Complete nuclear ribosomal DNA sequence amplification and molecular analyses of Bangia (Bangiales, Rhodophyta) from China

Filamentous Bangia,which are distributed extensively throughout the world,have simple and similar morphological characteristics.Scientists can classify these organisms using molecular markers in combination with morphology.We successfully sequenced the complete nuclear ribosomal DNA.approximately 13 kb in length,from a marine Bangia population.We further analyzed the small subunit ribosomal DNA gene(nrSSU) and the internal transcribed spacer(ITS) sequence regions along with nine other marine,and two freshwater Bangia samples from China.Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences(0-0.003;0-0.006,respectively) from each other,but high divergences(0.123-0.126;0.198,respectively) from freshwater samples.An exception is the marine sample collected from Weihai,which shows high divergence from both other marine samples(0.063-0.065;0.129,respectively) and the freshwater samples(0.097;0.120,respectively).A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades,with the two marine sample clades containing Bangia spp.from North America,Europe,Asia,and Australia;and one freshwater clade,containing Bangia atropurpurea from North America and China.

Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia

Fargesia,the largest genus within the temperate bamboo tribe Arundinarieae,has more than 90 species mainly distributed in the mountains of Southwest China.The Fargesia bamboos are important components of the subalpine forest ecosystems that provide food and habitat for many endangered animals,including the giant panda.However,species-level identification of Fargesia is difficult.Moreover,the rapid radiation and slow molecular evolutionary rate of Fargesia pose a significant challenge to using DNA barcoding with standard plant barcodes(rbcL,matK,and ITS) in bamboos.With progress in the sequencing technologies,complete plastid genomes(plastomes) and nuclear ribosomal DNA(nrDNA)sequences have been proposed as organelle barcodes for species identification;however,these have not been tested in bamboos.We collected 196 individuals representing 62 species of Fargesia to comprehensively evaluate the discriminatory power of plastomes and nrDNA sequences compared to standard barcodes.Our analysis indicates that complete plastomes have substantially higher discriminatory power(28.6%) than standard barcodes(5.7%),whereas nrDNA sequences show a moderate improvement(65.4%) compared to ITS(47.2%).We also found that nuclear markers performed better than plastid markers,and ITS alone had higher discriminatory power than complete plastomes.The study also demonstrated that plastomes and nrDNA sequences can contribute to intrageneric phylogenetic resolution in Fargesia.However,neither of these sequences were able to discriminate all the sampled species,and therefore,more nuclear markers need to be identified.

Phylogeny of Ptychostomum (Bryaceae,Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.

Phylogenetic analysis of Pectinidae (Bivalvia) based on the ribosomal DNA internal transcribed spacer region

The ribosomal DNA internal transcribed spacer （ITS） region is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, the molecular phylogenetic analysis of Pectinidae （ Mollusca： Bivalvia） was performed using the nucleotide sequences of the nuclear ITS region in nine species of this family. The sequences were obtained from the scallop species Argopecten irradians, Mizuhopecten yessoensis, Amusium pleuronectes and Mimachlamys nobilis, and compared with the published sequences of Aequipecten opercularis, Chlamys farreri, C. distorta, M. varia, Pecten maximus, and an outgroup species Perna viridis. The molecular phylogenetic tree was constructed by the neighbor-joining and maximum parsimony methods. Phylogenetic analysis based on ITS1, ITS2, or their combination always yielded trees of similar topology. The results support the morphological classifications of bivalve and are nearly consistent with classification of two subfamilies （Chlamydinae and Pectininae） formulated by Waller. However, A. irradians, together with A. opercularis made up of genera Amusium, evidences that they may belong to the subfamily Pectinidae. The data are incompatible with the conclusion of Waller who placed them in Chlamydinae by morphological characteristics. These results provide new insights into the evolutionary relationships among scallop species and contribute to the improvement of existing classification systems.

Molecular Characterization of Indian Species of Steinernema （Nematoda： Steinernematidae） Based on Restriction Fragment Length Polymorphism Profile of Internal Transcribed Spacer Region of Ribosomal DNA

Restriction fragment length polymorphism （RFLP） profiles of the amplified products of Internal Transcribed Spacer （ITS） region of rDNA using four restriction enzymes （Alul, Rsal, HinfI and HhaI） revealed distinctness of six Indian isolates of Steinernema one each from Maharashtra （IARI-EPN-mh）, Himachal Pradesh （IARI-EPN-hp）, Dehradun （IARI-EPN-dhdl）, Jharkhand （IARI-EPN-jhl） and two from Madhya Pradesh （IARI-EPN-bpll ＆ IARI-EPN-gwll）, when compared with the only native species Steinernema thermophilum. One of the restriction enzyme, Rsal could differentiate all the six species/strains from one another. The three restriction enzymes yielded patterns which were of diagnostic value but Rsal appeared to be the best diagnostic marker for differentiating these isolates. A tree constructed based upon the band sharing amongst the isolates, produced trichotomy which placed strains from Madhya Pradesh and Jharkhand in one group showing 94% homology, one strain from Bhopal （M.P） formed separate clade along with S. thermophilum with 72% similarity. These isolates, from Maharashtra, Himachal Pradesh and Dehradun, showed only 51% similarity with the S. thermophilum by forming separate clade.

Resolution of phylogenetic relationships of the major subfamilies of the Delphacidae （Homoptera： Fulgoroidea） using the mitochondrial ribosomal DNA

Delphacid relationships from the genus level to the subfamily have been completely resolved （among those taxa examined） using sequence data from the 3＇ end of the 12S gene. Monophyly of the non-asiracine subfamilies was strongly supported and the asiracine Ugyops was placed in the most basal position of the tree. Support levels for monophyly of the Delphacini increased after weighting transversions more heavily than transitions and after removing the cixiid outgroup from the dataset. Among the Delphacini, Conomelus and Megamelus were more closely related to each other than either was to Chloriona. These results are in agreement with the tree based on morphological characters. However, in contrast to morphological data our results strongly supported a sister group relationship between the Stenocraninae and the Kelisiinae. Although the 12S gene fragment gave some information about the species relationships within Chloriona, neither this fragment nor the 5＇ end of the 16S gene appear to be very useful for this level. Molecular evolutionary patterns provided evidence that there has been a shift in base composition from T to A during the early evolution of the non-Asiracinae. The non-Asiracinae also had comparatively fast substitution rates and these two observations are possibly correlated. In the ‘ modem＇ delphacid Chloriona, the AT content was comparatively low in regions free of constraints but this was not the case for ‘ non-modem＇ delphacids. The tRNA for valine has been translocated elsewhere, probably before the Delphacidae and Cixiidae diverged from each other.

Endodontic bacteria from primary and persistent endodontic lesions in Chinese patients as identified by cloning and 16S ribosomal DNA gene sequencing

Background Few literatures pertain to the 16S ribosomal DNA （16S rDNA） analysis of bacteria contributing to primary and persistent endodontic lesions, with no information available for the Chinese population. As such, we investigated endodontic bacteria associated with primary and persistent endodontic lesions in adult Chinese patients living in Beijing, China using 16S rDNA gene sequencing techniques. Methods Endodontic microbial samples were obtained from fourteen adult Chinese patients and subjected to DNA extraction. Pllymerase chain reaction （PCR） products were cloned and 100 clones from each generated library were randomly selected. Purified plasmid DNA with 16S rDNA gene inserts was sequenced, and the sequences were searched against GenBank databases using the BLASTN algorithm. Only significant identification with the highest-scored BLAST result and 99% minimum similarity was considered for phylotyping. Results More than 150 taxa were obtained. Primary endodontic infection was mainly associated with Burkholderia cepacia, Actinomyces, Aranicola spp. and Streptococcus sanguinis, whilst Burkholderia cepacia was predominant in the persistent endodontic infections. Conclusion There is a difference in the species profile associated with endodontic infections of Chinese patients living in Beijing in comparison to other geographical or ethnic reports.

PHYLOGENETIC RELATIONSHIPS AMONG GIANT PANDA AND RELATED SPECIES BASED ON RESTRICTION SITE VARIATIONS IN rDNA SPACERS

In this study, non radioactive Digoxigenin labeled ribosomal DNA(rDNA) probes were used for Southern blotting analysis to study the molecular phylogeny of the giant panda and related species. Restriction maps in the regions of rDNA spacers were compared between giant panda( Ailuropoda melanoleuca ), lesser panda( Ailurus fulgens ), Asiatic black bear( Selenarctos thibetanus ), sun bear( Helarctos malayanus ), raccoon( Procyon lotor ) and lynx( Felis lynx ). Phylogenetic trees for these species were constructed using maximum likelihood and parsimony method. The results show that in respect to rDNA RFLPs, the giant panda is more closely related to bear than to lesser panda; while the lesser panda is slightly related to the raccoon.

Genetic variation and phylogenetic relationship of Hypoderaeum conoideum(Bloch, 1782) Dietz, 1909(Trematoda: Echinostomatidae) inferred from nuclear and mitochondrial DNA sequences

Objective:To explore genetic variations of Hypoderaeum conoideum collected from domestic ducks from 12 different localities in Thailand and Lao PDR,as well as their phylogenetic relationship with American and European isolates.Methods:The nucleotide sequences of their nuclear ribosomal DNA(ITS),mitochondrial cytochrome c oxidase subunit 1(CO1),and NADH dehydrogenase subunit 1(ND1)were used to analyze genetic diversity indices.Results:We found relatively high levels of nucleotide polymorphism in ND1(4.02%),whereas moderate and low levels were observed in CO1(2.11%)and ITS(0.96%),respectively.Based on these polymorphisms,the 20 ND1,12 CO1,and 18 ITS haplotypes were classified,and several common haplotypes were observed in all samples.At least three major lineages,namely American,European and Asian lineages,have been classified by phylogenetic analyses based on ND1 sequences.Conclusions:Our report demonstrates that the ND1 gene is the most suitable genetic marker to explore genetic variation and phylogenetic relationship of Hypoderaeum conoideum.However,a combination of all loci for ND1,CO1 and ITS would be of great value toward further genetic investigation of this endemic worldwide parasite.Thus,comprehensive molecular genetic analyses of Hypoderaeum conoideum from its worldwide distribution is needed to further understanding of the evolutionary and systematic relationships of this parasite.

Hmo1:A versatile member of the high mobility group box family of chromosomal architecture proteins

Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures,which is facilitated by linker histone H1.Formation of chromatin compacts and protects the genome,but also hinders DNA transactions.Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions.The high mobility group box(HMGB)proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion.They play a major role in chromatin dynamics.The Saccharomyces cerevisiae(yeast hereafter)HMGB protein Hmo1 contains two HMGB motifs.However,unlike a canonical HMGB protein that has an acidic C-terminus,Hmo1 ends with a lysine rich,basic,C-terminus,resembling linker histone H1.Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions.For instance,Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones.Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome.This minireview reviews the functions of Hmo1 and the underlying mechanisms,highlighting recent discoveries.

Isolation and identification of Sclerotinia stem rot causal pathogen in Arabidopsis thaliana

A new stem rot disease is found to occur naturally on Arabidopsis plants in greenhouses of Fuzhou, China. In order to identify its pathogen, we conducted a series of fungal isolation and purification, plant reinoculation, and ascus and ascospore induction from the sclerotia. The isolate caused typical water-soaked lesions after reinoculation and produced sclerotia both on Arabidopsis plants and culture medium plates, and the sclerotia could be induced to produce discal apothecia and 8 binucleate ascospores per ascus. These disease symptom and fungal morphology data revealed that the fungus Sclerotinia sclerotiorum (Lib.) de Bary was the pathogen for Arabidopsis stem rot. To confirm this, we further amplified its large subunit ribosomal DNA (LSU rDNA) by polymerase chain reaction (PCR), and compared the sequence with the known LSU rDNA sequences in GenBank. The results show that the sequence shares the highest identities with the LSU rDNAs of different S. sclerotiorum strains. Taking all these data together, we concluded that the fungus that caused the Arabidopsis stem rot is S. sclerotiorum (Lib.) de Bary. This is the first report that Arabidopsis is naturally infected by S. sclerotiorum.

Construction of Saccharomyces cerevisiae Strain Stably Expressing a Fusion Protein Containing Ten Tandem Recombinant Human Glucagon-like Peptide-1 Analogues

The recombinant Saccharomyces cerevisiae strain stably expressing recombinant human glucagon-like peptide-l（rhGLP-1） analogue, as a potential oral drug delivery system for diabetes type II treatment, was successfully constructed by the homologous recombination between chromosomal DNA and yeast and integrating vector pNK-GLP containing yeast ribosomal DNA fragments. The amount of rhGLP-I analogue fusion protein in transformant SG2 reached ca. 0.84 mg per gram of packed cells when SG2 was grown for 24 h in the YPD medium with a inoculum and medium ratio of 1：1. Oral administration of 5 g lyophilized SG2/kg to hyperglycemic rats decreased serum glucose from （24.8±1.40） to （21.2±1.36） mmol/L.

Nuclear and chloroplast genome diversity revealed by low-coverage whole-genome shotgun sequence in 44 Brassica oleracea breeding lines

Whole-genome shogun sequence(WGS)data generated by next-generation sequencing(NGS)platforms are a valuable resource for crop improvement.We produced 5–6×WGS coverage of 44 Brassica oleracea breeding lines representing seven subspecies/morphotypes:cabbage,broccoli,cauliflower,kailan,kale,Brussels sprout,and kohlrabi to systematically evaluate the nuclear and chloroplast(Cp)diversity in the 44 B.oleracea breeding lines.We then exploited the impact of low-coverage NGS by evaluating nuclear genome diversity and assembly,annotation of complete chloroplast(Cp)genomes and 45 S nuclear ribosomal DNA(45S nrDNA)sequences,and copy number variation for major repeats.Nuclear genome diversity analysis has revealed a total of 496463 SNPs and 37493 indels in the nuclear genome across the 44 accessions.Interestingly,some SNPs showed subspecies enrichment at certain chromosomal regions.The assembly of complete Cp genomes contained 153361–153372 bp with 37 variants including SNPs and indels.The 45S nrDNA transcription unit was 5802 bp long with a total of 31 SNPs from the 44 lines.The phylogenetic tree inferred from the nuclear and Cp genomes coincided and clustered broccoli,cauliflower,and kailan in one group and cabbage,Brussels sprout,kale,and kohlrabi in another group.The morphotypes diverged during the last 0.17 million years.The Cp genome diversity reflected the unique cytoplasm of each subspecies,and revealed that the cytoplasm of many breeding lines was replaced and intermingled via inter-subspecies crosses during the breeding process instead.The polymorphic Cp markers provide a classification system for the cytoplasm types in B.oleracea.Furthermore,copy numbers of major transposable elements(TEs)showed high diversity among the 44 accessions,indicating that many TEs have become active recently.Overall,we demonstrated a comprehensive utilization of low-coverage NGS data and might shed light on the genetic diversity and evolution of diverse B.oleracea morphotypes.

Sensitive and rapid detection of two toxic microalgae Alexandrium by loop-mediated isothermal amplification

A loop-mediated isothermal amplification （LAMP） assay was designed and evaluated for rapid de- tection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning （PSP）. Two sets of four specific primers targeting these two species were derived from the sequence of internal transcribed spacer （ITS） of ribosomal DNA. The method worked well in less than an hour under isothermal conditions of 65℃. LAMP specificity was validated in closely related algae as a comparison, suggesting the strict specificity of the LAMP primers. Two visual inspection approaches were feasible to interpret the positive or negative results. The detection lim- its of A. catenella and A. minutum samples using the LAMP assay were found to be 5.6 and 4.5 pg DNA, respectively. The sensitivity of this LAMP assay was 10 or 100-fold higher than Polymerase Chain Reaction （PCR） method in detecting the two microalgae. These characteristics of species specificity, sensitivity, and rapidity suggest that this method has the potentiality in the monitoring of red tide caused by A. catenella and A. minutum.

Morphological and molecular characterizations of cereal cyst nematode Heterodera avenae Wollenweber, 1924 from the Czech Republic

The cereal cyst nematode,Heterodera avenae Wollenweber,1924,is a major pest of cereal crops throughout the world and causes serious yield losses,especially of wheat.Previous studies have shown that this species is widely distributed in the Czech Republic.In this study,seven populations of H.avenae were molecularly studied,and one population was morphologically described.Three regions（18S,28S,and internal transcribed spacer 1）of ribosomal DNA were sequenced and the analysis of the 18S gene of six populations did not reveal any variation,whereas the internal transcribed spacer 1and 28S sequences of six populations differed by only two nucleotides from a population in？ilina.Precise and quick identification of cereal cyst nematodes is important for effective control measures and ribosomal sequence analyses of seven populations in this study will be useful in future phylogenetic studies of Heterodera spp.occurring in the Czech Republic.

Isolation and molecular characterization of entomopathogenic nematode,Heterorhabditis sp.from an arable land in Nigeria

The occurrence of entomopathogenic nematodes(EPNs)in arable soil samples from Nigeria was investigated using Baermann extraction tray and insect-bait(White’s trap)techniques.Isolates were tested for infectivity using the larvae of Galleria mellonella(greater moth)and Tenebrio molitor(mealworm).The study revealed a new species of Heterorhabditis(MT371593)in soil samples that were randomly collected from an arable farmland cultivated with cassava TMS-30572 at the Teaching and Research Farm of Landmark University,Nigeria.Amplification of the internal transcribed spacer region(ITS)of the ribosomal DNA produced a nucleotide sequence of 933 base pairs(bp).A BLASTN search of GenBank showed that the sequence of the Nigerian isolate is identical at 99%similarity to that of Heterorhabditis sp.from Thailand.Infectivity test of the isolate showed 100%mortality against T.molitor larvae within 48 h of exposure while only 80%mortality was recorded for G.mellonella after 1 week of exposure.This is the first account of Heterorhabditis sp.in Nigeria.The varying degrees of infectivity against mealworm and greater moth observed in this study proved that the Nigerian isolate of Heterorhabditis sp.could potentially be an attractive option in the management of insect pests of cash crops.

Species diversity of thrips (Thysanoptera) in selected avocado orchards from Mexico based on morphology and molecular data

Avocado is one of the most important crops in the world, and Mexico is the largest producer of this fruit. Several insect pests affect its production, and thrips are amongst the most important. A key step in the design of control methods is accurate species identification. Despite this, formal reports on species diversity of thrips in Mexico are very scarce. Morphological identification can sometimes be time-consuming and inconclusive. Therefore, we explored the species diversity of thrips in Mexican avocado orchards （Michoacan state） based on partial sequences of the mitochondrial gene cytochrome oxidase subunit I （COl）. Forty-four specimens were analysed, which represented approximately 8% of all individuals collected from five localities distributed in three Municipalities. All specimens were analysed using the COl marker, and specimens within the genera Frankliniella were also analysed using a marker within the D2 domain of the 28S （28SD2） nuclear ribosomal DNA. Molecular identifications were confirmed using morphological taxonomy. Overall, six genera were found （Neohydatothrips, Scirtothrips, Frankliniella, Arorathrips, Caliothrips and Leptothrips）. AII genera contained only one species, except Frankliniella, for which there were six species. Data from the two molecular markers suggest the existence of cryptic species within Mexican F. occidentalis populations.

Succession of Bacterial Community Inhabited Acid Mine Drainage under High Fe（Ⅱ） Concentration

To reveal the effects of Fe2＋ on bacterial communities in the early stages of minerals dissolution, two different acid mine drainage （AMD） samples were collected at Dabaoshan Mine and Shenbu Mine. Community successions of AMD niches were analyzed by Amplified Ribosomal DNA Restriction Analysis （ARDRA）, sequencing, and phylogenetic analysis in original AMD samples and their subculture under Fe2＋ concentrations. Although geochemical properties and community structures were greatly different between the two original AMD samples, bacterial community successions were still very similar under high Fe2＋ concentrations. The results showed that Acidithiobacillus ferrooxidans have competitive relationship with other bacterial species living in the AMD, including species that were also capable of oxidizing ferrous ion. A competitive relationship among different At. ferrooxidans strains likewise existed. Some of At. ferrooxidans can grow first under conditions of high ferrous ion concentration, and other At. ferrooxidans species decreased gradually and disappeared. This suggested that these species of At. ferrooxidans are most acidophilic bacteria and afford Fe3＋ to leach other metallic ion in the early stages of minerals dissolution.

Molecular Identification of Hammerhead Shark Trunks from the Southern Gulf of California using Multiplex PCR

The demand for shark fins in Asiatic markets has resulted in excessive increases in shark catches,even for species that may be under protection or subject to management.As such,it has been necessary to develop and promote monitoring efforts for exploited species and taxonomic groups in order to improve fishing management strategies for elasmobranchs.Identifying species from landings is one of many fishing management problems because landed organisms have usually already been processed and are therefore incomplete,which makes identification problematic,impedes the generation of proper species records,and leads to poor fishery assessments.Tools that can correctly identify species,such as various molecular techniques,have become essential for accurate fishery assessments.In this study,30 hammerhead trunks from artisanal fisheries from the southern portion of the Gulf of California were identified using multiplex PCR(17 Sphyrna lewini and 13 Sphyrna zygaena).The total fee to identify each trunk with this technique was~$3.80 and the procedure required 2 to 5 days.When compared with other widely-used methods,such as PCR-RFLP or barcoding,multiplex PCR is fast,efficient,low-cost,and easy to implement in a laboratory.

Difference in DNA sequences in SSU rDNA variable regions among pathogens isolated from different epidemic foci of visceral leishmaniasis in China

To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L d ) isolates from different epidemic foci in China Methods Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM R T Easy Vectors After that, the specific fragments were sequenced by an automated DNA sequencer Results Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length All 5 point mutations were located in two unique sequence blocks (UQ Ⅰ and UQ Ⅱ), and no insertions or deletions were found The identities of comparison of Leishmania in GeneBank were more than 98% Conclusion Five point mutations exist in the SSU rDNA variable region of 5 L d isolates from different epidemic foci of visceral leishmaniasis (VL) in China Sequence differences of the SSU rDNA variable region exist among L d isolates from different foci