松材线虫与拟松材线虫rDNA中ITS区的比较研究

本研究利用PCR－RFLP和DNA测序技术分析了松材线虫和拟松材线虫rDNA中的ITS区。ITS区界于rDNA的18S和28S基因之间,并被5.8S基因分为ITS1和ITS2两个区。5种限制性内切酶酶切结果表明,松材线虫和拟松材线虫rDNA的ITS区存在着显著的差异;在松材线虫不同来源株系中,来自中国和日本的株系的酶切图谱一致,与来自加拿大的株系有着微小差别。对南京和日本的松材线虫及富阳的拟松材线虫株系ITS1区的序列分析显示在ITS1的280bp中,南京和日本的松材线虫株系之间,同源性高达99.7%,而来自富阳的拟松材线虫与南京和日本的松材线虫ITS1区序列的同源性相对较低,分别为90％和89.6%。     

基于核糖体DNA第二内部转录间隔区(ITS2)序列鉴定四季青及其近缘种和混伪品

中药材四季青(Ilicis Chinensis Folium)的基原植物冬青(Ilex chinensis)是重要的园林观赏物种。利用DNA条形码技术可以快速、准确鉴定四季青及其近缘种和混伪品。本研究以冬青、铁冬青(Ilex rotunda)、秤星树(Ilex asprella)、枸骨(Ilex cornuta)和女贞(Ligustrum lucium)等37份植物样本为实验材料,提取全基因组DNA,扩增核糖体DNA第二内部转录间隔区(internal transcribed spacer 2,ITS2)序列并进行双向测序。测序结果利用Codon Code Aligner 4.2.7进行序列拼接,获得高质量ITS2序列。同时从Gen Bank中获得28条冬青的同属近缘种以及混伪品女贞、四川山矾(Symplocos setchuensis)的ITS2序列。基于隐马尔科夫模型(hidden Markov model,HMM)注释5.8S和28S,获得完整的ITS2区域,进而应用MEGA6.0(molecular evolutionary genetics analysis)进行冬青种间和种内序列分析,计算种内种间K2P(Kimura 2-parameter)遗传距离,构建邻接树(neighbor-joining tree,NJ tree)。结果表明,琼脂糖凝胶电泳得到清晰明亮的均一条带,说明利用通用的ITS2引物可以成功扩增到用于测序的PCR产物。通过PCR产物测序、数据处理和序列分析可知,冬青种内ITS2长度经过比对后为239 bp,全部样品的种间ITS2比对后长度为269 bp。冬青种内有4个变异位点以及6个单倍型,种间有143个变异位点,远远多于其种内变异位点。MEGA6.0分析结果表明,种间最小K2P遗传距离(0.094)大于种内最大K2P遗传距离(0.013)。邻接树显示,女贞和四川山矾分别以100%的自展支持率分别聚类,表现出良好的单系性,冬青属物种聚为一大支。冬青在冬青属下可以单独聚为一支,可以进行区分。冬青属其他物种以同一亚属聚为一支。研究结果表明,作为一种快速、准确、简便的分子生物学鉴定方法,ITS2可以应用于冬青及其近缘种和混伪品的鉴定,同时对于准确鉴定冬青属物种亦有巨大潜力,可为冬青属植物在临床上的用药安全和开发应用提供科学依据。     

基于ITS序列和RAMS标记分析青枯雷尔氏菌的遗传多样性*

应用ITS序列和RAMS技术对福建省不同地域,不同寄主作物的青枯雷尔氏菌进行遗传多样性研究。结果表明,供试的21株青枯雷尔氏菌的ITS区序列有3种类型,这3种类型菌株的ITS序列只有1-2个碱基的差异,与参比菌株GMI1000的ITS区序列或者相同或者有1-2个碱基的差异。在此基础上,利用RAMS技术进一步分析表明,供试的青枯雷尔氏菌及参比菌株GMI1000的基因组DNA间存在丰富的多态性。聚类结果显示,供试菌株可聚为3个类群,同一寄主作物的菌株聚在同一类群中,同一地理来源的菌株聚在同一亚类群中,说明青枯雷尔氏菌的遗传分化与寄主作物和地理来源都存在相关性,与寄主作物的关系更密切。     

河南小麦黑胚病优势病原菌及其近似种rDNA-ITS序列分析

从河南省具有地域代表性的5个不同地区分离小麦黑胚病原菌Alternaria spp.菌株17株,经形态鉴定主要为链格孢属两个近似种A.alternata和A.tenuissima.通过与GenBank中登录的链格孢属中的9个种的rDNA-ITS序列比较分析,发现所有供分析菌株具有很高的同源性,其中与6个小孢子种A.alternata、A.tenuissima、A.mali、A.gaisen,A.citri、A.arborescens同源性为98%～100%,在聚类图上成为一支,而3个大孢子种A.radicina、A.porri和A.solani聚为另外一支,可明显地与其它小孢子种区分开.通过对Alternaria 58个菌株的rDNA ITS序列分析发现,同一个种或相似种内不分地域范围和寄主都有很高的保守性.与小孢子种相比,3个大孢子种在位点97-171、365-402、443-490处具有各自的特异位点,所以这些序列有可能作为它们分类鉴定、分子标记及系统发育的重要依据.分析结果表明,ITS1-5.8S-ITS2序列分析只能验证小麦黑胚病菌形态学鉴定的结果,但不能用于链格孢近似种的分类鉴定.     

与黄瓜感花叶病毒病基因连锁的分子标记

本研究以黄瓜(Cucumis sativus L.)高感黄瓜花叶病毒(CMV)和高抗CMV亲本组合(HZL04-1×F-3)的F2分离群体为试材,采用极端个体分组法和AFLP技术筛选与黄瓜抗CMV基因连锁的分子标记.AFLP引物组合E22M88在抗病组和感病组间约200 bp处表现多态性.经F2单株分析,在感病亲本和感病单株中扩增出了约为200 bp的特异片段,而抗病亲本和抗病个体无此条带.该特异标记与CMV抗性基因相连锁,遗传距离为9.74 cM.测序结果显示,目标片段的大小为202 bp,并将该标记转换为了序列特征化扩增区域(SCAR)标记.提供了黄瓜材料CMV抗性鉴定的分子方法和手段,可用于分子标记辅助育种.     

种植模式对当归根际土壤真菌群落与功能类群的影响

再植障碍问题限制了当归产业健康发展。为了建立高效种植方式,在甘肃渭源县设置5种种植方式[A：豌豆-小麦-当归;B：豌豆-蒙古黄芪-当归;C：豌豆-马铃薯-当归;D：豌豆-当归-当归（对照）;E：豌豆-休耕-当归],采挖期通过Illumina Hisqe 2500高通量测序平台测定了不同种植模式下当归根际土壤真菌ITS1变异区的基因序列,分析不同种植方式对当归根际土壤真菌群落和功能差异性的影响。结果表明：（1）5种种植方式下当归根际真菌多样性差异不大,较对照D处理,A、B、C和E处理的Chao1指数、Ace指数和Shannon指数较低,Simpson指数较高。（2）5种种植方式下当归根际土壤真菌群落隶属于11门167属,其中,子囊菌门（Ascomycota）、被孢菌门（Mortierellomycota）、担子菌亚门（Basidiomycota）和壶菌门（Chytridiomycota）为优势门;被孢霉属（Mortierella）、四枝孢属（Tetracladium）为优势属。（3）冗余度分析发现：在门水平影响土壤真菌群落结构组成的主要因子为速效钾、电导率、pH值,在属水平影响土壤真菌群...     

休耕和种植作物对黑麻土壤肥力的影响

为了明确不同茬口土壤养分的固持残留水平,建立高效合理的轮作顺序以解决连作再植障碍问题。2018年在甘肃定西渭源县莲峰镇绽坡村随机区组设计、“S”形取样休耕1季、种植1季春小麦、马铃薯、蒙古黄芪和当归后0-20 cm层次的黑麻土壤,测定土壤全量、速效养分和阴阳离子含量。结果表明:休耕1季和种植1季蒙古黄芪增加了0-20 cm层次土壤的有机质、全氮、速效磷、速效钾、阴阳离子、Mg^2+、SO4^2-和Cl^-含量,种植1季当归降低了0-20 cm层次土壤的有机质、全氮、速效磷、速效钾、阴阳离子、Mg^2+、SO4^2-和Cl^-含量。方差分析显示,休耕1季和种植1季蒙古黄芪后0-20 cm层次黑麻土壤的有机质、全氮、速效磷、速效钾、阴阳离子、Mg^2+、SO4^2-和Cl^-含量显著高于种植1季当归。主成分回归分析发现,休耕1季和种植1季蒙古黄芪后0-20 cm层次黑麻土壤肥力综合指数为0.98和0.56,种植1季当归的综合指数得分仅为0.07。休耕1季是黑麻土壤用养结合的简便有效方法,种植1季蒙古黄芪是培肥黑麻土壤肥力的适宜作物。     

抗白粉病糯性小麦的多重PCR分子鉴定技术

为同时鉴定小麦糯性和白粉病抗性,本研究利用已知3对引物建立多重PCR体系,分别扩增WxA1、Wx-B1、Wx-D1和Pm21基因的分子标记,其目的片段大小分别为:230/265、854、204和1400 bp。经过对供试品种和部分F2分离群体的Wx蛋白进行SDS-PAGE电泳检测,其结果与多重PCR结果一致,证明该多重PCR体系准确可靠,可提高小麦糯性兼抗白粉病的选育效率。     

ITS鉴定木材种类：一例司法鉴定研究实例

本研究选取一例非法收购国家重点保护植物油丹的司法鉴定案作为研究实例,在确认鉴定技术可行的前提下,应用ITS序列分析对待检测的原木进行了分子鉴定。本研究采用CTAB法提取原木DNA,对其提纯后进行ITSrDNA的PCR扩增,再对PCR阳性产物进行克隆、测序,将测序结果进行Blast分析和构建系统发育树。结果表明,送检的47根原木中有9根被鉴定为油丹。本研究采用ITS序列系统发育分析方法,成功鉴定了原木种类,为今后类似司法鉴定案件的审理提供了可靠的鉴定意见。     

斑茅杂种后代分子检测

利用RAPD随机引物和斑茅5S rRNA间隔序列(ITS)引物对斑茅与甘蔗的杂种F1、F2代进行了分子鉴定，获得了与含有斑茅血缘有关的RAPD分子标记3个及5S rRNA间隔序列标记1个；其中，5SrRNA间隔序列标记可用于斑茅杂种后代碱法DNA模板的快速检测。     

Phylogeny of Astragalus in China: molecular evidence from the DNA sequences of 5S rRNA spacer, ITS, and 18S rRNA

Radix Astragali (root of Astragalus; Huangqi) is a traditional Chinese medicine commonly used as an immunostimulant, hepatoprotective, diuretic, antidiabetic, analgesic, expectorant, and sedative drug. Although the species of Radix Astragali have been defined as Astragalus membranaceus and A. membranaceus var. mongholicus in Pharmacopoeia of China, their taxonomy remains controversial. The phylogenetic relationships among 10 Astragalus taxa, which are commonly found in China including A. membranaceus, A. membranaceus var. mongholicus, Astragalus propinquus, Astragalus lepsensis, Astragalus aksuensis, Astragalus hoantchy, Astragalus hoantchy subsp. dshimensis,Astragalus lehmannianus, Astragalus sieversianus, and Astragalus austrosibiricus, were determined using the DNA sequences of the 5S ribosomal RNA (5S rRNA) spacer, internal transcribed spacer region (ITS), and 18S rRNA coding region. The 5S rRNA spacer, ITS, and 18S rRNA, amplified by polymerase chain reaction from the isolated genomic DNAs, were sequenced. By using neighbor-joining and maximum parsimony analyses, phylogenetic trees were mapped by their sequence diversity. A. membranaceus and A. membranaceus var. mongholicus shared the greatest sequence homology. In addition, A. propinquus shared a closer relationship with A. membranaceus and A. membranaceus var. mongholicus, while other Astragalus species were less closely related. This is the first paper to show the phylogenetic relationship of Astragalus species related to Radix Astragali in China by the molecular genetic approach.     

ITS1-rDNA-based methodology to identify world-wide hake species of the Genus Merluccius

Species-specific DNA-based tags are valuable tools for the management of both fisheries and commercial fish products. In this study, we have developed a two-step molecular tool to detect the presence of hake DNA (Merluccius spp.) and to identify the exact hake species present in an blind sample. The first test involves PCR amplification of an ITS1-rDNA fragment of 193 bp using nested primers that are interspecifically conserved in Merluccius spp. and Atlantic cod, Gadus morhua. The second test consists of the PCR amplification of a 602-659 bp DNA fragment spanning part of the ribosomal cluster 18S-ITS1-5.8S and digesting it with four restriction enzymes whose targets map at interspecifically nonconserved sites of the ITS1. Alternatively, the identification of hake species can be achieved by FINS or BLAST, using the nucleotide sequence of either the whole ITS1 sequence or its nested fragment of 193 bp. Because of their high reproducibility and ease of execution, these procedures allow for routine analysis and constitute high reliable tools for the rapid identification of 12 species of hake.     

Identification of Chinese medicinal fungus Cordyceps sinensis by PCR-single-stranded conformation polymorphism and phylogenetic relationship

Fungi belonging to the Cordyceps species have long been used as food and herbal medicines in Asia and are especially popular as commercially available powdered supplements. Despite this acceptance and use, little is known of the phylogenetic relationships of the genus. Presently, the neighbor-joining method based on the ITS1, 5.8S rRNA, and ITS2 regions was used to construct a phylogenetic tree of 17 Cordyceps isolates. Five major groups were evident. Cordyceps sinensis was less closely related to 15 Cordyceps species but shared a closer relationship with Cordyceps agriota. PCR-single-stranded conformational polymorphism was applied to differentiate seven Cordyceps isolates: five were different from those used to construct the phylogenetic tree, based on differences in the internal spacer 2 (ITS2). The length of ITS2, amplified by primers 5.8SR and ITS4, vary between 334 and 400 bp. This segment could be used for intraspecies classification or detection of mutations and represents potential novel means of identification of this fungal genus in herbal medicines and in quality control applications in the fermentation industry.     

Efficiency of DNA typing methods for detection of smoked paprika "pimenton de la vera" adulteration used in the elaboration of dry-cured Iberian pork sausages

The purpose of this work was to develop a PCR method for the identification of smoked paprika "Pimento?n de la Vera" adulteration with paprika elaborated from varieties of pepper foreign to the la Vera region, in central western Spain. Three autochthonous varieties of pepper, Jaranda, Jariza, and Bola, and the varieties Papri Queen, Papri King, Sonora, PS9794, and Papri Ace, foreign to the La Vera region, were used in the study. Analyses of the ITS and 5.8S rDNA, RAPD-PCR, SSR, and ISSR were tested. RAPD-PCR, SSR, and ISSR analyses allowed differentiation among the varieties of paprika analyzed. There was no difference in the sequence of ITS1-5.8S rDNA-ITS2. In addition, with the RAPD-PCR primers S13 and S22, two molecular markers were obtained of 641 and 704 bp, respectively, which allowed all of the smoked paprika varieties to be differentiated from paprikas elaborated with the five foreign varieties. These two molecular markers were investigated as a basis for detecting the adulteration of smoked paprika with paprika elaborated from foreign varieties of pepper.     

ITS sequence-based identification and utilization evaluation of <Emphasis Type="Italic">“</Emphasis>Nanjiang” <Emphasis Type="Italic">(Lonicera similis</Emphasis> Hemsl.), a local cultivar in Sichuan,China

The internal transcribed spacer (ITS) region was employed to analyze phylogeny of 20 populations, including the local cultivar “Nanjiang”, belonging to 7 species of Lonicera L. 20 ITS sequences that we obtained range from 614 to 618 bp, and the G+C content was higher than 60% with the average value 63.5%. The sequences of 5.8S nrDNA was highly conservative in both length and component, while ITS2 had more variation than ITS1 in sequence component and length. For the ITS complete sequence, the mutation and information sites were 139 and 117 with the content of 22.38 and 18.84%, respectively. The dendrogram analysed by neighbor-joining method showed that “Nanjiang” and Lonicera similis Hemsl. comprised a same cluster with a higher bootstrap support, indicating “Nanjiang” would be a variation or subspecies of L. similis Hemsl. The results of antibacterial activity test and active compounds identification showed that “Nanjiang” had the best antibacterial activity and the highest yield of chlorogenic acid, the active component of Flos Lonicerae, in the six species of Lonicera L.     

河北安国7种中药材AM真菌遗传多样性研究

为探明药用植物根围AM真菌遗传多样性,于2010年8月在河北省安国市3个样地(中药材种植基地、霍庄、大户村)用随机抽样法采集7种中药材根围土壤样品,分离筛选双网无梗囊霉(Acaulospora bi-reticulata)为试验菌种,进行孢子DNA提取、PCR扩增、序列测定及聚类分析,研究了双网无梗囊霉遗传多样性与土壤因子的关系,并选取两条代表菌株所测序列,连同从NCBI数据库中下载的4属28种AM真菌相应序列构建系统发育树。结果表明,双网无梗囊霉可侵染不同样地不同宿主植物,3个样地7种中药材双网无梗囊霉DNA相似性达99.2%以上,其遗传特征保持了高度稳定性,双网无梗囊霉在样地和植物间具有广谱性。同一样地不同中药材根围土壤因子相似性很高,AM真菌DNA碱基序列相似系数也很高,而不同样地同一中药材根围AM真菌DNA碱基序列相似系数低于前者,可见遗传多样性与土壤因子密切相关。土壤因子对双网无梗囊霉基因序列的影响大于宿主植物的影响。     

不同施肥方式与保水剂互作对河西地区黄芪土壤微环境及产量的影响

土壤干旱缺水和有机质亏缺是限制甘肃河西地区黄芪产业发展的关键因素。为探索促进河西地区土壤蓄水保肥和土壤微环境改善的施肥措施，通过连续2年大田试验，设置保水剂+有机肥+普通尿素(B+OM+N)、保水剂+有机肥+缓释尿素(B+OM+HN)、保水剂+普通尿素(B+N)、有机肥+普通尿素(OM+N)、有机肥+缓释尿素(OM+HN)和单施普通尿素(CK)6个处理，探讨了不同施肥方式与保水剂互作对黄芪土壤水分、养分等微环境指标的影响，并分析了其与产量之间的相关性。结果表明：随着土层深度的加深，土壤水分含量先升后降，有机质、速效氮磷钾及微生物生物量碳、氮(MBC、MBN)含量逐渐降低，NH₄⁺-N和NO₃^–-N含量逐渐增加，MBC/MBN比先增后减；随着生育期的推进，土壤水分、NH₄⁺-N和NO₃^–-N含量逐渐降低，有机质、MBC、MBN含量和MBC/MBN比逐渐增加，碱解氮、速效钾含量先升后降，有效磷含量波动式下降；不同土层...     

Phylogenetic relationships in the North African genus <Emphasis Type="Italic">Hedysarum</Emphasis> as inferred from ITS sequences of nuclear ribosomal DNA

Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were analysed to precise their length (637–643 bp) and resolve phylogenetic relationships among eight Mediterranean species of the genus Hedysarum (Fabaceae). The infra-specific variability levels of the ITS sequences of spontaneous population of H. coronarium proved a lack of polymorphism both in the length and in the sequences examined in this species. Hence, a consensus ITS sequence characterising each Hedysarum species has been investigated for analysis of inter-specific polymorphisms. The level of variation of ITS sequence was high enough to make the ITS1 and ITS2 a useful tool for phylogenetic reconstruction. However, ITS2 seems to be relatively more polymorphic and more informative than ITS1 regarding length or GC percent. The phylogenic relationships in the genus Hedysarum based on ITS1 and ITS2 sequences taken independently or together, are discussed in the context of current work in molecular biosystematics. Results exhibited the distinctiveness of the two H. spinosissimum subspecies (i.e. H. spinosissimum ssp. capitatum and H. spinosissimum ssp. spinosissimum). In addition, the great similarity of the ITS sequences between H. coronarium (the only cultivated species of the genus) and H. carnosum suggests the usefulness of the latter in selection programmes to improve pastoral production in semi-arid areas.     

Assessment of genetic diversity, and phylogenetic relationships based on ribosomal DNA repeat unit length variation and Internal Transcribed Spacer (ITS) sequences in chickpea (Cicer arietinum) cultivars and its wild species

Repeat unit length variation and internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA were used to assess genetic diversity, and phylogenetic relationships in chickpea (C. arietinum) cultivars, and its related wild species. Total genomic DNAs of 76 accessions of 10 Cicer species, belonging to three sections of the genus, were restricted with seven enzymes and the restriction fragments were hybridized to heterologous ribosomal clones of wheat pTa71 and Vicia faba probes Ver 6-5 and Ver18-6. A single repeat unit length class of 11.4 kb or 10.5 kb was recognized across Cicer accessions with pTa71. The intraspecific variation was negligible in those species where more than one accession was studied, except the four C. judaicum accessions, which were different from the rest. EcoRI and DraI digests gave two and one-two fragments, respectively. All the accessions produced three and three-five bands with BamHI and SacI, respectively. Both the accessions of C. yamashitae differed in their rDNA repeat unit length as well as restriction site variation. Maximum likelihood tree with rDNA RFLP recognized five clades which were more or less congruent with the previous data. Length of ITS-1 region was more variable (235–239 bp) than the ITS-2 region (212–213 bp). Cladistic analysis of ITS data revealed two major clades, clade I consisting of C. arietinum, C. reticulatum and C. echinospermum, and clade II comprised of C. judaicum, C. chorassanicum, C. bijugum and C. cuneatum. C. microphyllum grouped with the above four species. C. pinnatifidum was present as a separate branch. C. yamashitae emerged as the most distinct species.     

Phylogenetic relationships among species of Citrullus and the placement of C. rehmii De Winter as determined by Internal Transcribed Spacer (ITS) sequence heterogeneity

The internal transcribed spacer regions (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA from the four recognized species of Citrullus (C. lanatus var. lanatus (Thunb.) Matsum. & Nakai., C. lanatus var. citroides (Bailey) Mansf., C. colocynthis (L.) Schrad., C. ecirrhosus Cogn., and C. rehmii De Winter) and Acanthosicycos naudinianus (Sond.) C. Jeffrey were amplified by PCR, and direct sequenced. Within the taxa examined, the length of ITS1 varied from 216 bp to 219 bp, and ITS2 varied from 239 bp to 249 bp. The average %CG content ranged from 59 to 64% and from 62 to 66% for ITS1 and ITS2, respectively. The greater length variation observed in ITS2 was primarily attributable to the occurrence of a (CC)_n microsatellite. Cladistic (PAUP) and phenetic (MEGA) analyses resulted in highly resolutive trees. ITS sequence analysis placed the recently described C. rehmii adjacent to the cultivated watermelon and supported the validity of the species classification of this taxa.