莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

吴茱萸的AFLP指纹图谱的初步研究

首次报道中草药植物吴茱萸基因组DNA指纹图谱的研究。吴茱萸是贵州省内经济价值极高的中草药之一,采用扩增片段长度多态性（AFLP）技术来分析来自不同地区的石虎、疏毛、大花吴茱萸3个品种的DNA指纹图谱,从18对引物中筛选出3对引物对19份材料的DNA检测,共得到93条带,其中多态性片段57条（平均61.3%）。3对引物组合从DNA指纹图谱上将19份材料完全区分开,结果表明AFLP技术是鉴别吴茱萸相近品种的有效方法,是形态学鉴定方法的有益补充;UPGMA方法聚类分析显示19份种质材料间的相似系数为0.235～0.941,表明吴茱萸种质间的遗传多样性丰富;余庆地区种植基地的石虎和疏毛样本聚为一类,提示人工栽培影响到吴茱萸的遗传特性。     

应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。     

应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。     

RAPD条件优化及天麻基因组DNA多态性分析

建立了RAPD扩增条件快速优化程序与方法．并应用于天麻基因组DNA扩增条件的优化及多态性的测定：获得了天麻基因组DNA的RAPD扩增优化条件和DNA指纹图谱;分析了模板DNA、引物、dNTP、Taq DNA聚合酶等的浓度和退火温度对RAPD扩增的影响．结果表明：天麻基因组DNA用引物S1扩增的片段具有更明显的多态性,这种指纹图谱更适合于天麻遗传分化研究;而用引物S12扩增的DNA指纹图谱具有更大的相似性,这种指纹图谱更适合于天麻真伪鉴别．该方法使RAPD扩增条件优化过程实现了程序化和数量化,是获得RAPD优化条件的简便快速、经济实用方法．应用该方法进行RAPD扩增,可获得图谱清晰、稳定可靠的实验结果．     

华南瓜类疫霉的RAPD遗传多样性分析

为了解来自广东和广西的瓜类疫霉的遗传多样性,利用从180条RAPD引物中所筛选出的多态扩增性强、重复性好的12条引物,对分离自两省区的96株瓜类疫霉进行了全基因组DNA遗传多样性分析和指纹图谱构建。通过对供试菌株的RAPD-PCR扩增,共获得135条DNA标记谱带,其中124条为多态性谱带,多态检测率为91.9%。利用NTSYSpc Version2.1软件对供试菌株间的遗传距离进行聚类分析并构建系统树,以遗传相似系数0.81为阈值,将96个供试菌株划分为12个RAPD群,多数分离物之间遗传相似性较低,在DNA水平上存在显著的遗传变异,具有较丰富的遗传多样性。不同地区间菌株的遗传分化程度不同,分离自黄瓜的菌株遗传分化明显高于分离自冬瓜的菌株。RAPD群与菌株地理来源、分离寄主、致病力、交配型及甲霜灵抗性均无明显的相关性。     

DNA指纹图谱鉴别双歧杆菌的研究

采用RAPD技术选用10条引物对7种9株双歧杆菌基因组DNA进行PCR扩增,根据在优化条件下所得DNA指纹图谱分析了双歧杆菌菌株的遗传多样性,并构建了相似性指数矩阵和树状图.结果表明,不同序列的随机引物可扩增不同形式的RAPD图谱,但并非所有图谱都具有分类学意义,其中引物S256对双歧杆菌种及同种不同菌株均具有良好的区分能力,由该引物扩增的RAPD图谱计算出的相对性指数矩阵以及由此构建的聚类树状图均能正确地反映出双歧杆菌的系统发育关系,同时对RAPD图谱作为工业双歧杆菌分子标记的可能性进行了探讨.     

基于ISSR标记的62个朱顶红品种的遗传关系分析及指纹图谱构建

采用ISSR标记方法分析了62个朱顶红(Hippeastrum spp.)品种(包含60个引自荷兰的品种和2个苏州本地品种)的遗传多样性,并采用UPGMA法对62个朱顶红品种进行聚类分析.在此基础上,通过特异性条带的比较及筛选,采用黑白方格示意图法构建了供试品种的ISSR指纹图谱.扩增结果显示:用11条引物从62个朱顶红品种的基因组DNA中共扩增出118条带,其中多态性条带109条,多态性条带百分率达92.4％,有5条引物扩增条带的多态性条带百分率达100.0％.62个品种间的遗传相似系数变幅较大,为0.371 4～0.842 9,表明各品种间存在丰富的遗传变异和遗传多样性.聚类分析结果显示:在遗传相似系数0.63处62个品种被分为7组,多数形态相似的品种被聚在一起;其中形态相似的白色单瓣品种间遗传相似系数较高(约0.8),大多聚在一起,表明它们同源性较高;而2个苏州本地品种间遗传相似系数最高(0.842 9),表明它们可能具有同一来源.品种‘小红星’在引物UBC873扩增图谱的450 bp处有1条特异性条带,而品种‘精灵’在引物UBC835扩增图谱的3 000 bp处有1条特异缺失条带,这2条特异性条带可分别用于品种‘小红星’和‘精灵’的鉴定.基于引物UBC835和UBC873的ISSR扩增条带组合构建了供试的62个朱顶红品种的ISSR指纹图谱,采用这一指纹图谱可对供试的所有朱顶红品种进行鉴定.     

稗草致病菌——尖角突脐孢菌菌株RAPD指纹图谱的分析

以我国主要稻区的稗草植株上分离的17株尖角突脐孢菌菌株为试验材料,采用改良的SDS法提取其基因组DNA,并运用优化的RAPD分析体系对其进行了分子标记遗传差异研究。从25个随机引物中筛选出20个扩增效果好的引物,对全部试验材料进行了RAPD扩增,共得到239条有效带,其中多态性带229条（占95.8%）。依据扩增结果建立了17株尖角突脐孢菌基因型的DNA指纹图谱并对其进行了有效区分。根据RAPD分析结果计算了菌株间的遗传距离,分析了它们的遗传差异并进行了聚类分析,结果表明,RAPD分子标记技术是能够用于杂草致病菌资源的鉴定的,并可以进一步应用于特定性状的基因标记研究。     

应用RAPD技术对不同基因组组合生物型遗传多态性的研究

利用RAPD技术对不同基因组合的鱼类进行了基因组指纹图谱构建,在DNA水平上对基因组成分进行了分析,探讨陈春遗传多态性。PARD结果发现,在26个随机引物扩增的产物中,平均每个个体观察到约142个RAPD标记,单个的扩增图谱（Fig.1)可将红鲫（RA）与其它组合区分开,还可将鲫鲫杂种二倍体（CA)鲫鲤杂种三倍体（CAA）和人工复合三倍体鲤（CCA）区分开;S－8引物（Fig.2)可区分开红鲤（RC）和镜锂（MC）;S－45引物（Fig.3)可区分开RC和CA;S－22引物则可区分开CAA和CCA。六种生物型均存在基因组特异性的图谱即各种独特的“诊断性”图谱,作者由此建立了详细的分子标记检索表（Table1）。通过对RAPD图谱的量化分析。利用UPGMA构建了不同生物型的遗传关系树图,反映了鲤鲫及各种组合生物型之间的遗传相似关系:RC的MC属同一种系,聚为一族;CAA和CA基因组类型相同,聚为一族;CCA虽自成一体,但可与各种生物型内个体间的遗传相似率均大于群体间的（P＜0．05）。此外,本研究还对基因剂量效应对PAPD扩增图谱的影响进行了探讨。综合以上结果,RAPD技术有简捷,灵魂、花费少等特点、在鱼类品系鉴定和遗传多态性研究方面具有血型血清学和蛋白质多态性等其它技术无可比拟的优势。     

Probiotic properties of lactic acid bacteria isolated from stool samples of longevous people in regions of Hotan, Xinjiang and Bama, Guangxi, China

A total of 567 lactic acid bacteria (LAB) strains were isolated from the stool samples of longevous people in regions of Hotan, Xinjiang and Bama, Guangxi, China. In order to reduce the number of strains for further examinations, 36 isolates were screened out for further examination whilst the other strains, which had lower probiotic properties, were not suitable for yogurt production due to the absence of growth in pH 3.5 MRS medium and no curding during fermentation, and so were excluded. The result of identification by API, sequence analysis of 16S rRNA and random amplified polymorphic DNA (RAPD) analysis showed that there were three strains of Lactobacillus acidophilus, 10 strains of Lactobacillus rhamnosus, three strains of Lactobacillus casein, three strains of Lactobacillus brevis, two strains of Enterococcus faecium, two strains of Enterococcus faecalis, four strains of Bifdibacterium infantis, three strains of Bifdibacterium brevise, three strains of Bifdibacterium bifidium, two strains of Bifdibacterium adolecentis and one strain of Bifdibacterium longam among the 36 isolates. These strains were evaluated by in vitro methods including survival upon exposure to pH 2.0, 3.0 and/or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. The results presented here show that L. rhamnosus LV108, L. rhamnosus F, B. brevise R39 and B. infantis R42 are acid and bile tolerant, adhere to the cultured human intestinal Caco-2 cell line, antagonistic activity against potential pathogenic bacteria infection in vitro, and so are potential strains for probiotic use.     

Genetic diversity (RAPD-PCR) of lactobacilli isolated from "Almagro" eggplant fermentations from two seasons

Genetic diversity of 323 strains of lactobacilli isolated from an Almagro eggplant manufacturing plant was analyzed by using random amplified polymorphic DNA (RAPD). Thirty-four distinct RAPD patterns were obtained with 95% of isolates grouped into 18 main clusters. Genetic diversity was higher in brines from season II, Lactobacillus plantarum and Lactobacillus fermentum/cellobiosus being the species with the greatest number of genotypes. A single L. fermentum/cellobiosus genotype comprised isolates from both seasons and could be considered endemic to that factory.     

Characterization of non-starter lactic acid bacteria from Italian ewe cheeses based on phenotypic, genotypic, and cell wall protein analyses

Non-starter lactic acid bacteria (NSLAB) were isolated from 12 Italian ewe cheeses representing six different types of cheese, which in several cases were produced by different manufacturers. A total of 400 presumptive Lactobacillus isolates were obtained, and 123 isolates and 10 type strains were subjected to phenotypic, genetic, and cell wall protein characterization analyses. Phenotypically, the cheese isolates included 32% Lactobacillus plantarum isolates, 15% L. brevis isolates, 12% L. paracasei subsp. paracasei isolates, 9% L. curvatus isolates, 6% L. fermentum isolates, 6% L. casei subsp. casei isolates, 5% L. pentosus isolates, 3% L. casei subsp. pseudoplantarum isolates, and 1% L. rhamnosus isolates. Eleven percent of the isolates were not phenotypically identified. Although a randomly amplified polymorphic DNA (RAPD) analysis based on three primers and clustering by the unweighted pair group method with arithmetic average (UPGMA) was useful for partially differentiating the 10 type strains, it did not provide a species-specific DNA band or a combination of bands which permitted complete separation of all the species considered. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis cell wall protein profiles clustered by UPGMA were species specific and resolved the NSLAB. The only exceptions were isolates phenotypically identified as L. plantarum and L. pentosus or as L. casei subsp. casei and L. paracasei subsp. paracasei, which were grouped together. Based on protein profiles, Italian ewe cheeses frequently contained four different species and 3 to 16 strains. In general, the cheeses produced from raw ewe milk contained a larger number of more diverse strains than the cheeses produced from pasteurized milk. The same cheese produced in different factories contained different species, as well as strains that belonged to the same species but grouped in different RAPD clusters.     

Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.     

Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus zeae isolates identified by sequence signature and immunoblot phenotype

Species taxonomy within the Lactobacillus casei group of bacteria has been unsettled. With the goal of helping clarify the taxonomy of these bacteria, we investigated the first 3 variable regions of the 16S rRNA gene, the 16S-23S rRNA interspacer region, and one third of the chaperonin 60 gene for Lactobacillus isolates originally designated as L. casei, L. paracasei, L. rhamnosus, and L. zeae. For each genetic region, a phylogenetic tree was created and signature sequence analysis was done. As well, phenotypic analysis of the various strains was performed by immunoblotting. Both sequence signature analysis and immunoblotting gave immediate identification of L. casei, L. rhamnosus, and L. zeae isolates. These results corroborate and extend previous findings concerning these lactobacilli; therefore, we strongly endorse recent proposals for revised nomenclature. Specifically, isolate ATCC 393 is appropriately rejected as the L. casei type strain because of grouping with isolates identified as L. zeae. As well, because all other L. casei isolates, including the proposed neotype isolate ATCC 334, grouped together with isolates designated L. paracasei, we support the use of the single species L. casei and rejection of the name L. paracasei.     

Diversity and dynamics of lactobacilli populations during ripening of RDO Camembert cheese

The diversity and dynamics of Lactobacillus populations in traditional raw milk Camembert cheese were monitored throughout the manufacturing process in 3 dairies. Culture-dependent analysis was carried out on isolates grown on acidified de Man - Rogosa - Sharpe agar and Lactobacillus anaerobic de Man Rogosa Sharpe agar supplemented with vancomycin and bromocresol green media. The isolates were identified by polymerase chain reaction - temperature gradient gel electrophoresis (PCR-TGGE) and (or) species-specific PCR and (or) sequencing, and Lactobacillus paracasei and Lactobacillus plantarum isolates were characterized by pulsed field gel electrophoresis (PFGE). Milk and cheese were subjected to culture-independent analysis by PCR-TGGE. Presumed lactobacilli were detected by plate counts throughout the ripening process. However, molecular analysis of total DNA and DNA of isolates failed to detect Lactobacillus spp. in certain cases. The dominant species in the 3 dairies was L. paracasei. PFGE analysis revealed 21 different profiles among 39 L. paracasei isolates. Lactobacillus plantarum was the second most isolated species, but it occurred nearly exclusively in one dairy. The other species isolated were Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, a Lactobacillus psittaci/delbrueckii subsp. bulgaricus/gallinarum/crispatus group, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Lactobacillus brevis, Lactobacillus kefiri, and Lactobacillus perolens. Lactobacilli diversity at the strain level was high. Dynamics varied among dairies, and each cheese exhibited a specific picture of species and strains.     

Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns

AIMS: The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques. METHODS AND RESULTS: Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process. Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used. RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out. The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates. In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates. CONCLUSIONS: The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle. The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.     

Phenotypic and genotypic characterization of non-starter lactic acid bacteria in mature cheddar cheese.

Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of Lactobacillus-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II) Lactobacillus spp. Phenotypically, the isolates comprised 96.4% L. paracasei, 2.1% L. plantarum, 0.3% L. curvatus, 0.3% L. brevis, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic Lactobacillus spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the L. paracasei complex.     

Strain identification of probiotic Lactobacillus casei-related isolates with randomly amplified polymorphic DNA and pulsed-field gel electrophoresis methods

Typing of reference strains and isolates identified as Lactobacillus casei, Lactobacillus paracasei or Lactobacillus rhamnosus was carried out using randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analyses. Strains of L. paracasei were mainly grouped in the same cluster as those of L. casei. The RAPD fingerprints of strains ATCC 393 and ATCC 15820 differ from those of the L. rhamnosus and L. paracasei/casei strains further supporting classification of these strains as a separate group. The RAPD profiling could be used for classification and discrimination of isolates belonging to the L. casei group.     

Lactic acid bacteria diversity in corn silage produced in Minas Gerais (Brazil)

The diversity of lactic acid bacteria (LAB) in silages produced in warm climate countries is not well known. This study aimed to identify and characterise the metabolic and genotypic aspects of autochthonous LAB isolated from corn silage produced in the state of Minas Gerais, Brazil. Eighty-eight LAB were isolated. To evaluate their performance at the strain level, all isolates were distinguished among strains using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) and repetitive extragenic palindromic PCR (REP-PCR) techniques. The organic acid and ethanol production were determined by high-performance liquid chromatography (HPLC). The fingerprints obtained by RAPD-PCR with a M13 primer were more discriminatory than those obtained with the REP-PCR technique using a (GACA)4 primer. Moreover, 28 representative isolates were identified as Lactobacillus acidophilus, L. buchneri, L. casei, L. diolivorans, L. hilgardii, L. paracasei, L. parafarraginis, L. plantarum, L. rhamnosus, L. zeae and Pediococcus acidilactici. Different fingerprinting profiles between isolates within the same species were observed. However, some strains isolated from different silages showed the same band profile, thus suggesting the presence of clusters with high similar fingerprints in silages from various regions. A variation in LAB diversity was observed in the silages of the evaluated regions, with L. rhamnosus and L. buchneri showing the highest distribution. Differences in organic acid production were observed among the strains belonging to the same species. This research contributes to a better understanding of the LAB community present in corn silage produced in warm climates. These strains will be studied as potential silage starters.