牦牛奶酪中乳酸菌的随机扩增多态性研究

目的：探讨随机扩增多态性(RAPD)技术在牦牛奶酪中乳酸菌基因型研究方面的应用价值。方法：采用M13引物,对退火温度、Mg2+浓度、引物用量、Taq酶用量、模板量进行单因素梯度试验,建立最佳反应条件,然后对分离自牦牛奶酪的39株乳酸菌进行扩增,用NTsys 2.10e软件分析扩增图谱,计算遗传相似系数,进行聚类分析,并与16S rRNA测序得到的菌种鉴定结果进行对比。结果：39株乳酸菌相互间的遗传相似系数在0.47～0.98之间,当相似系数为0.78时,菌株被分成10个组,除X23、Y36和Y37之外,其余菌株均按照不同的菌种聚类,与16S rRNA测序结果基本一致,聚类有较强的地域相关性。结论：RAPD技术可用于牦牛奶酪中乳酸菌的基因型研究,可用于菌种鉴定和遗传亲缘关系分析。     

Genotyping of fig (Ficus carica L) via RAPD markers

The genetic diversity among 15 fig accessions (Ficus carica L), belonging to 9 renowned cultivars of the Calabrian fig collection, and 24 unidentified genotypes of figs also located in the Calabrian region of Southern Italy, was investigated by random amplified polymorphic DNA (RAPD) analysis. The genetic similarity values were calculated for the 39 samples, and a dendrogram was elaborated by cluster analysis according to the UPGMA algorithm. The generated DNA fragments grouped the samples into two main clusters of RAPD profiles. Most of the 24 unknown samples, coming from the Luzzi area, formed a unique cluster with high degree of genetic similarity. This indicates that it is possible to distinguish, at DNA level, the fig trees with an already well‐known potential to produce figs suitable for the drying process and possibly to specify cultivars with suitable features for industrial transformation. Copyright © 2005 Society of Chemical Industry     

RAPD在灰平菇菌株亲缘关系研究中的应用

采用RAPD技术分析了5个不同来源的灰平菇栽培菌株的DNA多态性。筛选出的8条随机引物共扩增得到107条带,其中多态性片段为69条,多态性比率为64.4%,说明收集到的灰平菇菌株具有一定的遗传多样性,它们之间存在一定的遗传差异。进一步聚类分析结果显示,当相性达到0.470的水平时,5个菌株聚成2组,第1组为1号(PL-72)、2号(PL-2)、3号(烟平2106)、4号(PL-A2);第2组为5号(PL-AD28)。当相似性达到0.570的水平时,第1组又被分为两个亚组,1号和3号菌株聚为一个亚组,而2号和4号菌株聚为一个亚组。本研究为灰平菇优良品种选育和亲缘关系的研究提供分子生物学依据。     

随机扩增多态性法分析西藏牦牛奶酪中乳酸菌的遗传多样性

目的：利用随机扩增多态性（randomly amplified polymorphic DNA,RAPD）法对西藏地区牦牛奶酪中27 株乳酸菌基因型进行同源性分析。方法：利用5 个随机引物对Mg2+浓度、dNTP用量、退火温度、模版用量/引物用量（ng/pmol）4 个条件做单因素梯度试验,建立最佳反应条件,筛选最佳引物,然后对27 株乳酸菌和4 株乳酸菌标准菌株进行随机扩增,用NTsys 2.10e软件对扩增条带进行聚类和遗传相似性系数分析,分析结果与16S rRNA测序得到的菌种鉴定结果进行对比。结果：31 株菌遗传相似系数在0.72～1.00之间,当相似性系数在0.82时,菌株被分成了8 组,菌株按照不同种属聚类,聚类结果同16S rRNA测序结果基本一致,同时成功将Lactobacillus casei和Lactobacillus paracasei两个亚种区分开。结论：RAPD技术可以较好地应用于西藏地区牦牛奶酪中乳酸菌亲缘性关系分析。     

白平菇不同菌株菌丝体的RAPD分析及系统进化关系的研究

本研究采用RAPD技术分析白平菇四个不同株系的DNA序列多态性,用15个随机引物对菌株基因组DNA进行PCR扩增,其中3个引物可以扩增出较好的多态性条带图谱,共产生66条清晰稳定的带。通过对供试菌株遗传相似系数的计算和系统聚类分析,构建了树状聚类图谱,在分子水平上分析了白平菇的种质遗传学差异,为白平菇菌株鉴定提供快速有效的技术和方法。     

Genetic analysis of Turkish rice varieties (Oryza sativa L.) using seed storage proteins and RAPD markers

Due to an increasing demand for rice in Turkey, assessing and maintaining genetic variability in rice cultivars is essential for the success of rice breeding programs. Genetic polymorphisms in seed storage proteins (SSP) and RAPD markers were evaluated among different Turkish cultivars of Oryza sativa L. Twenty-one rice cultivars of Turkish origin and 11 rice cultivars from Bulgaria, France, Italy and Russia were analyzed. SDS-PAGE profiles from SSP of 20 cultivars showed similar expression patterns. Seven polymorphic and reproducible RAPD primers produced 42 bands ranging from 250 to 2,500 bp; 29 were polymorphic (69%) and 13 monomorphic (31%). Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) was performed to generate a pairwise genetic distance (or similarity) matrix. A rooted dendrogram resulted in two main groups based on Jaccard’s coefficients. Sürek-95, Serhat-92, and Rocca were placed in one group, and all other cultivars in another group. Principle coordinate analysis was also conducted and the results supported UPGMA results, where Sürek-95, Serhat-92, and Rocca were distinct from all other cultivars. Our results suggest that the rice cultivars screened in this study exhibit narrow genetic diversity. In addition, RAPD-based genetic characterization can aid in both conservation and selection of desired rice cultivars in future breeding studies.     

RAPD 在酵母分类鉴定与育种研究中的应用

随机扩增多态性DNA(RAPD)因其简便、快速、灵敏等优点在酵母的分类鉴定,种属间亲缘关系确定及育种等方面有广阔的应用前景。本文综述近年来RAPD技术在酵母相关研究中的应用,并简单讨论该技术目前的研究热点。     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

RAPD-PCR快速鉴定啤酒污染菌的研究

本研究主要评价RAPD-PCR技术在快速鉴定啤酒污染菌中的应用。首先评价了CTAB法和试剂盒提取DNA模板对RAPD指纹稳定性的影响以及乳酸菌专一性BP引物扩增的16SrDNA的5’末端740bp序列用于鉴定菌种的可行性,采用PCR产物直接测序鉴定分离的污染菌,构建标准污染菌库。对分离菌M-13的RAPD指纹聚类分析表明,相同来源的同一种菌能很好地归类在一起。根据建立的快速鉴定流程和初步确定的菌种相似性阈值(SCC),对8株分离菌的鉴定表明,RAPD-PCR技术是一项简单、快捷、可靠性强的鉴定技术,为研究啤酒酿造过程的污染菌提供了一个非常有用的快速鉴定工具。     

Quality parameters and RAPD-PCR differentiation of commercial baker's yeast and hybrid strains

Baker's yeast, Saccharomyces cerevisiae, is a key component in bread baking. Total of 12 commercial baker's yeast and 2 hybrid strains were compared using traditional quality parameters. Total of 5 strains with high leavening power and the 2 hybrid strains were selected and evaluated for their alpha-amylase, maltase, glucoamylase enzymes, and compared using random amplified polymorphic DNA (RAPD). The results revealed that all selected yeast strains have a low level of alpha-amylase and a high level of maltase and glucoamylase enzymes. Meanwhile, the Egyptian yeast strain (EY) had the highest content of alpha-amylase and maltase enzymes followed by the hybrid YH strain. The EY and YH strains have the highest content of glucoamylase enzyme almost with the same level. The RAPD banding patterns showed a wide variation among commercial yeast and hybrid strains. The closely related Egyptian yeast strains (EY and AL) demonstrated close similarity of their genotypes. The 2 hybrid strains were clustered to Turkish and European strains in 1 group. The authors conclude that the identification of strains and hybrids using RAPD technique was useful in determining their genetic relationship. These results can be useful not only for the basic research, but also for the quality control in baking factories.     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

Identifying the geographical origin of protected sea cucumbers (<Emphasis Type="Italic">Apostichopus japonicus</Emphasis>) in China using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)

Dalian sea cucumber, Yantai sea cucumber, and Weihai sea cucumber, which belong to Apostichopus japonicus, are protected as geographical indications in China based on their high nutritional values and medical propertys. The 26 samples, including Dalian sea cucumbers (9 samples) in Liaoning province, Yantai sea cucumbers (9 samples), and Weihai sea cucumbers (8 samples) in Shandong province, were individually collected from the designated geographical sea areas and the genetic relationships and DNA polymorphisms were evaluated by random amplified polymorphic DNA technology and gene segments sequencing. The RAPD dendrogram showed that the genetic diversity of the three types of sea cucumbers was rich. The neighbor-joining tree shows that the genetic relationship of the samples from the adjacent sea areas is closer. It demonstrates that the gene characteristics of sea cucumbers from different sea areas were obvious and the genetic diversity analysis by RAPD-PCR can be used as a rapid method for geographical discrimination.     

Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Y. enterocolitica-like bacteria

A random amplified polymorphic DNA (RAPD) protocol was developed for interlaboratory use to discriminate food-borne Yersinia enterocolitica O:3 from other serogroups of Y. enterocolitica and from Y. enterocolitica-like species. Factors that were studied regarding the RAPD performance were choice of primers and concentration of PCR reagents (template DNA, MgCl(2), primer and Taq DNA polymerase). A factorial design experiment was performed to identify the optimal concentrations of the PCR reagents. The experiment showed that the concentration of the PCR reagents tested significantly affected the number of distinct RAPD products. The RAPD protocol developed was evaluated regarding its discrimination ability using 70 different Yersinia strains. Cluster analysis of the RAPD patterns obtained revealed three main groups representing (i) Y. pseudotuberculosis, (ii) Y. enterocolitica and (iii) Y. kristensenii, Y. frederiksenii, Y. intermedia and Y. ruckeri. Within the Y. enterocolitica group, the European serovar (O:3) and the North American serovar (O:8) could be clearly separated from each other. All Y. enterocolitica O:3 strains were found in one cluster which could be further divided into two subclusters, representing the geographical origin of the isolates. Thus, one of the subclusters contained Y. enterocolitica O:3 strains originating from Sweden, Finland and Norway, while Danish and English O:3 strains were found in another subcluster together with O:9 and O:5,27 strains. The repeatability (intralaboratory) and reproducibility (interlaboratory) of the RAPD protocol were tested using 15 Yersinia strains representing different RAPD patterns. The intralaboratory and the interlaboratory studies gave similarity coefficients of the same magnitude (generally >70%) for the individual strains. In the present study, it was shown that interreproducible RAPD results could be achieved by appropriate optimisation of the RAPD protocol. Furthermore, the study reflects the heterogeneous genetic diversity of the Y. enterocolitica species.     

A Comparative Study of Ascorbic Acid and Capsaicinoid Contents in Red Hot Peppers (Capsicum annum L.) Grown in Southeastern Anatolia Region

The aim of this study was to evaluate selected physico-chemical properties, ascorbic acid, and capsaicinoid (capsaicin, dihydrocapsaicin, norhydrocapsaicin, homodihydrocapsaicin, and total capsaicinoid) quantities of red hot pepper populations (Capsicum annum L.) grown in the South-Eastern Anatolia Region. Fresh and sun-dried red hot peppers and red hot pepper seed samples were collected from Kahramanmara?, pepper of Kahramanmara?; Gaziantep, pepper of Gaziantep; Kilis, pepper of Kilis; Diyarbak?r, pepper of Diyarbak?r; and ?anl?urfa, pepper of ?anl?urfa in the months of September through October 2010. The collected samples were analyzed in terms of certain quality parameters and capsaicinoid contents, while ascorbic acid amounts were determined only in fresh samples. All of the examined prameters were found to be significant statistically (P < 0.05). From the obtained results, it might be considered that these red hot pepper samples were suitable for cultivation due to their high-nutritional quality.     

RAPD with microsatellite as a tool for differentiation of Candida genus yeasts isolated in brewing

Fifteen wild yeast strains were isolated in two factories of a lager brewing company in Poland. Their identification with API 32C system showed mainly the presence of Candida sake species (7/15). To differentiate the isolates, randomly amplified polymorphic DNA (RAPD) with (GTG)(5), (GAC)(5), (GACA)(4) microsatellite primers and M13 core sequence (5'-GAG GGT GGC GGT TCT-3') were chosen. The results of patterns similarity are presented as dendrograms for each RAPD analysis and for overall patterns. On the overall patterns, all isolates identified as C. sake, except Strain No. 1, were regrouped in one cluster. Collection strain C. sake CBS 617 was similar in 46% to the cluster with six isolates (Strain Nos. 3, 6, 8, 11, 13, 14). The second reference strain C. sake CBS 159 and the Strain No. 1 were regrouped with other Candida species (collection strains) showing, respectively, only 20% and 42% of similarity to other C. sake strains. The similarity based on the overall dendrogram between isolate Nos. 3, 6, 8, 11, 13, 14 and C. sake CBS 617 was 49%. Between those strains and other Candida, the similarity was only 37%.     

部分烤烟种质遗传多样性与亲缘关系的RAPD分析

以22份烤烟材料为供试材料,应用RAPD(随即扩增多态性DNA)分子标记技术研究22份烤烟材料之间遗传多样性和亲缘关系.结果表明,第一等级划分(D=0.30)可以将22份材料划分为三大类群.第二等级划分(D=0.23)可以将22份材料划分为六个小类群,其中,一个是由云烟系列与亲本是特字401系选的品种及其杂交后代组成的类群;一个是由多数美国来源品种组成的类群;一个由9804、岩烟97和Coker176三个品种组成的类群;一个由CF965和K07两个品种组成的类群;红花大金元和C2分别独立成类.说明分子标记多态性基本上反映了品种间的遗传变异.本结果为烟草品种改良和杂交种选配,提高育种效率提供了理论依据.     

Discrimination of Tsingtaodahua from Other Hop Cultivars and Its Quality Control by Molecular Analysis

Random amplified polymorphic DNA (RAPD) was used for hop varietal identification, primarily to distinguish Tsingtaodahua, a fine Chinese variety. Eleven typical varieties, including four aroma hops, five bitter hops, Tsingtaodahua and Cluster, were successfully identified on the basis of 28 polymorphic RAPD bands amplified by five random primers. UPGMA analysis of RAPD data showed genetic relationship among analyzed varieties consistent with traditional hop classification. Subsequently, one specific RAPD fragment was converted to a sequence tagged site (STS) marker which can detect as little as a 5% admixture of the variety Kirin 1 in Tsingtaodahua. The RAPD and STS markers can be successfully used for Tsingtaodahua identification and quality control.     

草菇有性世代间rDNA序列变异分析及RAPD分析

草菇的遗传与有性生殖过程存在很多的特殊性和不确定性,本研究以草菇V23菌株的成熟子实体弹射孢子,单孢分离后得13个单株,分别摇瓶培养、收集菌丝,提取总DNA,用通用引物ITS4、ITS5进行PCR扩增后连接、转化并测序,将单孢间和单孢与亲本间的ITS序列进行比对,分析草菇ITS序列多样性及变异性;同时以相应单孢菌株DNA为模板,用20条随机引物进行RAPD扩增。结果表明:草菇单孢间和单孢与亲本间的ITS序列相似度均在99%以上,相较于亲本的ITS序列,单孢序列中发现了有5个位点出现碱基转换,但没有两个以上的单孢发生同一转换;20条随机引物分别与14个样本进行RAPD扩增,其中3个引物出现多态性条带,带型结果发现单孢菌株间的带型差异呈现一定规律。草菇ITS序列变异较少,不适于单孢间的变异分析,而RAPD方法对摸索草菇有性世代间的遗传分离规律有重要价值。     

中国猴头菇栽培菌株的系统进化研究

利用随机扩增多态性DNA(RAPD)标记对7 个猴头菇之间的亲缘关系进行研究,获得了猴头菇不同菌株的DNA 指纹图谱。结果显示：14 个随机引物中有3 个随机引物的扩增产物的DNA 条带表现出明显的多态性。这3个引物共扩增出44 条带,其中36 条为多态性条带,多态性位点比率为81.81%。同时利用NTSYSpc 2.1 生物软件分析7 个供试菌株之间的遗传相似性,并绘制系统进化树。     

克罗诺杆菌的随机多态性扩增DNA(RAPD)基因分型研究

研究了克罗诺杆菌标准菌株和样品中分离菌株的分子特征,为克罗诺杆菌的种间鉴别和分子溯源提供一种有效手段。采用随机引物扩增多态性DNA分析对38株菌株进行基因分型。筛选出1条随机引物,每一菌株可扩增出1～7条DNA片段,片段在400-3500bp,可将8株标准菌株的种间区分开来。以相似性50%为标准,38株克罗诺杆菌按照其遗传差异可以分为8个基因型类群。所建立的RAPD技术可用于克罗诺杆菌的种间鉴别和分子溯源研究。