 database with our reference sequences,we recovered an additional 6 mock community species bringing the tally closer to that obtained with 18S primers.In summary,our newly designed COX1 primers significantly improved species recovery and thus can be supplementary or alternative to the conventional 18S metabarcoding.

Molecular Identification of a Species in Genus Nannochloropsis

Nannochloropsis is a genus of marine eukaryotic unicellular algae,which belongs to class Eustigmatophyceae.The spe-cies of Nannochloropsis which are fine rotifer feed and rich in eicosapentaenoic acid(EPA)are economically important.Species in this genus are usually 2-5μm in size and are morphologically similar,which makes their identification difficult.We obtained a monoclone of Nannochloropsis with plating method in this study.DNA was extracted and the quality was determined by restriction enzyme digestion and spectrophotometer analysis.The DNA extracted was used to amplify the sequences of 18S ribosomal RNA gene,ITS region of ribosomal RNA transcription unit and rbcL gene.The phylogenetic analysis was carried out by constructing the neighbor-joining trees with Tamura-Nei distances.The phylogenetic analysis showed that the monoclone is N.oceanica.

Antibiotic resistance mechanisms of Myroides sp.

Bacteria of the genus Myroides （Myroides spp.） are rare opportunistic pathogens. Myroides sp. infections have been reported mainly in China. Myroides sp. is highly resistant to most available antibiotics, but the resistance mechanisms are not fully elucidated. Current strain identification methods based on biochemical traits are unable to identify strains accurately at the species level. While 16S ribosomal RNA （rRNA） gene sequencing can accurately achieve this, it fails to give information on the status and mechanisms of antibiotic resistance, because the 16S rRNA sequence contains no information on resistance genes, resistance islands or enzymes. We hypothesized that ob- taining the whole genome sequence of Myroides sp., using next generation sequencing methods, would help to clarify the mechanisms of pathogenesis and antibiotic resistance, and guide antibiotic selection to treat Myroides sp. infec- tions. As Myroides sp. can survive in hospitals and the environment, there is a risk of nosocomial infections and pandemics. For better management of Myroides sp. infections, it is imperative to apply next generation sequencing technologies to clarify the antibiotic resistance mechanisms in these bacteria.

Identification of Anamorph Yeast of Tremella aurantialba and Optimization of Medium Composition for Production of Exopolysaccharides

A yeast-like fungus strain B1 isolated from wild fungus Tremella aurantialba was identified and initially characterized. Two phylogenetic trees were generated based on the sequences of large subunit ribosomal RNA gene D1/D2 regions and internal transcribed spacer (ITS) regions of related fungi, respectively. The analysis of D1/D2 regions and ITS sequences showed that fungus B1 was clustered together with T. aurantialba, T. aurantia and T. microspore in the phylogenetic trees. Both the morphological characteristic and phylogenetic analysis established that fungus B1 was one of the anamorph strains of T. aurantialba and belongs to Tremella genus. A fermentation medium for exopolysaccharides (EPS) production by T. aurantialba B1 . Plackett-Burmen design was used to evaluate the effects of different components in the culture medium. Glucose and yeast extract have significant influence on the EPS production. The concentrations of two factors were optimized subsequently using central composite design and response surface analysis. The results showed that 49.2 g/L glucose and 10.4 g/L yeast extract could lead to the maximum production of EPS (4.99 g/L). The optimized medium led to a 1.5-fold enhancement of the production of EPS by T. aurantialba B1 , as compared with that without optimization.