Intergenic spacer 1(IGS1) polymorphism map: A marker for the initial classification of cultivated Lentinula edodes strains in China

China is currently the world＇s leading producer of Lentinula edodes and owns many cultivated strains of this species. This study was performed in order to investigate intergenic spacer I （IGS1） polymorphism and classification among 49 popular cultivated strains. The great majority of the 49 strains possessed two different IGS1 sequences, with distinct lengths and homologies. Based on the length and homology of the IGS1 sequences of the 49 strains, the strains were classified into two groups： A and B. Group A was subdivided into six subgroups. Forty-seven strains were homozygous or heterozygous among these six subgroups in group A, Cr01 was heterozygous between A and B, and Guangxiang 9 was homozygous in group B. An IGS1 polymorphism map of each cultivated L. edodes strain is reported for the first time and could be used as a marker for the initial classification and management of cultivated L. edodes strains in China.

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.

栽培稻种内rDNA基因IGS序列分析及系统学意义

以普通野生稻为对照,将rDNA基因间区(IGS)序列用于栽培稻种内不同亚种以及亚种内不同品种的亲缘关系分析。结果表明,栽培稻种内IGS序列长度为2130～2145bp,G+C含量为74.59%～75.29%,变异位点229个,占10.70%,信息位点76个,占3.51%。IGS序列中籼亚种和粳亚种之间有38个亚种标志性碱基差异,主要分布在IGS5′端近400bp的序列中。用IGS序列构建的系统树能将栽培稻的籼亚种和粳亚种分为两大类,亚种内不同亲缘关系的品种也能区分开。本研究结果支持爪哇稻为栽培稻中一个独立亚种的观点。

IGS在小麦叶锈菌中的多态性分析初探

由小麦叶锈菌引起小麦叶锈病是我国小麦重要病害之一。为研究小麦叶锈菌流行群体的毒力构成和亲缘关系,利用IGS(Intergenic spacer)特殊引物(L318和5SK)对71个小麦叶锈菌IGS进行扩增检测。结果表明小麦叶锈菌小种间的IGS存在多样性,但多样性与小种毒力之间不存在直接相关性,和小种的区域性有一定的相关性。进一步对差异带型进行分析,获得IGS-1、IGS-2的序列。IGS在小麦叶锈菌种内存在多态性,这有益于开发和建立IGS分子标记,并为研究锈病流行群体结构和追溯年度流行的病原来源,以及该病菌的生物学进化提供依据。同时利用IGS可以预测病害发生的流行群体,进而有针对性的种植抗病品种来防治叶锈病大发生。

黑龙江省瓜类白粉病菌IGS-RFLP分析

应用通用引物对采自黑龙江省不同地区的甜瓜、黄瓜、南瓜和西瓜等16份瓜类白粉病菌进行转录基因间隔区(IGS)分析,结果表明不同菌株的IGS区存在长度异质性和数量多态性。应用MspⅠ、AsuⅠ、Hinp1Ⅰ3个限制性内切酶对IGS扩增产物进行酶切(IGS-RFLP),不同菌株的酶切位点不同,电泳后产生多样性丰富的图谱,成为菌株特有的DNA指纹。利用NTSYS-PC软件对IGS1-RFLP酶切图谱进行数据分析,各菌株之间遗传相似系数的变化幅度为0.4600～0.8000,表明白粉病菌间有丰富的遗传多样性,以遗传相似系数0.60为阈值,供试菌株可区分为3个类群。初步确定葫芦科白粉病菌致病性与DNA多态性不形成对应关系,菌株的遗传多样性与菌株地理来源、寄主来源及设施类型亦无明显的直接关系。

格特隐球菌IGS5b基因亚型和a交配型菌株的鉴定

目的鉴定1株格特隐球菌VGⅢ基因型中的少见基因亚型IGS5b及a交配型菌株。方法采用PCR指纹法、基因内间隔区(IGS)和磷脂酶基因(PLB1)测序分析鉴定基因型。采用PCR特异扩增法和交配试验鉴定交配型。采用GEF1基因序列分析同步鉴定基因型和交配型。结果结合PCR指纹法和序列分析,鉴定受试株RV63979为VGⅢ基因型中的少见基因亚型。针对交配型位点内STE12和STE20基因的特异性引物均扩增阴性。交配试验证实为a交配型。GEF1位点测序准确鉴定其基因型和交配型。结论本文通过多种鉴定手段结合IGS序列分析,鉴定1株VGⅢ基因型中的少见基因亚型IGS5b,证实VGⅢ基因型中至少存在IGS5a和IGS5b2种基因亚型。交配型分析表明该菌为VGⅢ基因型中少见的a交配型。

一株嗜水气单胞菌Aeromonas hydrophila的16S-23S rDNA间区序列分析及应用

根据细菌的16SrDNA3′端和23SrDNA5′端的高度保守区设计引物,扩增了一株中华鳖Trionyxsinensis"红板病"病原菌嗜水气单胞菌的16S-23SrDNA间区,克隆到pGEM＿T载体上,测序。用BLAST和DNAstar软件对16S-23SrDNA间区序列以及其内tRNA基因与已发表的嗜水气单胞菌和近缘种的IGSG序列进行比较分析,设计出对嗜水气单胞菌特异的PCR引物,建立了检测嗜水气单胞菌的PCR方法,进行了特异性、灵敏性检测,并用此方法检测了不同的水样。

香菇菌株分子鉴别技术的分辨率比较

本研究运用酯酶同工酶、RAPD、IGS1和IGS24种方法鉴别2个野生香菇菌株和19个栽培香菇菌株,并比较这4种鉴别方法的分辨率,结果表明:16条酯酶同工酶带中有15条具有多态性,将21个供试菌株分成11个类型,分辨率为0.92;10个随机扩增引物共扩增出86个DNA片段,其中95.3%具有多态性,将21个供试菌株分成16个类型,分辨率为0.97;IGS1将21个供试菌株分成7个类型,分辨率为0.81;IGS2将21个供试菌株分成7个类型,分辨率为0.73。这4种鉴别方法中RAPD的分辨率最高,与这4种鉴别方法的综合分析结果相同,因此RAPD可以作为香菇菌株鉴别的可靠依据。

白茅45S rDNA IGS的克隆及序列分析

本研究以云南2009-48和广东2010-13为实验材料,通过PCR方法扩增克隆了2种不同的白茅的45S rDNA IGS。序列分析结果表明:云南2009-48的45S rDNA IGS序列长度为2 613 bp,GC含量为64.6%;广东2010-13的45S rDNA IGS序列长度为2 882 bp,GC含量64.4%;它们的IGS序列一致性为84.5%。45S rDNA IGS均由NTS和ETS组成:云南2009-48的NTS和ETS长度分别为1 441 bp和1 172 bp,GC含量分别为63.5%和66.1%;广东2010-13的NTS和ETS长度分别为1 719 bp和1 163 bp,GC含量分别为64.2%和65.3%;它们的NTS序列一致性为76.5%,而ETS的NTS序列一致性高达96.2%。NTS中存在4种类型的亚重复序列(SR1,SR2,SR3,SR4),ETS中存在1种类型的亚重复序列(SR5),序列一致性为74.0%~93.0%。通过软件辅助分析以及与其他植物的比较分析,我们发现了预测的转录起始位点和转录终止位点,并且也存在着大量的甲基化位点,它们在调控rRNA转录方面起着非常重要的作用。此外,聚类分析结果表明白茅与甘蔗的亲缘关系相对较近,为拓宽甘蔗遗传基础提供了理论依据。

正阳县花生根瘤菌遗传多样性及其共生特性研究

从河南省正阳县土壤中捕捉67株根瘤菌,根据IGS-RFLP结果分成9个基因内间隔区(intergenic spacer,IGS)类型,通过16S rRNA基因序列系统发育分析鉴定菌株为慢生根瘤菌属(Bradyrhizobium),并依据持家基因多位点序列分析(multilocus sequence analysis,MLSA)结果将其分成7个簇(Clusters)。其中,Clusters 1被鉴定为广东慢生根瘤菌(B.guangdongense),Clusters 2、3、4、5和6分别被鉴定为慢生根瘤菌属的疑似新种群B.genospeciesⅡ、Ⅲ、Ⅳ、Ⅴ和Ⅵ。选取11个代表菌株,结瘤基因nodA序列与广东慢生根瘤菌CCBAU 51649T和广州慢生根瘤菌(B.guangzhouense)CCBAU 51670T聚为相同分支,宿主均为花生。所有代表菌株均能与花生植株共生结瘤,且叶绿素含量、地上部干质量和地下部干质量、株高度、根长度与对照相比均有显著提升,其中接种ZB30的处理组的地上部干质量是对照组的2.7倍。

The Amplification and Application of Ribosomal RNA (rDNA) Gene Sequences of <i>Blidingia minima</i>(Chlorophyta, Blidingia)

The sequence of the ribosomal RNA gene (rDNA) plays an important role in species identification and phylogenetic analysis. However, the only published full-length sequence of a ribosomal gene of green algae is that of Ulva mutabilis. In this study, we amplified the full-length sequence of each ribosomal gene unit of the ribosomal gene of Blidingia minima. The full-length sequence of the ribosomal gene in Blidingia minima is 8676 bp, including the 1759 bp 18S rDNA, 576 bp internal transcribed spacer (ITS) + 5.8S rDNA, 3282 bp 28S rDNA, and 3059 bp intergenic spacer (IGS) region. We then carried out a series of genetic analyses based on the ITS and IGS sequences, to verify whether IGS sequences are useful for studying the genetic diversity of green algae from different locations. We amplified the IGS sequences of Blidingia minima from 10 different locations in the Yellow Sea. Multiple alignments of the IGS sequences of samples from these 10 different sites revealed varying degrees of base differences, and comparative analysis of the ITS sequences revealed that our amplified species was classified as Blidingia minima and distinct from other green algae. In conclusion, our full-length amplified ribosomal gene provides useful information to enrich the data on green algae ribosomal genes and provides an effective molecular marker for the analysis of the interspecies and intraspecies relationships of Blidingia minima.

国内临床分离阿萨希毛孢子菌3个新基因型分析

目的通过对IGS1区基因序列分析,明确国内临床分离8株阿萨希毛孢子菌(T.asahii)的基因型,为T.asahii感染的流行病学研究提供依据。方法以国内不同地区、不同患者临床分离的8株T.asahii为研究对象,PCR扩增、克隆IGS1区,寻找基因突变位点,构建系统发生树,明确国内T.asahii的基因型。结果 IGS1区长度均为643bp,部分菌株碱基发生突变;8株T.asahii共发现5个基因型,其中3株为国际上首次发现的新基因型,分别命名为基因型10(BZ702)、11(BZ704)、12(BZ901),3株为基因型1(BZ701、BZ705、BZ706),2株为基因型4(BZ703、BZ902)。结论中国的T.asahii菌株具有不同于其他国家的多种基因型,种内多样性更丰富,这可能与中国地域广阔有关,为T.asahii感染的流行病学研究提供了依据。

一株病原菌的API生化鉴定及其16S—23S rDNA间区序列分析

目的建立针对在生化鉴定结果不准确的情况下,利用分子生物手段对病原菌进行鉴定的方法。方法将分离出的一株细菌经过普通的形态学观察和氧化酶试验,用肠杆菌科生化试纸条(API20E)做生化鉴定。并利用细菌通用引物对细菌的16S—23S rDNA间区进行PCR扩增,测序,利用BLAST在线同源性查询软件查询所测片段的属性。结果 API20E生化鉴定结果可能是河流弧菌,BLAST比对结果为嗜水气单胞菌(GenBank accession No:FJ013143)。结论传统的表型分类在细菌鉴定中已逐步与各种基因型分类水平的细菌鉴定方法相结合,在实际工作中应注意有效使用。