用AFLP技术和16S rDNA PCR|RFLP分析毛苜蓿根瘤菌的遗传多样性

采用选择性扩增片断长度多态性(简称AFLP)DNA指纹技术对采自我国云南省与西藏交界的高山地区的野生型豆科植物毛苜蓿根际土样分离的291株毛苜蓿(Medicago edgeworthii)根瘤菌进行遗传多样性的研究。从AFLP图谱中,揭示出毛苜蓿根瘤菌有较显著的遗传多样性,从291株中选择出90个代表株用计算机进行树状图的分析。结果表明,所分析的菌株在79%的相似性水平上聚类成3个群。对这90个代表株进行多聚酶链反应(PCR)扩增的16S rDNA的4种限制性内切酶长度多态(简称16S rDNA PCR|RFLP)分析,得出2个不同的16S rDNA PCR|RFLP类型的菌株。分别选出这2个类型的代表菌株与各种根瘤菌的参比菌株进行16S rDNA PCR|RFLP分析,再进行树状图的分析,初步得出了它们在根瘤菌系统分类中的地位。分析结果表明：毛苜蓿根瘤菌与根瘤菌属中的Rhizobium mongolense的相似性很高。     

斜茎黄芪根瘤菌的16S rDNA和23S rDNA PCR-RFLP比较分析

在表型性状数值分析和ＡＦＬＰ指纹图谱分析的基础上,选取５４株斜茎黄芪根瘤菌的代表菌株及已知根瘤菌参比菌株,进行１６ＳｒＤＮＡ和２３ＳｒＤＮＡ的ＰＣＲ－ＲＦＬＰ比较分析。结果表明斜茎黄芪根瘤菌具有极大的系统发育多样性,分别具有２４个１６ＳｒＤＮＡ遗传图谱类型和２２个２３ＳｒＤＮＡ遗传图谱类型,１６ＳｒＤＮＡ与２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ聚类分析树状图谱有较好的一致性,但也存在一些差异。在对较大类群的划分上,它们的结果与表型性状数值分析结果有较好的一致性。将１６ＳｒＤＮＡ和２３ＳｒＤＮＡＰＣＲ－ＲＦＬＰ分析     

用AFLP技术研究花生根瘤菌的遗传多样性

采用AFLP分子标记技术,对分离自中国、津巴布韦、以色列的133株慢生花生根瘤菌(%Bradyrhizobium %sp.%arachis)%和13个代表菌株(%Bradyrhizobium japonicum. Bradyrhizobium elkanii)%的DNA扩增长度多态性进行了分析,并根据各供试菌株的遗传相似性进行了数值聚类。结果表明慢生花生根瘤菌群体内存在很高的遗传多样性,每个菌株的AFLP带谱均与其它菌株完全不同。AFLP技术简便、快速、重现性极高,能表现高信息量的DNA长度多态性,是目前研究生物群体内遗传多样性的最有效办法。     

西北地区天蓝苜蓿根瘤菌16S rDNA RFLP分析

利用RFLP和序列测定方法,对分离自西北地区的67株天蓝苜蓿根瘤菌16S rDNA进行了分析研究。结果表明:所有供试菌株分别归属于中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)和土壤杆菌属(Agrobac-terium)。以CCNWNX0042-2为代表的大部分天蓝苜蓿根瘤菌属于草木樨中华根瘤菌(Sinorhizobium meliloti),其余菌株在分群上表现出了较为明显的地域特征。     

毛乌素沙地中间锦鸡儿根瘤菌遗传多样性及16S rDNA全序列分析

野生豆科植物中间锦鸡儿是毛乌素沙地的优势种。从蛋白质和DNA水平分析与其共生的根瘤茵的遗传多样性。在蛋白质水平上,24株中间锦鸡儿根瘤菌和9株参比菌株分为2组,A组包含95．8％供试中间锦鸡儿根瘤菌,参比菌株聚为B组,菌株GH72不与其余供试根瘤菌聚类。应用16S　rDNA　PCR—RFLP方法将供试菌株分为22种基因型,中间锦鸡儿根瘤菌组成12种,其余10种由参比菌株构成。表明中间锦鸡儿根瘤菌具高水平遗传多样性。选取代表菌株GH2001进行16S　rDNA全序列测定。与已知相关根瘤菌菌株16S　rDNA进行同源性比较,构建系统发育树状图。GH2001位于Rhizobium分支,与且Agrobacterium radiobacter,Ag.rubi,Rhizobium giardinii,R.mongolense,R.yanglingense,R.galegae和R.huautlense的序列同源性分别达到99％、98．3％、96．3％、95．5％、95．6％、95．27％和95．7％。     

西部某些根瘤菌的数值分类和16S rDNA PCR-RFLP分析

选用61株分离自我国西北地区的野豌豆、棘豆、苜蓿和草木樨根瘤菌和4株已知参比菌株,进行了营养利用、抗生素抗性和耐逆性等13个表型性状研究,通过MINTS软件分析,得到了数值分类树状图,发现全部供试菌株在79％的相似性水平上,分为5个群。对57株未知菌株和10株参比菌株16SrDNAPCR－RFLD分析,发现共具有20个遗传图谱类型,聚类分析树状图表明所有菌株共分为5个系统发育分支,与数值分类结果有较好的一致性。     

西部某些根瘤菌的数值分类和16SrDNA PCR-RFLP分析

选用 61株分离自我国西北地区的野豌豆、棘豆、苜蓿和草木樨根瘤菌和 4株已知参比菌株 ,进行了营养利用、抗生素抗性和耐逆性等 1 3 2个表型性状研究 ,通过MINTS软件分析 ,得到了数值分类树状图 ,发现全部供试菌株在 79%的相似性水平上 ,分为 5个群。对 5 7株未知菌株和 1 0株参比菌株进行了 1 6SrDNAPCR RFLP分析 ,发现共具有 2 0个遗传图谱类型 ,聚类分析树状图表明所有菌株共分为 5个系统发育分支 ,与数值分类结果有较好的一致性。     

我国豇豆和绿豆根瘤菌的数值分类及16S rDNA PCR-RFLP研究

对分离自中国14个不同省(自治区)的79株豇豆和绿豆根瘤菌及12株参比菌株进行了唯一碳、氮源利用,抗生素抗性,抗逆性和酶活性等128个表型性状的测定,并用MINTS软件进行聚类分析。表型性状测定结果发现,所有菌株都有极其广泛的碳、氮源利用谱,大多数菌株可在较宽的pH(pH5·0~11·0)值范围内生长,大部分菌株能在37℃高温条件下生长,个别菌株能耐受60℃高温较长时间(20~45min)的热激。聚类分析结果表明,全部供试菌株在63·5%的相似性水平上分为两大群:一个群为慢生菌群,另一群为快生和中慢生菌群;在79%的相似性水平上分为7个亚群。在数值分类的基础上,又将参比菌株增加到22株,对79株待测菌株进行了16SrDNAPCR-RFLP分析,16SrDNAPCR产物经HaeⅢ、HinfⅠ、MspⅠ和AluⅠ4种内切酶酶切共产生34种遗传图谱类型,经GelComparⅡ软件聚类后,在79%的相似性水平上也可划分为7个亚群,与数值分类的结果有很好的一致性。     

用AFLP技术分析紫茎泽兰的遗传多样性

利用AFLP技术,选择EcoRI/MesI这一酶切组合,应用6对E 3/M 3引物组合进行选择性扩增,检测了24个地区的紫茎泽兰种群的基因组DNA多态性。共扩增出509个遗传位点,其中392个多态位点,多态率为77.01%。有效等位基因数(Ne)为1.50,Nei’s基因多样性指数(H)为0.29,Shannon信息指数(I)为0.42。并通过Jaccard的方法将电泳带矩阵转化为遗传相似性系数矩阵,进行了UPGMA聚类分析。结果表明:被检测的24个紫茎泽兰种群遗传多样性丰富;丰富程度与入侵定植时间有关,最早受紫茎泽兰入侵的云南省遗传多样性最丰富,变异最大,大致分为9个类群,紫茎泽兰的遗传基因可能对该省多样的气候条件或生境产生了明显的适应性变化;新入侵地区的则遗传多样性相对较低,并与云南省临近地区具有明显的种源地的地源性亲缘关系。子实的风媒传播是紫茎泽兰入侵的主要途径,样点间均具有明显的地源性亲缘关系;水媒也可能是该草传播的途径之一。紫茎泽兰可能首先从缅甸、越南、老挝经风媒入侵我国云南南部,逐渐扩散入侵中北部,又从云南北部和东部传入四川、贵州、广西、湖北等省。     

大麻品种遗传多样性的AFLP分析

利用POPGENE 3.2软件对13个不同来源的大麻群体进行遗传多样性分析。结果显示:云南地区的大麻群体具有最高的遗传多样性水平(PPB=88.82%,He=0.3000,I=0.4571),其次为黑龙江群体(PPB=75.66%,He=0.2572,I=0.3897)。13个大麻群体的多态位点百分率(PPB)为92.11%,Nei’s总遗传多样性(Ht)为0.3837,Shannon’s信息指数I=0.5374。群体内遗传多样性(Hs)为0.1640,群体间的遗传分化系数(Gst)为0.5725,总的遗传变异中有57.25%发生在群体间,42.75%发生在群体内。根据Nei’s(1978)的方法计算了13个大麻群体间的遗传距离和遗传一致度。结果显示:各群体间的遗传一致度在0.6556～0.9258之间,其中四川群体和广西群体间具有最高的遗传一致度(0.9258);云南群体与贵州群体和四川群体间遗传一致度分别为0.9196、0.9173。所有群体中甘肃群体和山西群体遗传一致度最低为0.6556,说明大麻种内具有较大的遗传变异。     

糖单孢菌16S rDNA的PCR-RFLP分析

ＰＣＲ－ＲＦＬＰ分析适用于种和种间水平的多相分类研究。文章对ＰＣＲ－ＲＦＬＰ方法应用于糖单孢菌属分类有具体方法进行了探讨;报道了一组适于糖单孢菌属ＰＣＲ－ＰＦＬＰ分析的限制性内切酶,并用这组酶对６株糖单孢菌属分离株进行了研究,进一步探讨了实验菌株在该属的分类地位。同时指出了ＰＣＲ－ＲＦＬＰ方法应用中应注意的问题。     

用rep—PCR技术研究中国花生根瘤菌的多样性

采用细菌基因组重复序列ＰＣＲ技术（简称ｒｅｐＰＣＲ）中常用的ＲＥＰＰＣＲ和ＥＲＩＣＰＣＲ,对从中国１１个省、市的２３个点、２４个花生品种采集的根瘤中分离的５９株花生根瘤菌Ｂｒａｄｙｒｈｉｚｏｂｉｕｍｓｐ．（Ａｒａｃｈｉｓ）进行多样性研究,同时对来自国外的６株花生根瘤菌及１４株参比慢生根瘤菌也进行了比较。得到的低相似性结果表明中国花生根瘤菌基因组存在显著的多样性。ＲＥＰＰＣＲ揭示,在相似性５０％上分为１１个群,而ＥＲＩＣＰＣＲ却得到２４个分群。这两种结果对菌株的分群有差异,暗示这两种短重复序列在慢生根瘤菌基因组中的分布的不同。没有发现菌株间基因组的多样性分布与花生品种、地理来源之间的必然联系。将两者电泳图谱结合并分析,得到介于上述两者间的结果。此结果进一步反映了菌株基因组间存在的多样性。同时还表明ｒｅｐＰＣＲ不仅是研究生物多样性的快速简便方法,还可应用于菌株的鉴别和生态学研究。     

13种石斛属植物遗传多样性的AFLP分析

采用AFLP技术对13种石斛属植物的遗传多样性进行了分析。选择性扩增引物组合E ACT/M CAC、E AAC/M CAC和E ACA/M CAC分别对这13种材料进行扩增,得到丰富的条带。在100-300bp共得到346条带,多态性带342条,多态性百分率为98.8%。聚类分析结果表明,在相似系数0.54处,可将13种材料分为Ⅰ、Ⅱ、Ⅲ类。Ⅰ类包括:串珠石斛、铁皮石斛、广东石斛、重唇石斛、晶帽石斛、细叶石斛、滇桂石斛、报春石斛、玫瑰石斛、球花石斛;Ⅱ类包括:鼓槌石斛;Ⅲ类包括:美花石斛(花,浅红)、美花石斛(花,淡白)。AFLP分析结果与传统分类学的结果基本一致,表明该标记技术对石斛属植物的遗传多样性和分类研究是可行的。     

商洛多花胡枝子根瘤菌16S rDNA-RFLP分析及系统发育研究

利用16S rDNA-RFLP和全序列测定方法,对分离自商洛地区5个分布点的59株多花胡枝子根瘤菌进行了RFLP分析和系统发育研究.结果表明:(1)42株供试菌株归属根瘤菌属(Rhizobium)、11株归属中华根瘤菌属(Sinorhizobium).其余6株非根瘤菌中3株是嗜麦芽黄单胞菌(Stenotrophomonas maltophilia)、3株是解淀粉类芽孢杆菌(Paenibacillus amylolyticus),说明胡枝子根瘤内生菌较为丰富且类型多样.(2)结合供试菌株的地理生境分析,发现来自不同采集点的菌株有些具有同样的遗传类型,而来自同一采集点的菌株遗传类型却有差异,证明胡枝子根瘤菌在分群类别上与地理环境之间没有明确的对应关系,地理环境并非根瘤菌多样性形成的主要因素.建议今后对根瘤菌多样性研究应从根瘤菌与寄主植物物之间的共生选择进化,特别是对共生体系中基因的横向转移方面进行深入探讨.     

IDENTIFICATION OF GROUP- AND STRAIN-SPECIFIC GENETIC MARKERS FOR GLOBALLY DISTRIBUTED ALEXANDRIUM (DINOPHYCEAE). I. RFLP ANALYSIS OF SSU rRNA GENES1

Two distinct small-subunit ribosomal RNA genes (SSU rDNAs), termed the “A gene” and “B gene,” were recently found in the toxic dinoflagellate Alexandrium fundyense Balech. A restriction fragment length polymorphism (RFLP) assay was developed to rapidly detect the A and B genetic markers. SSU rDNA from 58 cultures with species designations of A. tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense, A. affine (Fukuyo et Inoue)Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were screened. These cultures represent toxic and non-toxic isolates from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo ships. The RFLP assay revealed five distinct groups. Three subdivided the A. tamarense/catenella/fundyense“species complex” into clusters defined by geographic origin, not by morphospecies designations. The fourth group consisted of A. affine, whereas the fifth group was represented by A. minutum, A. lusitanicum, and A. andersoni. The B gene was only found in A. tamarense, A. catenella, and A. fundyense, but not in all isolates. However, all North American isolates of this closely related group harbored this gene, and it also was found in some A. tamarense from scattered locations in Japan and in the ballast water of one ship that operated exclusively between Japan and Australia. Isolates without the B gene appeared to have only a single class of SSU rDNA. The B sequence was not essential for toxin production, but thus far those organisms harboring it were toxic. The A. tamarense/catenella/fundyense complex is composed of genetically distinct populations, within which may exist two or all three of the mophotypically defined species. The B gene is a promising taxonomic and biogeographic marker and may be useful for tracking the regional and/or global dispersal of particular populations.     

鸡眼草根瘤菌的１６SrDNA全序列分析

Based on the previous studies on numerical taxonomy, SDS-PAGE of whole-cell protein and DNA hybridization, the rhizobial strains isolated from Kummerowia sp. in semi-arid area of North-west constituted a new subgroup, the 16S rDNA sequence of representative strain SH714 were tested. The unrooted phylogenetic tree was produced. In this tree, the strain SH714 with Sinorhizobium xinjiangensis, S. fredii, S. meliloti, S. medicae, S. saheli and S. teranga constituted a branch of Sinorhizobium. Within this branch, the similarity valuse of 16S rDNA sequence between strain SH714 and S. xinjiangesis, S. fredii, S. meliloti, S. medicae, S. saheli and S. teranga were 97.4%, 97.5%, 96.8%, 96.7%, 97.2% and 95.6% respectively, the values were more than 95%, this indicated that these known species should belong to the same genus. The values of DNA homology between type strains of these species were less than 70%. Thus, the strain SH714 represented a new rhizobial species, and there were some diversity between SH714 and known rhizobial species in phenotypic feature and composition of protein.     

翘嘴红鲌雌雄基因组差异及太湖野生群体遗传多样性现状的AFLP分析

为发展翘嘴红鲌Erythroculter ilishaeformis(Bleeker)性别控制育种和单性养殖技术, 利用AFLP技术对翘嘴红鲌太湖野生群体中雌、雄各20个个体的基因组进行了遗传多样性和雌雄差异分析。用筛选出的8对AFLP多态性引物在太湖野生群体基因组中共检测到319个位点, 其中多态性位点185个。通过对所有多态性位点在个体中的分布进行分析, 发现一个只在雄性个体中稳定存在、在雌性个体中缺失的差异位点。在人工雌核发育翘嘴红鲌群体只有雌性个体, 其基因组中也检测不到该差异位点的存在。这些结果表明翘嘴红鲌性别分化可能受到严格的遗传调控, 该差异位点是雄性特有的性别分子标记。翘嘴红鲌太湖野生群体目前的多态位点比率P=57.99%, 观测等位基因数N_a=1.5799±0.4943, 有效等位基因数N_e=1.3859±0.3971, Shannon’s信息指数I=0.3221±0.2987。这些统计分析结果与10年前对该群体的AFLP研究结果(P=51.21%, N_a=1.512±0.500,N_e=1.252±0.371,I=0.218±0.275)相比没有显著差别(P>0.05), 说明该群体目前还具有适度的遗传多样性。     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.