Identification and rDNA-ITS Sequence Analysis of a New Anthracnose Pathogen of Dracaena fragrans in Yunnan Province

In this study, identification and rDNA-ITS sequence analysis of an anthracnose pathogen on Dracaena fragrans were carried out. [Method] D. fra-grans leaves with lesions were used as experimental materials to isolate anthrac-nose pathogen. Morphological observation, rDNA-ITS amplification and sequence analysis were performed to identify the pathogen strain. [Result] Caonidia of the iso-lated anthracnose pathogen were straight or curved, el iptic to crescent, with 2-5 oil droplets, 7.5-20 × 4.5-5 μm. According to molecular phylogenetic analysis, the iso-lated pathogen strain was identified as a new species, which was named Col-letotrichum dracaena-fragrantis sp. nov. [Conclusion] This study provided theoretical basis for the prevention and control of anthracnose.

4种乳菇属真菌的形态学与rDNA-ITS鉴定

为明确内蒙古赤峰地区常见有毒乳菇种类,准确、快速地识别有毒乳菇,将采集的4个乳菇属Lactarius真菌标本采用传统形态学分类方法进行鉴定,同时提取子实体DNA,使用真菌通用引物ITS1/ITS4进行PCR扩增,用Blast对扩增产物纯化测序所得的序列进行比对,采用MEGA5.1软件构建系统发育树确定属和种。结果表明,2种方法鉴定结果一致,标本CFSZ10167为毛头乳菇[Lactarius torminosus(Schaeff.)Pers.],标本CFSZ10216为绒边乳菇[Lactarius pubescens(Fr.ex Kronbh.)Fr.],标本CFSZ13021为灰褐乳菇[Lactarius pyrogalus(Bull.)Fr.],3种均为胃肠炎型毒菌,CFSZ10044为皱乳菇(Lactarius ruginosus Romagn),为中国新记录种,主要特点是菌盖棕褐色、浅褐色,有绒质感,盖缘呈齿状,孢子球形具小刺,食毒不明。对4个种的形态学特征进行了详细描述,并附有生境图片和显微图像,研究结果为乳菇属物种鉴定和资源分布提供了重要补充,并为常见毒菌识别提供了理论依据。

The Application of Nicotiana benthamiana as a Transient Expression Host to Clone the Coding Sequences of Plant Genes

Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.

Pig pangenome graph reveals functional features of non‑reference sequences

Background The reliance on a solitary linear reference genome has imposed a significant constraint on our compre-hensive understanding of genetic variation in animals.This constraint is particularly pronounced for non-reference sequences(NRSs),which have not been extensively studied.Results In this study,we constructed a pig pangenome graph using 21 pig assemblies and identified 23,831 NRSs with a total length of 105 Mb.Our findings revealed that NRSs were more prevalent in breeds exhibiting greater genetic divergence from the reference genome.Furthermore,we observed that NRSs were rarely found within coding sequences,while NRS insertions were enriched in immune-related Gene Ontology terms.Notably,our investigation also unveiled a close association between novel genes and the immune capacity of pigs.We observed substantial differences in terms of frequencies of NRSs between Eastern and Western pigs,and the heat-resistant pigs exhibited a substantial number of NRS insertions in an 11.6 Mb interval on chromosome X.Additionally,we discovered a 665 bp insertion in the fourth intron of the TNFRSF19 gene that may be associated with the ability of heat tolerance in South-ern Chinese pigs.Conclusions Our findings demonstrate the potential of a graph genome approach to reveal important functional features of NRSs in pig populations.

Phylogenetic, phylogeographic and divergence time analysis of Anopheles subpictus species complex using ITS2 and COI sequences

Objective:To address the phylogenetic and phylogeographic relationship between different lineages of Anopheles(An.)subpictus species complex in most parts of the Asian continent by maximum utilization of Internal Transcriber Spacer 2(ITS2)and cytochrome C oxidase I(COI)sequences deposited at the GenBank.Methods:Seventy-five ITS2,210 COI and 26 concatenated sequences available in the NCBI database were used.Phylogenetic analysis was performed using Bayesian likelihood trees,whereas median-joining haplotype networks and time-scale divergence trees were generated for phylogeographic analysis.Genetic diversity indices and genetic differentiation were also calculated.Results:Two genetically divergent molecular forms of An.subpictus species complex corresponding to sibling species A and B are established.Species A evolved around 37-82 million years ago in Sri Lanka,India,and the Netherlands,and species B evolved around 22-79 million years ago in Sri Lanka,India,and Myanmar.Vietnam,Thailand,and Cambodia have two molecular forms:one is phylogenetically similar to species B.Other forms differ from species A and B and evolved recently in the above mentioned countries,Indonesia and the Philippines.Genetic subdivision among Sri Lanka,India,and the Netherlands is almost absent.A substantial genetic differentiation was obtained for some populations due to isolation by large geographical distances.Genetic diversity indices reveal the presence of a long-established stable mosquito population,at mutation-drift equilibrium,regardless of population fluctuations.Conclusions:An.subpictus species complex consists of more than two genetically divergent molecular forms.Species A is highly divergent from the rest.Sri Lanka and India contain only species A and B.

On power series statistical convergence and new uniform integrability of double sequences

In the present paper,we mostly focus on P_(p)^(2)-statistical convergence.We will look into the uniform integrability via the power series method and its characterizations for double sequences.Also,the notions of P_(p)^(2)-statistically Cauchy sequence,P_(p)^(2)-statistical boundedness and core for double sequences will be described in addition to these findings.

Secure Transmission of Compressed Medical Image Sequences on Communication Networks Using Motion Vector Watermarking

Medical imaging plays a key role within modern hospital management systems for diagnostic purposes.Compression methodologies are extensively employed to mitigate storage demands and enhance transmission speed,all while upholding image quality.Moreover,an increasing number of hospitals are embracing cloud computing for patient data storage,necessitating meticulous scrutiny of server security and privacy protocols.Nevertheless,considering the widespread availability of multimedia tools,the preservation of digital data integrity surpasses the significance of compression alone.In response to this concern,we propose a secure storage and transmission solution for compressed medical image sequences,such as ultrasound images,utilizing a motion vector watermarking scheme.The watermark is generated employing an error-correcting code known as Bose-Chaudhuri-Hocquenghem(BCH)and is subsequently embedded into the compressed sequence via block-based motion vectors.In the process of watermark embedding,motion vectors are selected based on their magnitude and phase angle.When embedding watermarks,no specific spatial area,such as a region of interest(ROI),is used in the images.The embedding of watermark bits is dependent on motion vectors.Although reversible watermarking allows the restoration of the original image sequences,we use the irreversible watermarking method.The reason for this is that the use of reversible watermarks may impede the claims of ownership and legal rights.The restoration of original data or images may call into question ownership or other legal claims.The peak signal-to-noise ratio(PSNR)and structural similarity index(SSIM)serve as metrics for evaluating the watermarked image quality.Across all images,the PSNR value exceeds 46 dB,and the SSIM value exceeds 0.92.Experimental results substantiate the efficacy of the proposed technique in preserving data integrity.

Spatial transcriptomics combined with single-nucleus RNA sequencing reveals glial cell heterogeneity in the human spinal cord

Glial cells play crucial roles in regulating physiological and pathological functions,including sensation,the response to infection and acute injury,and chronic neurodegenerative disorders.Glial cells include astrocytes,microglia,and oligodendrocytes in the central nervous system,and satellite glial cells and Schwann cells in the peripheral nervous system.Despite the greater understanding of glial cell types and functional heterogeneity achieved through single-cell and single-nucleus RNA sequencing in animal models,few studies have investigated the transcriptomic profiles of glial cells in the human spinal cord.Here,we used high-throughput single-nucleus RNA sequencing and spatial transcriptomics to map the cellular and molecular heterogeneity of astrocytes,microglia,and oligodendrocytes in the human spinal cord.To explore the conservation and divergence across species,we compared these findings with those from mice.In the human spinal cord,astrocytes,microglia,and oligodendrocytes were each divided into six distinct transcriptomic subclusters.In the mouse spinal cord,astrocytes,microglia,and oligodendrocytes were divided into five,four,and five distinct transcriptomic subclusters,respectively.The comparative results revealed substantial heterogeneity in all glial cell types between humans and mice.Additionally,we detected sex differences in gene expression in human spinal cord glial cells.Specifically,in all astrocyte subtypes,the levels of NEAT1 and CHI3L1 were higher in males than in females,whereas the levels of CST3 were lower in males than in females.In all microglial subtypes,all differentially expressed genes were located on the sex chromosomes.In addition to sex-specific gene differences,the levels of MT-ND4,MT2A,MT-ATP6,MT-CO3,MT-ND2,MT-ND3,and MT-CO_(2) in all spinal cord oligodendrocyte subtypes were higher in females than in males.Collectively,the present dataset extensively characterizes glial cell heterogeneity and offers a valuable resource for exploring the cellular basis of spinal cordrelated illnesses,including chronic pain,amyotrophic lateral sclerosis,and multiple sclerosis.

猕猴桃果实贮藏期主要真菌病害的rDNA-ITS鉴定及序列分析

【目的】对陕西省周至县猕猴桃主栽品种‘华优’、‘海沃德’及‘秦美’贮藏期霉烂果实中的病原菌进行分离与鉴定,分析比较病原菌rDNA-ITS序列的差异和种内与种间群体分化状况,确定优势菌,为病害防治提供依据。【方法】采用病原菌形态鉴定与rDNA-ITS鉴定相结合的方法,确定引起猕猴桃霉烂的病原菌种类;通过构建系统发育树,分析病原菌种之间的亲缘关系。【结果】从贮藏期霉烂的猕猴桃果实中共分离出30株病原菌,主要为青霉属(Penicillium)、木霉属(Trichoderma)、交链孢霉属(Alternaria)、毛霉属(Mucor)、拟青霉属(Simplicillium)等5个属。其中青霉属(Penicillium)19株占63.3%,为优势菌,包括Penicillium sp.、Penicillium chrysogenum、Penicillium paneum、Penicillium purpurogenum和Penicillium commune;木霉属(Trichoderma)8株占26.7%较次之,包括Trichoderma longibrachiatum和Trichoderma sp;交链孢霉属(Alternaria)、毛霉属(Mucor)、拟青霉属(Simplicillium)各1株,各占3.3%;聚类分析表明,30株病原聚为五大类群。【结论】引起陕西省周至县‘华优’、‘海沃德’和‘秦美‘猕猴桃贮藏期果实霉烂优势病原菌为Penicillium sp.、Penicillium chrysogenum、Penicillium paneum、Penicillium purpurogenum和Penicilliumcommune;rDNA-ITS区序列在青霉菌属内种间差异鉴别具有较高的参考价值。

中国红麻炭疽病病原菌的分离鉴定及rDNA-ITS序列分析

【目的】明确中国红麻炭疽病病原菌种类。【方法】从河南、安徽、浙江、福建4省红麻主产区采集红麻炭疽病病原菌样本,进行分离、纯化,共获得16个菌株,在致病性测定及形态学特征鉴定的基础之上,从中选取AH、HN、ZJ、FJ 4个典型病原菌菌株,对rDNA-ITS区域进行基因序列分析。【结果】AH、HN、ZJ与FJ炭疽病菌株在形态特征及致病力有明显差异;rDNA-ITS序列实际长度分别为541、545、541、535 bp,AH、HN、ZJ与FJ炭疽病菌株同源性达90%—91%,而AH、HN和ZJ 3个菌株的同源性达96%—98%。【结论】从分子水平上对中国麻区红麻炭疽病病原菌种类进行鉴定分析,证实中国红麻主产区近年炭疽病发生的病原菌类型主要有黑线炭疽菌(Colletotrichum dematium)和胶孢炭疽菌(C.gioeosporioides),其中以黑线炭疽菌致病力较强,胶孢炭疽菌致病力较弱。

不同地区微小按蚊rDNA-ITS2序列差异

目的 比较我国云南、海南省、广西壮族自治区及泰国等不同地区微小按蚊核糖体 DNA第 2内转录间隔区 (ITS2 )序列差异。 方法 取单只雌蚊蚊腿消化提取 DNA,PCR特异扩增 r DNA- ITS2片段 ,并对其扩增产物纯化、测序及分析。 结果 发现 2种不同的 ITS2序列 (Gen Bank登录号 :AF4 16 783,AF4 16 784 ) ,分别与微小按蚊 A和 C同源 ,ITS2序列长度及 GC含量分别为 4 81bp,5 4 .0 4 %和 4 83bp,5 4 .2 3% ,两者无显著性差异 ;但 2 2个固定位点存在碱基置换和插入 /缺失 ,序列差异为 5 .8%。 结论 研究区内微小按蚊存在 A和

rDNA-ITS序列分析法鉴定桑椹菌核病病原菌

采集四川南充、绵阳、乐山等地果桑园中的真菌12株,利用ITS引物扩增并进行测序,构建系统进化树,分析12个真菌样品与已知物种的亲缘关系。结果表明,12个真菌样品扩增出的目的条带长度为500 bp,J1、J2、J3、J4、J5、J7、J8、J12菌株与桑实杯盘菌(Ciboria shiraiana)的序列相似度最高,J6、J9菌株与小核盘菌(Sclerotinia minor)的序列相似度最高,J10菌株与肉阜状杯盘菌(Ciboria carunculoides)的序列相似度最高,J11菌株与桑椹核地杖菌(Scleromitula shiraiana)的序列相似度最高,且序列比对分析结果同传统形态学鉴定结果完全一致。2种方法相结合最终得出结论:J1、J2、J3、J4、J5、J7、J8、J12菌株为桑实杯盘菌;J6、J9菌株为小核盘菌;J10菌株为肉阜状杯盘菌;J11菌株为桑椹核地杖菌。与传统形态学鉴定法相比,rDNA-ITS序列分析法具有准确度高,不受真菌样品生长情况、地理环境影响等优点,可避免因形态特征掌握不足而导致的不确定性,2种方法相结合在真菌鉴定中会有更广泛、更深入的应用。

rDNA-ITS在植物病原真菌分子检测中的应用

文章综述了rDNA序列结构特点及ITS序列在植物病原真菌的分类鉴定、分子检测、病害诊断及土壤中病原真菌的检测方面的应用,并对其应用前景进行了评述。

内蒙古中东部草原羽茅Epichloё属内生真菌的分布及rDNA-ITS序列系统发育

对内蒙古中东部地区分布的羽茅6个地理种群的染菌率进行了调查,采集种子并从中分离得到不同形态型的内生真菌,选取其中的19株进行rDNA-ITS片段的扩增、克隆、测序和系统发育分析。结果表明:(1)6个样地羽茅种群内生真菌感染率除西乌旗为96.7%外,其他5个样地均为100%,表明内生真菌侵染羽茅并非偶然现象,二者之间存在一种稳定的共生关系。(2)ITS和5.8S序列得到的N-J树显示,相对于Epichloё属的其他参考菌株,不同地理种群羽茅中的内生真菌聚为一类,形成一个具有97%支持强度的分支。由此推测,不同地理种群羽茅中的内生真菌具有相同的起源点。(3)结合形态观察结果和rDNA-ITS序列分析结果可以看出,羽茅内生真菌种群的优势种亲缘关系较近,可能起源于同一种内生真菌;但由于其地理分布广、气候差异大、群落类型差别也较大,从而造成不同地理种群内生真菌形态上的分化以及种群间明显的遗传分化和较高的遗传多样性。

橡胶树炭疽病菌rDNA-ITS区序列分析

应用核糖体rDNA-ITS区的序列分析研究18个不同地区来源的橡胶树炭疽病病原菌。结果表明:测试菌株的ITS扩增区(ITS3-ITS4)约为300bp,无太大的长度变异。ITS核酸序列聚类分析的结果表明:18个测试菌株被聚为2个类群,群与群之间差异不明显,2组之间同源率大约为99.3%,通过在NCBI网站的blast比对分析,结果显示2组均为胶孢炭疽菌(Colletrichum gloeosporioides)。

豇豆根腐病病原分离鉴定及其核糖体rDNA-ITS序列分析

采用形态学及分子生物学的方法对采集自武汉的豇豆根腐病分离物进行形态学和分子生物学鉴定。结果表明,菌株JGF形态学上为茄腐皮镰孢菌;对JGF菌株的核糖体RNA基因内转录间隔区(rD-NA-ITS)进行了PCR扩增和序列测定,并在GenBank中进行了Blast搜索和比对分析,根据rDNA-ITS序列分析结果结合豇豆病株症状和病菌的形态学特征,认为武汉地区豇豆根腐病的病原菌为茄腐皮镰孢菌(Fusarium solani Schl.)

旱稻孢囊线虫在广西的发生及其rDNA-ITS异质性分析

2011年,从广西龙胜梯田的水稻根系和稻田土壤中分离到一种孢囊线虫。该线虫形态特征与已报道的旱稻孢囊线虫（Heterodera elachista）相符,其rDNA-ITS序列与GenBank中旱稻孢囊线虫的rDNA-ITS序列相似性为97.7%~99.3%。rDNA-ITS分子发育树表明该线虫与旱稻孢囊线虫以100%的置信度及较小的遗传距离形成一个单系,据此将该线虫鉴定为旱稻孢囊线虫。进一步对本研究获得的10条旱稻孢囊线虫rDNA-ITS区序列及Tanha Maafi等报道的1条旱稻孢囊线虫rDNA-ITS序列（AF498391）的酶切位点进行分析,结果发现AluⅠ、AvaⅠ、MvaⅠ和RsaⅠ4个内切酶的酶切位点在11条序列中完全一致,而Bsh1236Ⅰ、BsuRⅠ和CfoⅠ3种酶的酶切位点存在变异,其中Bsh1236Ⅰ和BsuRⅠ有2种变异型,CfoⅠ则有3种变异型,表明旱稻孢囊线虫的rDNA-ITS序列存在DNA异质性。

不同地理种群美洲斑潜蝇及近缘种的rDNA-ITS1序列分析和比较

目的通过对美洲斑潜蝇Liriomyzasativae Blanchard不同地理种群及近缘种间的核糖体DNA第一内转录间隔区（rDNA-ITS1）进行比较,分析美洲斑潜蝇不同地理种群间的遗传分化情况,并为美洲斑潜蝇与近缘种间提供分子鉴别标记。方法用PCR产物直接测序法及克隆测序法对我国美洲斑潜蝇8个地理种群的rDNA-ITS1序列进行测序,并调用GenBank中3个近缘种的rDNA-ITS序列,运用软件MEGA3.1对美洲斑潜蝇不同地理种群及近缘种间的rDNA-ITS1序列进行分析。结果美洲斑潜蝇8个地理种群间的分化程度较低,只有8个变异位点,遗传距离都在0.02以下,但4个近缘种间的碱基差异显著,遗传距离为0.149-0.390,有126个变异位点,12个美洲斑潜蝇特异性识别位点。结论虽然基于rDNA-ITS1序列所显示的美洲斑潜蝇各地理种群之间的遗传分化很小,但是其分化趋势与地理分布基本相吻合;得到的12个特异性识别位点不仅可以作为美洲斑潜蝇与其近缘种间鉴别的分子标记,而且可为今后设计鉴别性PCR引物提供重要的参考依据。

剑麻茎腐病菌的rDNA-ITS序列分析

应用核糖体rDNA-ITS区的序列分析研究13个不同地区来源的剑麻茎腐病菌,测序结果表明,参试菌株的ITS扩增区约600 bp,无太大的长度变异,同时通过在GenBank中搜索其同源性序列并构建它们的系统发育树,结果表明,引起剑麻茎腐病的病原为黑曲霉菌Aspergillus niger。

浙江4地桃花水母的rDNA-ITS序列分析

采用PCR和DNA测序技术,对浙江省4个地点桃花水母(Craspedacusta)的核糖体RNA基因内转录间隔区(rDNA-ITS)进行了扩增与测序,并与GenBank中已有的桃花水母ITS区基因序列进行比对分析,计算了它们的遗传距离,利用MEGA 4.1构建了系统发育树。结果显示:这4个地点的桃花水母与索氏桃花水母(Craspedacusta sowerbyi)的ITS区基因相似度极高,同源性都在97%以上,遗传距离保持在0～0.008之间,在进化树中与索氏桃花水母聚为同一支。研究结果表明,这4地的桃花水母都属于索氏桃花水母(C.sowerbyi)。