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.

Application of the first internal transcribed spacer(ITS-1)of ribosomal DNA as a molecular marker to population analysis in farrer's scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.

Role of transcribed ultraconserved regions in gastric cancer and therapeutic perspectives

Gastric cancer(GC)is the fourth leading cause of cancer-related death.The occurrence and development of GC is a complex process involving multiple biological mechanisms.Although traditional regulation modulates molecular functions related to the occurrence and development of GC,the comprehensive mechanisms remain unclear.Ultraconserved region(UCR)refers to a genome sequence that is completely conserved in the homologous regions of the human,rat and mouse genomes,with 100%identity,without any insertions or deletions,and often located in fragile sites and tumour-related genes.The transcribed UCR(T-UCR)is transcribed from the UCR and is a new type of long noncoding RNA.Recent studies have found that the expression level of T-UCRs changes during the occurrence and development of GC,revealing a new mechanism underlying GC.Therefore,this article aims to review the relevant research on T-UCRs in GC,as well as the function of T-UCRs and their regulatory role in the occurrence and development of GC,to provide new strategies for GC diagnosis and treatment.

Phylogenetic Relationships of <i>Termitomyces aurantiacus</i>Inferred from Internal Transcribed Spacers DNA Sequences

Fungus-growing termites cultivate species of the mutualistic basidiomycete genus Termitomyces on a substrate called the fungal comb. Identification of fungal species based on morphological features is complicated, tedious, and prone to errors. As an alternative, nuclear ribosomal DNA sequences consisting of the internal transcribed spacers (ITS1 and ITS2) and 5.8S rDNA were used to identify Malaysian isolates of Termitomyces sp. The morphological characteristics and molecular data indicate that Malaysian Termitomyces isolated is clearly monophyletic and belongs to the Tricholomataceae family. The Malaysian isolates analyzed in this study represent the termite fungus species called T. aurantiacus.

DNA Barcoding of <i>Ricinus communis</i>from Different Geographical Origin by Using Chloroplast <i>matK</i>and Internal Transcribed Spacers

Ricinus communis have attracted considerable attention because of its specific industrial and pharmacological activities. DNA barcodes can be used as reliable tools to facilitate the identification of medicinal plants for the safe use, quality control and forensic investigation. In this study, the differential identification of eight accessions of R. com-munis was investigated through DNA sequence analysis of two candidate DNA barcodes. The nucleotide sequence of internal transcribed spacers (ITS2) and chloroplast maturase gene (matK) have been determined to construct the phylogenetic tree. The phylogenetic relationships of accessions based on the nrITS2 region and partial matK region showed that all accessions in this study were related to three geographical origins. Based on sequence align-ment and phylogenetic analyses we concluded that the ITS2 sequences can distinguish R. communis accessions from different geographical distributions.

基于SCAR标记和DNA条形码技术的苍术基原鉴别研究

目的开发出能同时鉴别北苍术和关苍术的分子标记方法,并探究不同种质资源苍术的遗传进化关系。方法对不同地区北苍术Atractylodes chinensis(Bunge)Koidz及关苍术A.japonica Koidz.ex Kitam基因组DNA的差异片段进行测序,结合SRAP、ISSR、DAMD分子标记方法,优化PCR反应体系,筛选并转换成特异性标记,同时,采用条形码方法分析种间序列差异。结果通过SRAP、ISSR、DAMD三种分子标记方法的PCR扩增,共筛选出198对能稳定扩增且重现性好的引物,转换出7对能稳定、快速鉴别北苍术和关苍术的SCAR引物。条形码方法检测出北苍术ITS2序列长度为454 bp,关苍术ITS2序列长度为453 bp,与其他苍术属植物之间遗传距离较远。NJ树结果显示,北苍术、关苍术及其他苍术属植物均各自聚为一支,表现出良好的单系性。依据ITS2二级结构,4种苍术属植物在螺旋区的茎环数目、大小、位置均有明显差异,可以直观地进行区分。结论所开发的特异性SCAR标记为苍术属植物优良品种的筛选提供了新方法,DNA条形码能稳定、准确鉴别北苍术。

Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.

Natural selection maintains the transcribed LTR retrotransposons in Nosema bombycis

Eight intact LTR retrotransposons （Nbr1-Nbr8） have been previously characterized from the genome of Nosema bombycis, a eu- karyotic parasite with a compact and reduced genome. Here we describe six novel transcribed Nbr elements （Nbr9-Nbr14） identified through either cDNA library or RT-PCR. Like previously determined ones, all of them belong to the Ty3/Gypsy superfamily. Retrotransposon diversity and incomplete domains with insertions （Nbr12）, deletions （Nbrll） and in-frame stop codons in coding regions （Nbr9） were detected, suggesting that both defective and loss events of LTR retrotransposon have happened in N. bornbycis genome. Analysis of selection showed that strong purifying selection acts on all elements except Nbr11. This implies that selective pressure keeps both these Nbrs and their functions in genome. Interestingly, Nbrll is under positive selection and some positively selected codons were identified, indicating that new functionality might have evolved in the Nbrll retrotransposon. Unlike other transposable elements, Nbrll has integrated into a conserved syntenic block and probably resulted in the inversion of both flanking regions. This demonstrates that transposable element is an important factor for the reshuffling and evolution of their host genomes, and may be maintained under natural selection.

Metataxonomics of Internal Transcribed Spacer amplicons in cerebrospinal fluid for diagnosing and genotyping of cryptococcal meningitis

Background:Cryptococcal meningitis is a severe infectious disease associated with high morbidity and mortality.Rapidity and accuracy of diagnosis contribute to better prognosis,but readily available tools,such as microscopy,culture,and antigens do not perform well all the time.Our study attempted to diagnose and genotype cryptococcus in the cerebrospinal fluid(CSF)samples from patients with cryptococcal meningitis using the approach of metataxonomics of Internal Transcribed Spacer(ITS)amplicons.Methods:The CSF samples were collected from 11 clinically suspected cryptococcal meningitis patients and four non-infectious controls.Samples were recruited from the First Affiliated Hospital of Fujian Medical University Hospital,Fuzhou Fourth Hospital and the 476th Hospital of Chinese People's Liberation Army from December 2017 to December 2018.ITS1 ribosomal deoxyribonucleic acid(rDNA)genes of 15 whole samples were amplified by universal forward primer ITS1(CTTGGTCATTTAGAGGAAGTAA)and reverse primer ITS2(GCTGCGTTCTTCATCGATGC),sequenced by Illumina MiSeq Benchtop Sequencer.The results were confirmed by sanger sequencing of ITS1 region and partial CAP59 gene of microbial isolates from 11 meningitic samples.Pair-wise comparison between infectious group and control group was conducted through permutational multivariate analysis(PERMANOVA)in R software.Results:The 30,000 to 340,000 high-quality clean reads were obtained from each of the positively stained or cultured CSF samples and 8 to 60 reads from each control.The samples from 11 infected patients yielded detectable cryptococcal-specific ITS1 DNA with top abundance(from 95.90%to 99.97%),followed by many other fungal groups(each<1.41%).ITS genotype was defined in 11 CSF samples,corresponding to ITS type 1,and confirmed by Sanger sequencing.A statistically significant difference(r2=0.65869,P=0.0014)between infectious group and control group was observed.Conclusions:The metataxonomics of ITS amplicons facilitates the diagnosis and genotype of cryptococcus in CSF samples,which may provide a better diagnostic approach of cryptococcal infection.

Internal transcribed spacer guided multiplex PCR for species identification of Convolvulus prostratus and Evolvulus alsinoides

Shankhpushpi is a reputed drug from an Indian system of medicine for treating mental disorders and enhancing memory. Two herbs, namely Convolvulus prostratus Forssk. and Evolvulus alsinoides(L.) L., are commonly known as Shankhpushpi. Ambiguous vernacular identity can affect the scientific validity of the Shankpushpi-based herbal drug therapy. In the present investigation, a novel and sensitive multiplex PCR method based on polymorphism in the internal transcribed spacer(ITS) region was developed to establish the molecular identity of C. prostratus and E. alsinoides. DNA was isolated and the ITS region was amplified, sequenced and assembled. Sequences were aligned to identify variable nucleotides in order to develop plant-specific primers. Primers were validated in singleplex reactions and eventually a multiplex assay was developed. This assay was tested for sensitivity and validated by amplifying DNA isolated from the simulated blended powdered plant material. Primers developed for C. prostratus resulted into a 200 bp amplicon and 596 bp for E. alsinoides. The assay was found to be sensitive enough for amplification of low quantities of DNA. The method can detect 10% of the mixing of plants with each other in blended material. This PCR assay can be used for rapid botanical identification of Shankhpushpi plant materials and will improve evidence-based herbal drug therapy.

Mutational screening of BASP1 and transcribed processed pseudogene TPΨg-BASP1 in patients with Mbius syndrome

Moebius syndrome is a rare disorder primarily characterized by congenital facial palsy, frequently accompanied by ocular abduction anomalies and occasionally associated with orofacial, limb and musculoskeletal malformations. Abnormal development of cranial nerves Ⅴ through Ⅻ underlines the disease pathogenesis. Although a genetic etiology for Moebius syndrome was proposed, molecular genetic studies to identify the causative gene（s） are scarce. In this study, we selected two candidate genes. One is BASP1 residing in a human chromosome 5p15.1-p15.2, syntenic to mouse chromosome 15qA2-qB2, to which a mouse model with facial nerve anomalies was mapped. The other is transcribed processed pseudogene TPψg-BASP1, which is located on chromosome 13q flanking the putative locus for Moebius syndrome and might be involved in the regulation of the transcripts encoded by BASP1. Mutation analyses in nineteen patients excluded these genes as being candidates for Moebius syndrome.

TELS: A Novel Computational Framework for Identifying Motif Signatures of Transcribed Enhancers

In mammalian cells, transcribed enhancers(TrEns) play important roles in the initiation of gene expression and maintenance of gene expression levels in a spatiotemporal manner. One of the most challenging questions is how the genomic characteristics of enhancers relate to enhancer activities. To date, only a limited number of enhancer sequence characteristics have been investigated, leaving space for exploring the enhancers’ DNA code in a more systematic way. To address this problem, we developed a novel computational framework, Transcribed Enhancer Landscape Search(TELS), aimed at identifying predictive cell type/tissue-specific motif signatures of TrEns.As a case study, we used TELS to compile a comprehensive catalog of motif signatures for all known TrEns identified by the FANTOM5 consortium across 112 human primary cells and tissues.Our results confirm that combinations of different short motifs characterize in an optimized manner cell type/tissue-specific TrEns. Our study is the first to report combinations of motifs that maximize classification performance of TrEns exclusively transcribed in one cell type/tissue from TrEns exclusively transcribed in different cell types/tissues. Moreover, we also report 31 motif signatures predictive of enhancers’ broad activity. TELS codes and material are publicly available at http://www.cbrc.kaust.edu.sa/TELS.

Repeats in the transcribed regions: comprehensive characterization and comparison of Citrus spp.

A large number of expressed sequences tags are available for Citrus spp., which provides an opportunity to understand genomic organization of the transcribed regions. Here, we report a detailed analysis of repetitive elements including tandem repeats(TRs) and transposable elements(TEs) in the transcribed region of the Citrus spp.On average, 22% of the expressed sequence tags(ESTs)contain TRs. The relative density of TR classes is highly taxon-specific. For instance, Citrus limonia has a high relative density of mononucleotide repeats, whereas dinucleotide repeats are rare. The proportions of 2–6,7–30 and 31–50 bp repeats were almost identical in all studied species except for C. limonia and C. limettioides.We found that < 1% of the citrus ESTs have a similarity with transposable elements. Transcriptional activity of transposable element families varied even within the same class of elements. A high proportion of transcriptional activity was observed for gypsy-like TEs compare to other TE classes. While TEs are relatively rare, TRs are abundant elements in ESTs of citrus. The high proportion of TRs that have a unit size longer than 6 bp raises the question about a possible functional or evolutionary role of these elements.

腐烂茎线虫不同地理种群ITS区序列比对及系统发育

为弄清我国腐烂茎线虫(Ditylenchus destructor)不同地理种群ITS(Internal transcribed spacer)区之间的碱基差异及系统发育关系,本研究通过网络软件Clustalw1.83对腐烂茎线虫的核糖体ITS区核酸序列进行了比对,结果发现腐烂茎线虫的8个种群中,DEll、DEly、DEAY987007三个种群(I组)内部之间的碱基差异在1%以内,其它的5个种群(II组)之间的差异为0～1%,而I组与II组种群之间的差异达到了15%(根据ITS1+5.8s+ITS2序列)或20%(根据ITS1+ITS2序列)。这说明我国腐烂茎线虫很可能是1个复合种。根据ITS区核酸序列构建的系统发育图同样显示,我国腐烂茎线虫的7个地理种群明显分为2个分支A和B。

云南羊肚菌rDNA的ITS序列与亲缘关系分析

采用ITS（Internal Transcribed Spacers）序列比对技术分析了根据形态挑选的14个羊肚菌子实体（其中11个来自云南，3个来自浙江）。结果表明，云南的11个羊肚菌子实体（M1～M11）可分为4个种；高羊肚菌（Morchella elata），尖顶羊肚菌（Morchella conica），粗柄羊肚菌（Morchella crassipes）和大孔（宽肋）羊肚菌（Morchella costata）。浙江的3个羊肚菌子实体（M12～M14）经鉴定为未知羊肚菌种。14个羊肚菌子实体样品和GenBank中黑脉（小顶）羊肚菌[Morchella angusticeps（AJ698476）]、美味羊肚菌[Morchella esculenta（AJ698475）]以及羊肚菌科常见属钟菌属Verpa conica（AJ544206）的ITS序列聚类，以钟菌（AJ544206）作为外类群，可分为三大聚类群：高羊肚菌、大孔（宽肋）羊肚菌和黑脉（小顶）羊肚菌为第1类群（Group1）；美味羊肚菌和尖顶羊肚菌为第2类群（Group2）；粗柄羊肚菌和浙江的3个羊肚菌为第3类群（Group3）。

蜡梅科(Calycanthaceae)若干植物nrDNA ITS序列PCR反应条件分析

该文旨在探讨适合于扩增蜡梅科植物ITS序列的PCR反应条件。试验中设计了2个新引物来扩增整个核糖体RNA基因(nrDNA)的ITS序列,进行了4种引物组合、2种扩增程序、7种Mg^(2+)浓度、4种dNTP浓度和3种二甲基亚砜浓度的比较。结果表明:当反应体系中Mg^(2+)浓度为1.8 mmol/L,dNTP为2 mmol/L,含5%二甲基亚砜,扩增时先进行两步预扩增,反应效果最好。文中还对引物的设计进行了探讨,认为当所选用的植物可能有寄生真菌时,最好不用与真菌同源的引物。最后提出了几点引物设计的注意事项。

哈密瓜(Cucumis melo L.)致腐真菌的鉴定

研究从哈密瓜表面分离出引起其腐败的菌株A和菌株B,并采用显微镜形态学观察、表型特征和分子生物学三种方法对其进行鉴定。结果发现菌株A可利用碳源包括杏苷、糊精、D-半乳糖、D-葡萄糖酸、麦芽糖、肝糖、L-鼠李糖、L-山梨糖、L-乳酸等72种碳源,其ITS rDNA序列与尖孢镰刀菌甜瓜转化型(Fusarium oxysporum f.sp.melonis)的同源性为99%,分支长度为0.00。菌株B可利用碳源包括L-阿拉伯糖、D-果糖、丙三醇、蔗糖和木糖醇等54种碳源,其ITS rDNA序列与链格孢菌(Alternaria alternata)的同源性为100%,分支长度为0.00。因此,推断菌株A为尖孢镰刀菌甜瓜转化型,菌株B为链格孢菌。

First observation of Karlodinium venef icum from the East China Sea and the coastal waters of Germany

Harmful algal blooms often occurred in the East China Sea （ECS） and the German coastal waters of the North Sea but Karlodinium veneficum had not been taxonomically reported. Two strains of Karlodinium （LAMB090611 and LAMB010601） were isolated from the two areas in 2009. The morphological characteristics and molecular phylogeny of two strains are compared on the basis of observation of a light microscope, a scanning electron microscope （SEM）, a laser scanning micro-scope （LSM） and an internal transcribed spacer （ITS） sequence data. The mean cell length of strain LAMB090611 is （14.2 ± 1.8） μm （range 11.1–18.7 μm） and mean width is （10.8 ± 1.5） μm （range 8.2– 14.7 μm）. The mean cell length of strain LAMB010601 is （15.1 ± 1.2） μm （range 12.7–17.9 μm） and the mean width is （11.4 ± 1.1） μm （range 9.1–14.7 μm）, respectively. The two strains are similar in morphological characteristics, including a straight apical groove, distinct ventral pore, sulcal extension, cingulum displacement, two or four irregular shaped chloroplasts within the cell and almost equal sized epicone and hypocone. The large and round nucleus is located at the center or at the hypocone of the cell. The sequence length of the ECS strain LAMB090611 and the German strain LAMB010601 is 640 and 646 bp, respectively. The GC content is 49%. The nucleotide similarity of the two strains is 98.1%. The sequence divergence is 0.003. Both strains are confirmed as Karlo-dinium veneficum （D. Ballantine） J. Larsen and this is the first taxonomic report from China and Germany coastal waters. The population dynamics of this toxic species in the ECS and German coastal waters needs to be investigated in the near future.

The identities and anti-herpes simplex virus activity of Clinacanthus nutans and Clinacanthus siamensis

Objective:To distinguish the difference among the Clinacanthus nutans(Burm.f.)Lindau(C.nutans)and Clinacanthus siamensis Bremek(C.siamensis)by assessing pharmacognosy characteristics,molecular aspect and also to evaluate their anti-herpes simplex virus(HSV)type 1 and type 2 activities.Methods:Macroscopic and microscopic evaluation were performed according to WHO Geneva guideline.Stomatal number,stomatal index and palisade ratio of leaves were evaluated.Genomic DNA was extracted by modified CTAB method and ITS region was amplified using PGR and then sequenced.Dry leaves were subsequently extracted with n-hexane,dichloromethane and methanol and antiviral activity was performed using plaque reduction assay and the cytotoxicity of the extracts on Vero cells was determined by MTT assay.Results:Cross section of midrib and stem showed similar major components.Leaf measurement index of stomatal number,stomatal index and palisade ratio of C.nutans were 168.32±29.49,13.83±0.86 and 6.84±0.66,respectively,while C.siamensis were 161.60±18.04,11.93±0.81and 3.37±0.31,respectively.The PCR amplification of ITS region generated the PGR product approximately 700 bp in size.There were 34 polymorphisms within the ITS region which consisted of 11 Indels and 23 nucleotide substitutions.The IC_(50)values of C.nutans extracted with n-hexane,dichloromethane and methanol against HSV-1 were(32.05±3.63)μg/mL,(44.50±2.66)μg/mL,(64.93±7.00)μg/mL,respectively where as those of C.siamensis were(60.00±11.61)μg/mL,(55.69+4.41)μg/mL,(37.39±5.85)μg/mL,respectively.Anti HSV-2 activity of n-hexane,dichloromethane and methanol C.nutans leaves extracts were(72.62±12.60)μg/mL,(65.19±21.45)μg/mL,(65.13±2.22)μg/mL,respectively where as those of C.siamensis were(46.52±4.08)μg/mL,(49.63±2.59)μg/mL,(72.64±6.52)μg/mL,respectively.Conclusions:The combination of macroscopic,microscopic and biomolecular method are able to authenticate these closely related plants and both of them have a potency to be an anti-HSV agent.

A re-investigation of the bloom-forming unarmored dinoflagellate Karenia longicanalis(syn.Karenia umbella)from Chinese coastal waters

The dinoflagellate genus K arenia is known for recurrent harmful blooms worldwide. However, species diversity of the genus is generally overlooked owing to the difficulty of identifying small unarmored dinoflagellates. We have established four clonal cultures of K arenia longicanalis isolated from the type locality, Hong Kong harbor(strain HK01) and other three locations along the Chinese coasts(strains YB01, DT01, and NJ01). The morphology of the strain was studied by light and scanning electron microscopy(LM and SEM) and the pigment composition analyzed by high-performance liquid chromatography. We provide the first molecular data of K. longicanalis based on the large subunit(LSU) rRNA gene sequence and internal transcribed spacer(ITS). The four strains showed identical LSU rDNA sequences with a similarity of 99.4% to the holotype of K arenia umbella(strain KUTN05) from Australia. In the ITS phylogeny, the sequence of K. umbella branched between the Chinese strains of K. longicanalis. A careful comparison of the morphology of K. longicanalis and K. umbella reveals the similarity in the diagnostic characters. Diff erences may appear due to the sample treatment for SEM. We conclude that K. umbella is a junior synonym of K. longicanalis.