Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer(ITS)region of peritrich ciliates in environmental samples

Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifi cations of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specifi c PCR primers were newly designed to amplify a fragment including the internal transcribed spacer(ITS) region of ribosomal rDNA from environmental samples. The primers showed high specifi city in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28 S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18 S rDNA in peritrichs. The newly designed specifi c primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in dif ferent systems.

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.

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.

Characterization of Fusarium Oxysporum Isolates Obtained from Wax Gourd and Chieh-qua in China by Pathogenicity, RAMs and Sequence Analysis of the rDNA Internal Transcribed Spacers (ITS1 and ITS2)

Wax gourd (Benincasa hispida Thumb. Cogn) is called white gourd, winter melon, Chinese preserving melon, Chinese squash, and don kwa. It has been cultivated in China for over 2 300 years. It probably

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.

The homology analysis of internal transcribed spacer sequence of ribosomal DNA in common dermatophytes

In order to analyze the sequences of the internal transcribed spacer （ITS） including the 5.8 S ribosomal DNA （rDNA） of common dermatophytes, so as to obtain a rapid and accurate method to identify the species of dermatophytes and to establish the phylogenetic tree of these species to understand their relationship, 16 strains of dermatophytes were collected and preliminarily identified by morphological characteristics. General primers for fungi ITS1 and ITS4 were used to amplify the ITS rDNA of each strains with PCR. The PCR products after purification were sequenced directly and were analyzed through internet. In the results, 11 strains were identified by means of morphological features, among which 5 strains were Trichophyton, 5 strains were Microsporum and 1 was Epidermophytoa, which was consistent with the results by molecular biology. In the 5 unidentifiable strains, 1 strain was proved to be Chrysosporium by molecular biology. These strains studied could be divided into 3 different classes as indicated in the analysis of the phylogenetic tree of the sequences in ITS, which were quite different from those of morphological classification. It is evident from the above observations that the molecular method of analysis on the ITS sequences is a rapid, highly sensitive and accurate approach for the detection of dematophyte species, however, it still exhibits some limitations needing the supplementation with morphological identification.

云南蚕区家蚕微孢子虫遗传多样性分析

家蚕微孢子虫(Nosema bombycis)是蚕业生产上一种重要病害——微粒子病的病原体。探讨家蚕微孢子虫种内的遗传多样性,可为云南蚕区家蚕微粒子病的防控提供参考依据。从云南省不同养蚕地区收集了感染微孢子虫的病蚕样品,分离纯化家蚕微孢子虫并提取基因组,克隆SSU rDNA(small subunit ribosomal DNA)和ITS(internal transcribed spacer)序列并进行生物信息学分析。结果发现,云南蚕区Nosema bombycis分离株SSU rDNA序列同源性高达99%以上,遗传距离小于0.006,它们在长度和多个位点存在差异,呈现不同程度的多态性;ITS遗传差异较为显著,序列中存在多碱基的插入或缺失、单碱基的转换和颠换。基于SSU rDNA和rDNA-ITS序列构建系统发生树,结果显示,云南蚕区家蚕微孢子虫分离株系间存在遗传分化,种群间亲缘关系与地理位置无直接联系。研究结果丰富了云南蚕区家蚕微孢子虫的种内遗传多样性。

基于ISSR-PCR体系鉴别樟芝单核体交配型

通过原生质体单核化技术获得樟芝单核体,基于内转录间隔区(internal transcribed spacer,ITS)序列进行鉴定,采用14个引物对简单重复序列区间(inter-simple sequence repeats,ISSR)进行多态性扩增,筛选条带清晰、重复性好的引物用于樟芝单核体的交配型鉴定。结果表明,通过原生质体单核化技术获得31个樟芝单核体,经ITS序列分析确定获得的单核体为樟芝。以单核体S2、S10、S14、S27的DNA为模板,对14条ISSR引物进行初步筛选,得到4个条带清晰、重复性好的引物P7、P9、P21、P25用于交配型鉴定。单核体S9和S25为同一交配型,两者与S2为不同交配型,通过镜检进一步验证S9、S25可以与S2形成具有锁状联合的双核菌株。该方法可明显缩短樟芝单核体交配型的鉴定时间。

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.

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.

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。

黑龙江省扶余市麦穗鱼舌状绦虫裂头蚴的分子鉴定和系统发育分析

为鉴定从黑龙江省扶余市麦穗鱼腹腔内收集的绦虫裂头蚴的种类,本试验采用PCR方法分别对其核糖体内转录间隔区(ITS)和线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因进行扩增和序列测定。运用DNASTAR软件的MegAlign程序分析核苷酸同源性,采用邻接法构建基于ITS和COⅠ基因序列的系统进化树,以此分析分离虫株种内与种间的系统发育关系。结果显示,各分离虫株的ITS1和ITS2基因与舌状绦虫(AY121751)的核苷酸序列同源性最高,分别为98.8%~100%和99.6%~99.7%;各分离虫株的COⅠ基因与肠舌状绦虫(EU241273)的核苷酸序列同源性最高,为91.2%~94.7%。基于ITS序列构建的系统进化树显示,舌状绦虫和双线绦虫的ITS序列具有高度的保守性,ITS区域可能不适宜作为区分舌状绦虫属内部种类的可靠分子标记。基于COⅠ基因构建的系统进化树显示,分离虫株与土耳其、俄罗斯和欧洲的肠舌状绦虫分离株进化关系最近。根据序列同源性和COⅠ基因进化树结果,初步将本试验所分离虫株鉴定为肠舌状绦虫。结果表明,COⅠ基因更适合于舌状绦虫属种类的鉴别。本试验为舌状绦虫的分子流行病学调查提供了参考。

基于ITS基因序列的羊源食道口线虫PCR检测方法建立及应用

为了能对羊源食道口线虫病进行特异性诊断,基于食道口线虫ITS基因序列设计特异性引物,建立食道口线虫的特异性PCR检测方法,对其特异性、敏感性以及在临床羊粪便样本中食道口线虫虫卵的检测效果进行验证。结果显示,在对食道口线虫、仰口线虫、毛尾线虫、捻转血矛线虫等9种线虫的阳性DNA模板进行扩增时,只有在食道口线虫DNA阳性模板中扩增出650 bp的目的条带,而在其他线虫的DNA模板中无条带出现;将食道口线虫原始质粒模板进行10倍梯度稀释后进行敏感性检测,该PCR最低检出浓度为90 copies/μL;利用所建立的PCR检测方法对临床200份羊粪便样本进行检测,结果检出食道口线虫的阳性率为32%,高于显微镜镜检的阳性检测结果(阳性率为31%),但二者无显著性差异。研究表明,该研究所建立的食道口线虫PCR检测方法能够特异性地检测食道口线虫及其虫卵的阳性DNA样本,且敏感性良好,该方法为食道口线虫的种类鉴别以及食道口线虫病的诊断提供了一种可供选择的技术。

基于ITS和ISSR标记的灵芝遗传多样性和群体结构分析

以来自不同地理区域的48个灵芝种质资源为试材,采用ITS和ISSR分子标记的方法,研究了灵芝遗传多样性,以期利用遗传信息数据较理想地显示出不同灵芝菌株的遗传多样性。结果表明:通过进行ITS序列扩增测序,将48个灵芝菌株分为赤芝(35个)、无柄灵芝(11个)、四川灵芝(1个)、古巴栓孔菌(1个)。5条ISSR引物共扩增得到53条条带,多态性条带比率为96.23%,Nei′s基因多样性为0.27,Shannon′s信息指数为0.43。ISSR标记遗传相似系数约为0.70,将48个灵芝菌株分为3组,其中第1组为11个无柄灵芝,第2组为菌株29“盆景1”,其他菌株为第3组,说明2种标记结合分析能够更加准确分析不同菌株间的亲缘关系。菌株16和17同为赤芝,且ISSR分子标记遗传相似系数达0.98,推测可能为同一品种,为生产中出现的同物异名现象。该研究选取的用于PCR扩增的ISSR引物,多态性较好、稳定性较高,能有效鉴别出无柄灵芝与其他灵芝,并揭示种质的遗传多样性和群体遗传结构,可为灵芝种质资源保护及优质种质材料选育提供参考依据,以期更好地推动灵芝产业健康发展。

A preliminary analysis of phylogenetic relationships of Arundinaria and related genera based on nucleotide sequences of nrDNA （ITS region） and cpDNA （trnL-F intergenic spacer）

Phylogenetic relationships of Arundinaria and related genera (Pleioblastus, Pseudosasa, Oligostachyum, Bashania, Clavinodum, etc.) were assessed by analyzing the sequences of the nrDNA internal transcribed spacer (ITS) and the cpDNA trnL-F intergenic spacer (IGS). Comparison with trnL-F IGS sequence, the ITS region provided the higher number of parsimony informative characters, and the interspecific variation of the ITS sequence was higher than that of the trnL-F IGS sequence.The tree obtained by combining both sets of data showed that the species sampled in Arundinaria and the related genera were monophyletic and divided into two clades. The relationships and positioning of all the taxa surveryed (including A. oleosa, A. hsienchuensis, A. chino, A. amara, A. yixingensis, A. amabilis, A. fortunei, A. pygmaea, A. gramineus, A. fargesii, A. faberi, A. hupehense, Pseudosasa japonica cv. Tsutsumiana, P. japonica and Brachystachyum densiflorum) were also discussed. The results from the sequences were broadly consistent with morphological characters, appearing all these taxa sampled belong to the genus of Arundinaria. The topologies of the trees generated from individual data and the combined data were similar.

葡萄座腔菌Botryosphaeria dothidea胞内miRNA及siRNA的鉴定与分析

通过ITS-rDNA检测和形态学典型特征观察确定葡萄座腔菌Botryosphaeria dothidea的遗传背景,并对其胞内小RNA测序.新预测10个miRNAs和298个siRNAs,并分析了预测的miRNAs和siRNAs的序列信息、结构、长度分布、碱基偏好性、表达情况,为在分子水平了解葡萄座腔菌发育和致病机制提供了理论基础.

斑玉蕈种质资源评价及遗传多样性分析

利用体细胞不亲和性、核糖体基因转录间隔区域(internal transcribed spacer,ITS)序列和简单序列重复区间(inter-simple sequence repeat,ISSR)对67株斑玉蕈种质资源进行鉴定评价,并分析遗传多样性。结果表明,67株斑玉蕈菌株被分为15组,组内无拮抗反应,组间拮抗反应明显。褐色品系和白色品系间拮抗反应明显。采用邻接法(neighbor-joinging,NJ)构建系统进化树,将其分为3个类群,种内遗传距离为0~0.079 4,平均遗传距离为0.005 5±0.001 6。11条ISSR引物,扩增出51个多态性片段,菌株间Nei’s遗传距离为0.034 7~0.627 2。经非加权算数平均配对法(unweighted pair-group method with arithmetic mean,UPGMA)聚类分析,将其分为3个大类群,类群I中分为6个小类群。

山东桃褐腐病病原菌种群鉴定及致病性分析

【目的】明确山东省桃褐腐病病原菌种群结构及其致病力差异,为山东桃褐腐病病原菌的多样性研究及有效防控提供理论依据。【方法】采集烟台、威海、临沂等地桃褐腐病样本,利用形态学鉴定、rDNA-ITS序列分析、欧氏距离非加权组平均法(UPGMA)等技术手段,对桃褐腐病病原菌种类、致病力等进行分析。【结果】采集桃树叶片、果实、枝条褐腐病样品,通过组织分离获得41株桃褐腐病病原菌,这些菌株在菌落形态上存在较大差异,结合rDNA-ITS序列分析,分别鉴定为Monilinia fructicola、Monilia yunnanensis及Monilia polystroma,三者占比分别为80.48%、9.76%、9.76%。桃褐腐病病原菌菌丝生长速率为0.47~1.09 cm·d-1,UPGMA聚类分析证实,其生长速率可被划分为慢、中、快三大类。采用桃叶片有伤接种菌饼方法,确定桃褐腐病病原菌引起的病斑大小范围为0~2.32 cm,UPGMA聚类分析证实,其致病力可被划分为强、中、弱三类。桃褐腐病病原菌菌丝生长速率及产孢量与致病力相关性分析发现,相关系数r分别为0.297 5、0.030 0,表明菌丝生长速率及产孢量均与致病力无相关性。【结论】山东省桃褐腐病病原菌主要为Monilinia fructicola,Monilia yunnanensis及Monilia polystroma,其中Monilinia fructicola为优势菌种,Monilia yunnanensis是首次在山东省被鉴定,证实了山东省桃褐腐病病原菌趋于多样化,不同菌株间菌丝生长速率、产孢量及致病力存在较大差异,菌丝生长速率及产孢量均与致病力无相关性。