Genetic Diversity of Pleurotus pulmonarius Revealed by RAPD,ISSR, and SRAP Fingerprinting

Pleurotus pulmonarius is one of the most widely cultivated and popular edible fungi in the genus Pleurotus. Three molecular markers were used to analyze the genetic diversity of 15 Chinese P. pulmonarius cultivars. In total, 21 random amplified polymorphic DNA (RAPD), 20 inter-simple sequence repeat (ISSR), and 20 sequence-related amplified polymorphism (SRAP) primers or primer pairs were selected for generating data based on their clear banding profiles produced. With the use of these RAPD, ISSR, and SRAP primers or primer pairs, a total of 361 RAPD, 283 ISSR, and 131 SRAP fragments were detected, of which 287 (79.5 %) RAPD, 211 (74.6 %) ISSR, and 98 (74.8 %) SRAP fragments were polymorphic. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of these three methods were structured similarly, grouping the 15 tested strains into four clades. Subsequently, visual DNA fingerprinting and cluster analysis were performed to evaluate the resolving power of the combined RAPD, ISSR, and SRAP markers in the differentiation among these strains. The results of this study demonstrated that each method above could efficiently differentiate P. pulmonarius cultivars and could thus be considered an efficient tool for surveying genetic diversity of P. pulmonarius.     

Evaluation of genetic diversity in Lentinula edodes strains using RAPD, ISSR and SRAP markers

Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers were used to evaluate the genetic diversity among 23 elite Lentinula edodes strains in China. A total of 138, 77 and 144 bands were detected by 16 RAPD primers, 5 ISSR primers and 23 SRAP primer combinations, among which 58.8%, 73.5% and 56.3% was polymorphic, respectively. By UPGMA clustering, a dendrogram was constructed based on each analysis. The three dendrograms showed that 23 L. edodes strains were clustered into three or four groups. The grouping exhibited similar structure and was generally consistent with their pedigrees. Twenty-three L. edodes strains shared great similarity indicated that the low level of genetic diversity of L. edodes strains and their relationship between each other. The important source of breeding material, such as wild and exotic types, must be introduced in order to broaden genetic base and decreases genetic vulnerability of L. edodes.     

Analysis of genetic diversity among Chinese <Emphasis Type="Italic">Pleurotus citrinopileatus</Emphasis> Singer cultivars using two molecular marker systems (ISSRs and SRAPs) and morphological traits

To evaluate the genetic diversity of Pleurotus citrinopileatus Singer cultivars in China, 20 P. citrinopileatus strains were analyzed using morphological traits, inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) molecular markers. Eleven ISSR primers amplified a total of 116 DNA fragments of which 96 (82.91%) were polymorphic, whereas 8 SRAP primer pairs amplified 69 fragments of which 65 (93.47%) were polymorphic. Phylogenetic trees constructed on the basis of ISSR, SRAP, and combined ISSR/SRAP analyses using the Unweighted Pair-group Method with Arithmetic Averages method distributed the 20 strains into three or six major groups. The grouping exhibited great similarity and was generally consistent with their morphological characters and antagonism test, which indicated a high level of genetic diversity among P. citrinopileatus Singer and relationship between each other. Based on the genetic analysis, the primary mini-core strains were constructed with progressive sampling method of the smallest genetic distance. The mini-core germplasm collection included 4 strains (strain 2, 5, 7 and 11). Our findings will provide a scientific fundament for facilitating parent selection for broadening genetic base, accelerating the genetic breeding, identification of cultivated strains and the development of bioactive products from this commercially important medicinal mushroom.     

Analysis of Genetic Diversity Among Chinese Auricularia auricula Cultivars Using Combined ISSR and SRAP Markers

DNA polymorphism among 34 Chinese Auricularia auricula cultivars was analyzed using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Thirty ISSR primers amplified a total of 129 DNA fragments of which 125 (96.9%) were polymorphic, whereas 11 SRAP primer combinations amplified 154 fragments of which 148 (96.1%) were polymorphic. Both methods were highly effective in discriminating among the test strains. Phylogenetic trees constructed on the basis of ISSR, SRAP, and combined ISSR/SRAP analyses using the Unweighted Pair-group Method with Arithmetic Averages (UPGMA) method distributed the 34 strains into four or five major groups. Clustering analysis based on all the three data sets indicated a high level of genetic diversity among A. auricula, although the combined ISSR/SRAP data were more concordant with the main agronomic characters of strains and their geographical centers of cultivation. Our findings will facilitate future A. auricula breeding programs and the development of bioactive products from this commercially important medicinal mushroom.     

Evaluation of the Use of SCAR Markers for Screening Genetic Diversity of Lentinula edodes Strains

In this study, three molecular marker systems including sequence related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and inter-simple sequence repeats (ISSR) were screened to select polymorphisms of 24 main commercial strains of Lentinula edodes cultivated widely in China. Twenty-nine sequence characterized amplified region (SCAR) markers were developed to set up a dendrogram using UPMGA based on nucleotide sequences of some SRAP, RAPD, and ISSR polymorphic fragments. The grouping showed that the 24 strains were apparently clustered into five groups at a level of 0.68 similarity coefficient, and those that have similar breeding background clustered preferentially into the same subgroup. Results also revealed that the 24 strains had a low level of genetic diversity, and the breeding source of L. edodes should be broadened by exploiting wild types and introducing exotic strains. In addition, the tested strains of L. edodes could be clearly distinguished and identified from others by using different combinations of SCAR primers. Thus, results of this work demonstrated that SCAR was an excellent genetic marker system to characterize and investigate genetic diversity of L. edodes. Furthermore, this provided an alternative method to identify the genetic relationship of different strains of other fungi.     

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

Molecular Characterization of <Emphasis Type="Italic">Cyclamen</Emphasis> Species Collected from Different Parts of Turkey by RAPD and SRAP Markers

The genus Cyclamen (family Myrsinaceae) contains about 20 species, most of which occur in the Mediterranean region. Turkey has critically important Cyclamen genetic resources. Molecular characterization of plant materials collected from different regions of Turkey in which Cyclamen species grow naturally, namely Adana, Antalya, Ayd?n, Mu?la, ?zmir, Denizli, Kahramanmara?, Osmaniye, Eski?ehir, Trabzon, and Rize provinces, was performed using RAPD and SRAP markers. DNA was successfully amplified by 30 RAPD primers and 14 SRAP primer pairs. Among the 470 bands generated by the RAPD primers, 467 were polymorphic. The number of bands detected by a single primer set ranged from 11 to 22 (average of 15.6). The percentage polymorphism was 99.3 % based on the RAPD data. In the SRAP analysis, a total of 216 bands were generated, showing 100 % polymorphism. The number of bands detected by a single primer set ranged from 9 to 22 (average of 15.4). All data were scored and UPGMA dendrograms were constructed with similar results in both marker systems, i.e., different species from nine provinces of Turkey were separated from each other in the dendrograms with the same species being clustered together.     

Use of Randomly Amplified Polymorphic DNA as a Means of Developing Genus- and Strain-Specific Streptomyces DNA Probes

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.     

Evaluation of Genetic Diversity Among Chinese Pleurotus eryngii Cultivars by Combined RAPD/ISSR Marker

Pleurotus eryngii (DC. Ex. Fr.) Quél is a rare precious edible fungus which belongs to the family Pleurotaceae. This mushroom has highly nutritional, pharmaceutical, economic and ecological values. In the present study, combined randomly amplified polymorphic DNA (RAPD)/inter-simple sequence repeat (ISSR) was used to assess the genetic diversity of P. eryngii strains cultivated in China. For the RAPD and ISSR analyses, 404 and 392 polymorphic bands were obtained from 32 P. eryngii strains using 28 and 24 selected primers, respectively. A combined RAPD/ISSR dendrogram grouped the 32 strains into five clades with coefficient of 0.770. The comparison of RAPD and ISSR was also elucidated in the present study. The results of our study obtained by combined RAPD/ISSR analysis contributed to a better understanding of the genetic relationships among the P. eryngii strains and provide orientation for the strain improvement of P. eryngii species.     

Differentiation of Dextran-Producing Leuconostoc Strains by a Modified Randomly Amplified Polymorphic DNA Protocol

Seven dextran-producing Leuconostoc strains were differentiated by using a modified randomly amplified polymorphic DNA (RAPD) protocol that incorporated specific primers designed from conserved regions of dextransucrase genes. RAPD profiles showed intraspecies differences among the Leuconostoc mesenteroides strains tested. This modified RAPD protocol will aid in the differentiation of polymer-producing leuconostocs, which are currently distinguished by time-consuming analyses of the dextrans they synthesize.     

Genetic diversity assessment of a germplasm collection of Salvia miltiorrhiza Bunge. based on morphology,ISSR and SRAP markers

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.     

松花型花椰菜主要品种鉴定的分子标记分析

利用RAPD、ISSR和SRAP 3种分子标记对我国南方地区松花型花椰菜主栽品种进行鉴定,分析了品种间的遗传多样性。3种标记共产生370条扩增带,238条为多态性条带,其多态率为64.32%。其中只有SRAP标记的引物m e1/em1可将20个品种全部鉴别。遗传相似系数分析表明,松花型花椰菜品种之间的亲缘关系较近,遗传背景比较狭窄。聚类分析表明品种间的亲缘关系与熟性、地理分布相关。研究表明,分子标记能有效地应用于花椰菜品种鉴定,且综合多种分子标记分析品种间的遗传多样性将更加准确可靠。     

Genetic diversity,core collection and breeding history of Pleurotus ostreatus in China

Pleurotus ostreatus is one of the most widespread and favourably cultivated mushrooms in China. The cultivated strains of this species have been frequently exchanged domestically and internationally, but no detailed breeding history has been documented. The frequent domestic and international exchange of strains combined with a non-detailed historical documentation of breeding might have led to confusion about strain names and genetic background. In this study, 91 strains of P. ostreatus, including 57 cultivated and 34 wild strains, were analysed using 21 simple sequence repeat markers developed from the genomic sequence of “P. cf. floridanus”. Among the cultivated strains, 46 were found to possess different allelic patterns. The remaining 11 strains were clustered into five groups, each with their own private alleles, suggesting that 10.5% (6/57) of the cultivated strains were previously labelled with improper names. Our analyses indicate that wild strains harbored greater genetic diversity than the cultivated strains. With regard to the cultivation history of P. ostreatus, the cultivated strains in China have three sources: direct introduction from Europe, domestication from wild strains from China, or hybridisation of the European and Chinese strains. Furthermore, we propose a core collection of P. ostreatus with 34 strains, including 13 wild and 21 cultivated strains. The allele retention proportion of the core collection for the entire collection was 100%.     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Genetic Linkage Map of the Edible Basidiomycete Pleurotus ostreatus

We have constructed a genetic linkage map of the edible basidiomycete Pleurotus ostreatus (var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (est1); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of P. ostreatus, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of P. ostreatus.     

Genetic diversity and population structure of Butea monosperma (Lam.) Taub.- a potential medicinal legume tree

Three molecular marker systems, Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeats (ISSR) and Sequence-Related Amplified Polymorphism (SRAP) were employed to investigate the genetic structure and diversity among the 14 natural populations of Butea monosperma collected from different geographical regions of India. Detected by 17 RAPD, 15 ISSR and 11 SRAP primer combinations, the proportions of polymorphic bands were 84.2 %, 77.2 % and 91.9 %, respectively, and the mean Nei’s genetic distances among the populations were 0.13, 0.10 and 0.13, respectively. Partitioning of genetic variability by Analysis of molecular variance (AMOVA) revealed that the high genetic diversity was distributed within the populations. AMOVA also revealed that the coefficient of gene differentiation among populations based on F_ST was very high irrespective of markers used. The overall gene flow among populations (Nm) was very low. Cophenetic correlation coefficients of Nei’s distance values and clustering pattern by Mental test were statistically significant for all three marker systems used but poor fit for ISSR data than for RAPD, SRAP and combined data set of all three markers. For all markers, a high similarity in dendrogram topologies was obtained, although some differences were observed with ISSR. The dendrogram obtained by RAPD, SRAP and combined data set of all three markers reflect relationship of most of the populations according to their geographic distribution.     

Strain-Specific Identification of Probiotic Lactobacillus rhamnosus with Randomly Amplified Polymorphic DNA-Derived PCR Primers

In the present work, strain-specific PCR primers for Lactobacillus rhamnosus Lc 1/3 are described. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. They were screened for specificity by hybridization with DNA from 11 L. rhamnosus strains. A 613-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific to L. rhamnosus Lc 1/3 was constructed based on the sequence. The primer pair was tested with 11 Lactobacillus species and 11 L. rhamnosus strains and was found to be strain specific. The nucleotide sequence of the specific RAPD marker was found to contain part of a protein encoding region which showed significant similarity to several transposases for insertion sequence elements of various bacteria, including other lactic acid bacterium species.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

Characterization of enteroinvasive Escherichia coli and Shigella strains by RAPD analysis

Genetic variation of 33 enteroinvasive Escherichia coli (EIEC), 12 non-EIEC and 39 Shigella strains (representing the 4 species of this genus) was analyzed using the random amplified polymorphic DNA (RAPD) technique. Reproducible polymorphisms were generated and the combined data allowed us to construct a dendrogram using Jaccard's distance. Two main groups were obtained: one for Shigella and the other for EIEC and non-EIEC strains. The first group contained four clusters, one for each Shigella species. The second group contained one cluster for EIEC and another for non-EIEC strains. The main clusters encompassed many small clusters corresponding to different serotypes. It was possible to characterize each one of the 84 strains under study as well as the boundaries among Shigella species and between this genus and EIEC strains.