DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus.     

RAPD discrimination of Agaricus bisporus mushroom cultivars

Cultivars of the white button mushroom Agaricus bisporus are difficult to differentiate, which has made strain protection problematic for this crop species. We have used RAPDs to discriminate between 26 strains of A. bisporus, 24 of which were commercial cultivars, and to characterise the genetic relatedness of these strains. Using 20 primers, 211 RAPD markers were identified and used in hierarchical cluster, patristic distance and parsimony analyses. All strains could be differentiated using the aggregated primer data. Although no one primer could differentiate all 26 strains, several individual primers yielded unique fingerprints for a variety of strains. The greatest differences (up to 28% variation) were observed in comparisons with or between two wild collections of A. bisporus. Quondam cultivars, commercial brown and off-white varieties proved more variable than the widely grown 'hybrid' types. Of the 15 hybrid varieties analysed, only one differed substantially (20% or more variable). The patristic and parsimony analyses both demonstrated the gross similarity of the hybrids, many of which appear to be essentially derived varieties from two original hybrid cultivars. RAPD analyses can assist mushroom strain identification and could play a role in the protection of novel cultivars.     

Mitochondrial inheritance and the detection of non-parental mitochondrial DNA haplotypes in crosses of Agaricus bisporus homokaryons

This study evaluates mtDNA transmission in Agaricus bisporus, as well as the occurrence of non-parental haplotypes in heterokaryons produced by controlled crosses. Sixteen crosses were performed with blended liquid cultures, using different combinations of 13 homokaryotic strains. For each cross, different mtDNA haplotypes were present in each homokaryon. Heterokaryons generated from these crosses were subject to genetic analysis with RFLP markers to identify (i). karyotic status, (ii). mtDNA haplotype, and (iii). the occurrence of non-parental mtDNA haplotypes. These analyses generally supported the occurrence of uniparental mitochondrial (mt) inheritance in A. bisporus, with one mtDNA haplotype usually favoured in the new heterokaryon. The preponderance of one mtDNA haplotype in a new heterokaryon did not necessarily show a correlation with a greater mycelial growth rate for the parent homokaryon possessing that haplotype. Mixed mtDNA haplotypes and non-parental haplotypes were also identified in the heterokaryons from some crosses. Evidence for the occurrence of two mtDNA haplotypes in one heterokaryotic mycelium was observed in 8 of 16 crosses, suggesting the maintenance of true heteroplasmons after three successive subculturing steps. Non-parental mtDNA haplotypes were seen in heterokaryons produced from 7 of 16 crosses. The mating protocol described can be utilized to generate novel mtDNA haplotypes for strain improvement and the development of strain-specific markers. Mechanisms of mt selection and inheritance are discussed.     

Genetic relationship between Polish and Chinese strains of the mushroom Agaricus bisporus (Lange) Sing., determined by the RAPD method

The genetic relationship between twenty-six strains of Agaricus bisporus were analysed by the RAPD (random amplified polymorphic DNA) method. DNA amplification was performed with the use of twelve arbitrary 10-mer primers. Four primers, which gave polymorphic band patterns were chosen for RAPD analysis. In total, they gave 24 distinguishable bands, of which nine were polymorphic. The conducted research showed that there is a great genetic similarity among the examined strains. Low polymorphism of the strains may be a proof of a limited genetic pool used in the cultivation of those strains.     

Mitochondrial Haplotype Influences Mycelial Growth of Agaricus bisporus Heterokaryons

We evaluated the influence of mitochondrial haplotype on growth of the common button mushroom Agaricus bisporus. Ten pairs of heterokaryon strains, each pair having the same nuclear genome but different mitochondrial genomes, were produced by controlled crosses among a group of homokaryons of both wild and commercial origins. Seven genetically distinct mitochondrial DNA (mtDNA) haplotypes were evaluated in different nuclear backgrounds. The growth of heterokaryon pairs differing only in their mtDNA haplotypes was compared by measuring mycelial radial growth rate on solid complete yeast medium (CYM) and compost extract medium and by measuring mycelial dry weight accumulation in liquid CYM. All A. bisporus strains were incubated at temperatures similar to those utilized in commercial production facilities (18, 22, and 26(deg)C). Statistically significant differences were detected in 8 of the 10 heterokaryon pairs evaluated for one or two of the three growth parameters measured. Some heterokaryon pairs showed differences in a single growth parameter at all three temperatures of incubation, suggesting a temperature-independent difference. Others showed differences at only a single temperature, suggesting a temperature-dependent difference. The influence of some mtDNA haplotypes on growth was dependent on the nuclear genetic background. Our results show that mtDNA haplotype can influence growth of A. bisporus heterokaryons in some nuclear backgrounds. These observations demonstrate the importance of including a number of mitochondrial genotypes and evaluating different nuclear-mitochondrial combinations of A. bisporus in strain improvement programs.     

New available SCAR markers: potentially useful in distinguishing a commercial strain of the superior type from other strains of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in China

At present, more than 100 strains of Lentinula edodes are cultivated on a commercial scale in China. A simple, reliable, and effective method to distinguish some commercial strains of the superior type from other commercial strains is very important for the Lentinula industry. In this study, 23 commercial strains of L. edodes cultivated widely in China at present were collected and analyzed with randomly amplified polymorphic DNA (RAPD) technique. Three informative dominant sequence characterized amplified region (SCAR) markers were developed by designing three pairs of specific SCAR primers from three sequenced differential RAPD bands, respectively. Based on the three SCAR markers, three different multiplex polymerase chain reaction (PCR) phenotypes were detected among the 23 studied commercial strains and in which a multilocus phenotype characterizing a commercial strain Cr02 of the superior type could potentially be used to distinguish this strain from the other 22 studied commercial strains. To our knowledge, this study is the first to describe the development of a multiplex PCR technique based on SCAR markers for detecting the molecular phenotypes among commercial strains of L. edodes in China.     

双孢蘑菇遗传多样性分析

应用AFLP指纹技术对双孢蘑菇的20个野生菌株和5个栽培品种的遗传多样性进行了研究。AFLP指纹揭示出20个野生异核体菌株所固的基因型。5个商业品种表现出比较一致的AFLP指纹,但也显示出它们之间的一些差别。由单孢分离获得的同核体菌株携带着部分异核体菌株的AFLP指纹;由同一子实体分离得到的大部分单孢菌株是异核体菌株,它们具有与其亲本一致的AFLP指纹。UPGMA分析揭示出2个与地理分布（美洲、欧洲）和相对应的组。研究结果表明：（1）在野生菌株之间存在着明显的遗传差异;（2）大多数单孢分离的菌株具有与母本一致的遗传物质;（3）野生菌株间的遗传变异大于栽培品种间的变异;（4）在双孢蘑菇的遗传多样性分析中,AFLP技术是非常有效的工具。     

微卫星(TATG)n基序在香菇菌种中的验证

以（TATG）4重复序列为引物对香菇属的3个种13个菌株的微卫星区DNA进行PCR扩增,15%的琼脂糖凝胶电泳,获得了25个条带,并且在供试菌株上表现出多态性,可以实现遗传分类研究。为了验证微卫星分子标记实验准确性,又用RAPD技术对13个供试菌株进行了实验。7个引物在13个菌株上共获得了102条多态性条带。通过聚类分析,RAPD获得的分类结果与微卫星分子标记获得的结果一致。此外,为了证明微卫星分子标记获得的条带不是假阳性,在实验中回收了No.10菌株的PCR扩增产物,进行克隆测序。测序结果显示有（TATG）n基序存在,并且达到了微卫星基序重复数量的最低限度。通过本实验可知,香菇中是存在微卫星（TATG）n基序的, 且基序的多态性可以用于香菇的遗传分类研究。     

Random-amplified polymorphic DNA (RAPD) analysis shows intraspecies differences among Xanthomonas albilineans strains

Five strains of Xanthomonas albilineans , causal agent of leaf scald disease in sugarcane from various geographical regions, were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the DNA level. CsC1-purified genomic DNA from these strains were amplified by the polymerase chain reaction (PCR) using arbitrary 10-mer primers according to standard RAPD conditions and the amplification product profiles analysed by conventional agarose gel electrophoresis. Although most RAPD markers were common to all five strains, unique profiles for each strain were discernible using four 10-mer arbitrary primers individually. Reproducible DNA fingerprints indicate that RAPD analysis can be used to identify and differentiate the X. albilineans strains. This technique has the potential for use in monitoring the appearance of foreign strains of X. albilineans in various geographical regions and could be used for the construction of phylogenetic trees.     

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay

Abstract Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, Lentinula edodes . Seven primers produced polymorphisms in all 15 strains tested, producing 12–19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applications in mushroom breeding and strain improvement programs.     

球孢白僵菌种群野生菌株异核现象的分子验证*

球孢白僵菌28S rRNA基因D11域由于一内含子的存在与否,引起这一区域的扩增产物有两种片段类型(约520bp及120bp),将此作为分子标记分析了球孢白僵菌的117个菌株,发现17%菌株的扩增产物为双片段类型,即为异核体。异核体在不同种群中的分布频率因地理生态条件而异。单孢分离分析表明,异核体的分离频率约为35%。RAPD检测结果说明异核体在准性循环中发生了不同程度的遗传交换及遗传重组。球孢白僵菌异核体在继代培养中极不稳定。     

Assessment of the genetic diversity among strains of Xanthomonas cynarae by randomly amplified polymorphic DNA analysis and development of specific characterized amplified regions for the rapid identification of X. cynarae

The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.     

Strain-specific detection of introduced Beauveria bassiana in agricultural fields by use of sequence-characterized amplified region markers

Field studies on the efficacy and persistence of an introduced strain of Beauveria bassiana for insect control require detection assays to differentiate the non-native strain from indigenous populations. In this study we developed strain-specific molecular markers based on polymerase chain reaction amplification of sequence-characterized amplified regions (SCAR) in combination with dilution plating on semi-selective medium to detect and estimate density of propagules of a commercial strain of B. bassiana (strain GHA) in field samples. Using random amplified polymorphic DNA (RAPD) analysis, unique fragments that distinguished GHA from other strains of B. bassiana were obtained. Three amplicons, OPA-14(0.44), OPA-15(0.44), and OPB-9(0.67), generated with RAPD primers were cloned and sequenced and used as bases for designing SCAR primers OPA14 F/R(445), OPA15 F/R(441), and OPB9 F/R(677), respectively. All three SCAR primers were highly sensitive, capable of detecting 100pg B. bassiana GHA genomic DNA, and thus could be used to detect varying levels of the fungus in the field.     

双孢蘑菇杂交菌株As2796家系的分子遗传研究

应用PCR和凝胶电泳等技术,对双孢蘑菇杂交菌株As2796及其亲本和子代作分子遗传标记跟踪分析,结果如下: 1) 总DNA的RAPD分析表明,随着遗传代数的增加,杂种子代和出发异核体亲本间的遗传差异逐渐增大; 2) mtDNA的酶切图谱表明,亲本8213及其杂交子代具有相同的基因型,表明双孢蘑菇的mtDNA呈单亲遗传; 3) Est同工酶的PAGE图谱表明, 结合了亲本02高产特征和8213优质特征的杂交子代具有两个亲本的标记带型,证明Est同工酶标记是双孢蘑菇新菌株特性预测或鉴定的有效指标。     

Identification of Escherichia coli Recovered from Milk of Sows with Coliform Mastitis by Random Amplified Polymorphic DNA (RAPD) Using Standardized Reagents

A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden. Previously, the 58 E. coli strains were characterized serologically and profiled biochemically. They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts. The RAPD analysis was fast, easily performed, and required only a nanogram of DNA. The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E. coli strains that cause mastitis in sows. The results of the RAPD analyses demonstrated that E. coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique. The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type. No relationship between serotypes, virulence factors and RAPD types was found.     

双孢蘑菇杂交菌株As2796家系的分子遗传研究*

应用PCR和凝胶电泳等技术,对双孢蘑菇杂交菌株As2796及其亲本和子代作分子遗传标记跟踪分析,结果如下: 1) 总DNA的RAPD分析表明,随着遗传代数的增加,杂种子代和出发异核体亲本间的遗传差异逐渐增大; 2) mtDNA的酶切图谱表明,亲本8213及其杂交子代具有相同的基因型,表明双孢蘑菇的mtDNA呈单亲遗传; 3) Est同工酶的PAGE图谱表明, 结合了亲本02高产特征和8213优质特征的杂交子代具有两个亲本的标记带型,证明Est同工酶标记是双孢蘑菇新菌株特性预测或鉴定的有效指标。     

Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.     

Genomic diversity within the genus Pediococcus as revealed by randomly amplified polymorphic DNA PCR and pulsed-field gel electrophoresis

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.     

甜菜夜蛾抗高效氯氟氰菊酯近等基因系的RAPD标记

用随机引物扩增的方法对近等基因系抗高效氯氟氰菊酯种群和敏感种群甜菜夜蛾进行比较分析,从160条引物中筛选,引物S42(GGACCCAACC)和OPH13(GACGCCACAC)在抗性种群中扩增分别得到了1条特异扩增带,带型清晰、重复性较好,所以S42和OPH13产生的两条特异扩增带是分别与甜菜夜蛾对高效氯氟氰菊酯抗性相关的RAPD分子标记。     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.