利用SSR和RAPD标记分析中国部分地区晚疫病菌群体遗传多样性

利用SSR和RAPD对来自福建、黑龙江、河北和内蒙古4个地理群体的80个马铃薯晚疫病菌（phytophthora infestans）株进行了遗传多样性分析。13对SSR引物共扩增出76条谱带,多态性条带比率78.9%,相似系数变化范围0.00～0.42之间;筛选出的14条RAPD引物共扩增出189条谱带,多态性条带比率95.2%,相似系数变化范围0.04～0.66之间。遗传多样性分析表明,在4个群体中,福建群体的多样性更为丰富。遗传相似性分析显示,黑龙江和内蒙古两个群体间的遗传相似性最高,而福建和河北两个群体间的遗传相似性最低。聚类分析显示,来自南方福建的菌株与来自北方黑龙江、河北和内蒙古的菌株亲缘关系较远,且福建群体分布于更多的聚类组,显示出更高的遗传变异度。     

不同致病型水稻细条病菌的遗传多样性分析

采用引物BOX和ERIC对来自4省的35个细条病菌菌株进行Rep-PCR扩增,BOX引物扩增出14条指纹带,10种谱型,以致病型为单位计算遗传多样性值为0.63～1.00;ERIC引物扩增出19条指纹带,17种谱型,遗传多样性值为0.86～1;引物ERIC比BOX在遗传多样性方面有更好的分辨率;树状聚类图反映的遗传分簇差异,与菌株的致病型及其地理的来源之间没有相关性。     

嗜热菌和假单孢菌及其融合子质粒DNA的RAPD分析

本研究选用15个RAPD随机引物,对白琼海温泉分离的嗜热菌（HSR001）和海口琼山红城湖分离的假单孢菌（HSP002）,以及二者的融合子RH004、RH006、RH009、RH012等共6个样株无性系作了RAPD多态性分析。其中3个引物未扩增出质粒DNA的条带,其余12个引物扩增出了有效的谱带。并对各无性系的指纹图谱中的DNA条带进行了统计,利用Clustal-w对其进行分析建立系统树和相似系数比较。结果表明：6个样株均具有丰富的RAPD多态性,6个菌株可分为2个类群,两亲本为一类,4个融合子为另一个类群。     

小麦叶锈菌毒性及分子多态性分析

以来自河北、江苏两地的22株小麦叶锈菌作供试菌株，用27个小麦抗叶锈近等基因系品系作为鉴别寄主进行毒性测定。毒性多态性分析结果为：22个菌株通过聚类被分为两组，第一组17个菌株主醚自河北，第二组5个菌株全部来自江苏，表明毒性与地理来源密切相关；菌株间遗传相似系数较高并且差异不大，为0.6296-0.9259之间，说明两地的小麦叶锈菌所含的毒性基因差异不大，应用RAPD技术，从65个随机引物中筛选了12个扩增多态性较好的随机引物，用于分子多态性分析，共扩增出DNA条带102条，其中多态性条带54条，RAPD分析结果为：22个菌株被分为两组，第一组菌株主要来自河北，第二组菌株主要来自江苏，说明DNA多态性与地理来源间具有一定的相关性；菌系间遗传相似系数相差较大，为0.3889-0.9074之间，说明两地小麦叶锈菌群体遗传结构丰富而复杂，比较22株小麦叶锈菌在27个鉴别品系上的毒性特征和54个RAPD标记建立的聚类分析树状图，发现以RAPD标记为基础的分子多态性与毒性多态性相关性不强。     

基于RAPD标记的福建省稻曲病菌遗传多样性分析

为了解福建省稻曲病菌群体的遗传多样性和遗传组成,应用随机扩增多态性RAPD (random amplified polymorphic DNA)技术分析了来自福建省不同水稻种植区的102个稻曲病菌(Ustilaginoidea virens)的遗传多样性.从100条随机引物中筛选出10条扩增带清晰、重复性好的引物进行稻曲病菌多态性扩增,共扩增出157条带,多态性条带比率为82.17％,遗传距离变化范围为0.02～0.67.遗传多样性分析表明,福建省稻曲病菌具有丰富的遗传多样性,相对于其它地区而言,福建闽西地区分离的菌株遗传多样性水平最高PPB=76.43,H=0.2212,I=0.3383),晚稻分离的菌株群体遗传多样性(PPB=91.08,H=0.2402,I=0.3655)高于早稻群体(PPB=63.06,H=0.1892,I=0.2870).聚类分析显示,在遗传距离0.349水平上,供试的所有菌株可被划分成7个遗传聚类组(R1～R7),聚类组R1为优势聚类组,包含有80个菌株,其内又存有一些亚组.这些菌株的聚类与菌株的地理来源及水稻品种无明显相关性.但是在遗传距离0.330水平上,来自宁化的10个菌株可被明显划分成早稻群体和晚稻群体.初步分析认为菌株的地理来源、水稻品种及其生长季节是影响福建省稻曲病菌遗传多样性的主要因素,在稻曲病菌的遗传变异以及该病的发生和流行中可能起重要的作用.     

应用RAPD技术研究4种鲍的亲缘关系

应用RAPD技术研究 4种鲍的亲缘关系结果表明 ,4种鲍群体中 2 0个有效引物共扩增出 5 38条DNA带 ,平均每个引物扩增的条带数为 2 6 .9;扩增的多态性条带数 136 ,平均每个引物扩增的多态性条带数为 6 .8;多态位点百分数为 2 5 .3%。盘鲍群体和皱纹盘鲍群体之间遗传距离与遗传一致度分别为 0 .2 8和 0 .72 ,杂色鲍群体和九孔鲍群体之间遗传距离与遗传一致度分别为 0 .32和 0 .6 8。聚类分析把盘鲍群体和皱纹盘鲍群体聚为 1组 ,二者亲缘关系较近 ;杂色鲍群体和九孔鲍群体聚为 1组 ,二者亲缘关系也较近     

利用ISSR揭示不同类型月季遗传多样性

本研究应用正交设计法对ISSR反应体系中的各个主要影响因子进行了优化筛选,确立了适合月季ISSR-PCR反应的最佳体系。结果表明,25μL的ISSR反应体系中各组分的最适浓度分别为：1×PCR缓冲液、1U Taq DNA聚合酶、800pmol／L 引物、0.16mmol／L dNTPs、Mg^2＋ 1.5mmol／L。筛选了33个ISSR引物,共得到了11个多态性比较高的ISSR引物,占所筛引物的33.33％。利用筛选出的11条ISSR引物对3种月季类型的23份月季材料进行遗传多样性分析,共扩增出477条DNA带,其中多态性位点有14个,平均每条引物可以检测到4.5个多态性位点。用NTSYS软件对样品进行了UPGMA聚类分析,聚类结果表明丰花月季基本能聚为一类,切花月季与藤本月季交叉聚在一起。这表明月季种质的遗传差异与其应用分类的相关性不紧密。     

利用RAPD和SSR标记分析陆地棉种质资源的遗传多样性*

遗传多样性的量化与分类是收集和利用种质资源的重要前提。利用随机扩增多态性（RAPD）和简单重复序列(SSR)两种分子标记对31份陆地棉(Gossypium hirsutum L.)种质资源进行了遗传多样性分析,其中14份材料最近从美国和伊朗引进。研究的目标是对这些陆地棉种质资源进行遗传聚类分析,为制定引进资源的利用策略提供参考。从有多态性的21个RAPD引物和18对SSR引物中共获得117个多态性位点。利用NTSYS-pc2．10统计分析软件,采用Jaccard′s相似系数UPGMA法进行聚类。结果表明,中国的17份材料部分聚为一类,国外的14份材料大部分聚为另一类,其它材料相间排列。分别对31份材料的相似系数与中国的17份材料的相似系数进行了比较分析,中国材料相对于新引进的国外材料的遗传多样性水平稍高,与国外材料之间的遗传差异较大。这说明扩大不同地区间种质资源的交流和引进种质资源可以丰富本地材料的遗传多样性。     

虫生真菌座壳孢的RAPD与LSU rDNA序列分析

用17个引物对不同地理来源的4种11株座壳孢Aschersonia进行RAPD分析，结果表明，种间和种内具丰富的遗传多样性，菌株间差异与地理来源和寄主不相关。部分菌株和GenBank上的座壳孢属核糖体nrDNA大亚基序列进一步用于该属系统发育研究。系统发育树反映的种间和种内关系与形态分类结果基本一致，可以将不同地理来源的不同种区分开来，但从同科或同目寄主昆虫上分离到的不同种并不具有相近的亲缘关系。同时进化树还表明，所分离的菌株Aa是粉虱座壳孢(Aschersonia aleyrodis Webber)。此外，对相同的供试菌株两技术所反应的亲缘关系趋势相同，表明RAPD技术和LSU rDNA用于座壳孢的亲缘关系分析和种类鉴定均是可行的。     

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。     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

Isolation and identification of locally isolated bacterial strains effective against whitefly Bemisia tabaci

Potent bacterial strains effective against the whitefly, Bemisia tabaci, nymphs (second instar), were isolated from tomato cultivated fields at Fayoum governorate, Giza, Egypt. Of 72 isolates, 12 with the most morphologically distinct-looking bacterial colonies were selected and named A1, A2, A3, A6, A7, A9, A12, A13, A107, B37, B45 and B100. All isolates were preliminarily identified as members of the genus Bacillus based on morphological, physiological and biochemical characteristics. When tested for their pathogenicity against Bemisia tabaci, the 12 isolates revealed varying efficiencies with isolates A1 and A9 being superior, exhibiting maximum mortality of 92.2 and 90.8% on day 10, respectively. Isolate A7 recorded the lowest percentage at 18.3%. Further genetic characterization of the 12 isolates was performed using inter simple sequence repeat (ISSR), randomly amplified polymorphic DNA (RAPD) and 16S rDNA gene sequencing analysis. RAPD and ISSR results confirmed each other. The combined ISSR and RAPD phylogenetic tree showed two major clusters. With 16S rRNA gene analysis, isolate A1 and A12 sequences recorded 100% identity with Bacillus thuringiensis, while isolates A7 and B100 showed 95.7% and 95.6% identity with Bacillus cereus and Bacillus sphaericus, respectively.     

金福菇菌株的酯酶同工酶分析

本文采用酯酶同工酶分析和聚类分析技术,对15个金福菇菌株的遗传多样性和亲缘关系进行分析鉴定,以便为金福菇的遗传育种提供依据。试验结果表明：共检测到18条谱带,其中有2条谱带的分布频率为100%,是所有菌株的特征谱带;15个菌株共有14种酶带类型,不同菌株的酶带数在8条至12条之间。聚类分析结果表明,15个菌株间的遗传相似系数在0.33~1.00之间,在相似系数为0.62时,可将15个金福菇菌株划为3大类群。老挝金福菇TgLo菌株与国内金福菇的亲缘关系最远;同一菌株的不同分离株如Tg1.1与Tg1.2、Tg10.1、Tg10.2与Tg10.3间的遗传相似系数在0.94以上;菌株Tg3与Tg8,Tg12与Tg10.2或Tg10.3间的遗传相似系数也达到0.94,可认为是同物异名。上述结果表明,酯酶同工酶方法可有效应用于金福菇的菌株鉴定及亲缘关系分析。     

鸡传染性支气管炎病毒（IBV）广西流行株3＇端非编码区序列分析

本研究应用RT-PCR方法扩增出分离自广西的26株鸡传染性支气管炎病毒（IBV）以及参考株M41和疫苗株H120、Ma5和4/91的3＇端非编码区（3＇UTR）的cDNA,并对其进行了克隆、序列测定、比较及其系统进化分析。序列分析结果表明,与Beaudette株的3＇UTR序列相比较,26株分离的IBV中有1株和3株的分离株3＇UTR序列分别插入了23个和2个碱基,另外有12株、6株、1株、2株和1株的分离株3＇UTR序列分别缺失了1个、2个、22个、29个和84个碱基,不同毒株之间碱基数量最大相差达107个碱基。系统进化分析结果表明,26株分离株可分为4群,其中21株属于第Ⅰ群,与经典疫苗株H120的核苷酸同源性均较低（54.4%～75.5%）;3株与4/91同属于第Ⅱ群;1株与H120同属于第Ⅲ群;另外1株单独属于Ⅴ群。综上所述,广西流行IBV的绝大部分毒株在3＇UTR序列上与目前常用的疫苗株相比都发生了很大的变异,而且存在多种形式的碱基缺失和插入现象。由此,我们认为流行株的变异可能是疫苗不能提供有效保护的主要原因。     

RAPD and isozyme analysis of genetic relationships between Carica papaya and wild relatives

The genetic origin of cultivated papaya is not clear. Wild relatives of papaya (Carica papaya) from central southern America were investigated using isozyme and RAPD analysis. Seven other species (including six from the genus Carica) were found to be relatively distant from papaya providing no indication of the genetic origin of papaya. Isozyme and RAPD data gave similar measures of genetic similarity within this group. C. papaya was about 70% dissimilar to the other Carica species by both methods. The other Carica species had average dissimilarities around 50%. Two species, C. pubescens and C. stipulata were much closer to each other with similarities of 87% by isozyme analysis and 82% by RAPD analysis. Although both methods gave similar measures for genetic distance the large number of RAPD markers available made RAPD analysis more reliable for analysis of the extremes (e.g. closely related taxa may show no isozyme differences and distant taxa may show no isozyme similarities).     

Characterisation of Oil-Degrading Bacteria Isolated from Bilge Water

During an initial screening in samples of bilge water, four bacterial strains capable to oil degrade have been isolated. Two strains, named isolates BW-1 and BW-2, were clustered with Acinetobacter genus (a similarity of 100% and 99%, respectively) and two strains, named isolates BW-3 and BW-4 were related to Rhodococcus genus (a similarity of 99% and 98%, respectively). During growth (8?days) in M9 medium with Arabian Light Crude Oil (1%, w/v), measures of microbial abundance, surface tension, emulsification index (E ₂₄) and oil degradation, were carried out. During cultivation, isolates BW-1 and BW-2 are good biosurfactant producers and about 80% of crude oil is degraded. Also, isolates BW-3 and BW-4 show, during cultivation, production of biosurfactant but only 40% of total oil is degraded. Data obtained give important results in order to utilise these isolate native bilge waste microorganisms in the studies/process of bioremediation.     

Genetic diversity of fast-and slow-growing soybean rhizobia determined by random amplified polymorphic DNA analysis

The genetic relationships among six strains of rhizobia, including three strains of Rhizobium fredii and three strains of Bradyrhizobium japonicum, was determined using random amplified polymorphic DNA (RAPD) technique. In this study, 46 arbitrary 10mer primers were employed for RAPD, generating a total of 251 informative fragments. A dendrogram of phylogenetic relationships among the six strains was constructed. The results indicated that geographical distribution may affect phylogeny, as there were closer relationships among the four Taiwanese strains, SB138, SB562, SB368 and SB651, than between these strains and USDA192, which originated from mainland China. The strain USDA110, obtained from the United States, was used in the parsimony analysis. The greatest similarity (55.6%), existed between two strains of B. japonicum, SB562 and SB138, which both, and the lowest R. fredii (44.4%) between two strains of R. fredii, SB368 and USDA192. We also found a RAPD marker specific to the four Taiwanese SB strains used in the study. The RAPD technique is a potential tool for the identification of the genetics and systematics of different populations. Received: 23 January 1997     

大豆种质资源RAPD标记遗传多样性研究

为深入研究并充分利用野生大豆资源,本文利用RAPD分子标记对40份大豆材料加以分析,旨在从DNA分子水平上探索野生大豆、地方品种和育成品种之间的遗传多样性状况。结果表明:50个RAPD引物筛选出具有多态性且扩增条带清晰的引物38个,共检测出407条带,其中多态谱带309条,多态性程度为75.92%。每个引物可扩增出2～14条多态性带,平均产生多态性谱带8.1条;平均多样性指数为2.3377,变幅范围为0.5865～4.2133。遗传相似系数变幅范围为0.44～0.92,平均为0.75。野生大豆的多态比例(94.35%)、多样性指数(2.2336)分别高于育成品种(87.47%、1.7331)和地方品种(83.54%、1.6198)。遗传相似系数为野生大豆(0.6498)地方品种(0.7015)育成品种(0.7177),育成品种与地方品种间为0.6599,育成品种与野生大豆间为0.6487,地方品种与野生大豆间为0.6045。UPGMA聚类分析结果表明,40份大豆材料聚为6类,育成品种和地方品种各自聚为一类,野生大豆聚为4类。野生大豆特异等位基因数远远高于育成品种和地方品种二者的相加之和。本研究从分子水平上揭示了野生大豆与栽培大豆区别明显,宜作为一个独立的种,同时野生大豆变异幅度大,遗传基础广,是大豆育种实践中的优良基因资源。     

Genetic diversity of resident soil rhizobia isolated from nodules of distinct hairy vetch (Vicia villosa Roth) genotypes

Hairy vetch (Vicia villosa Roth, HV) is widely grown as a legume cover crop throughout the U.S.A., with biological nitrogen fixation (BNF) through symbiosis with Rhizobium leguminosarum biovar viciae (Rlv) being one of the most sought after benefits of its cultivation. This study determined if HV cultivation history and plant genotype affect genetic diversity of resident Rlv. Soil samples were collected from within farmers’ fields at Graham, Cedar Grove and Ivanhoe sites in North Carolina and pairs of genetically similar hairy vetch genotypes used as trap hosts. A total of 519 Rlv strains were isolated from six paired field soils, three with and three without histories of HV cultivation. A total of 46 strains failed to PCR-amplify the nifH gene; however nodC PCR amplification of these nifH-negative strains resulted in amplification of 22 of the strains. Repetitive element polymerase chain reaction (rep-PCR) with BOX-A1R primer and redundancy analysis showed rhizobial diversity to vary greatly within and between fields, with over 30 BOX banding patterns obtained across the six fields. Cluster analysis of BOX-PCR banding patterns resulted in 36 genetic groups of Rlv at a similarity level of 70%, with 15 of the isolates from fields with HV history not belonging to any of the clusters. Site was found to be the main driver of isolate diversity overall, explaining 57%, of the total variation among rhizobia occupying HV nodules, followed by history of hairy vetch cultivation. Evidence of a HV host genotype influence on the populations of rhizobia that infect hairy vetch was also observed, with plant genotype explaining 12.7% of the variation among all isolates. Our results show that second to site, HV cultivation history was the most important driver of rhizobial nodule community structure and increases the genetic diversity of resident Rlv in soils.