Species of Gleditsia show considerable morphological variability that makes them difficult to distinguish using either vegetative or floral characters. Honeylocusts, especially the thornless cultivars, are popular ornamental, shade, street, attractive landscape trees. In this study the ISSR technique was used to evaluate the range of genetic variability between seven genotypes of Gleditsia cultivated in Polish dendrological collections [Gleditsia caspica Desf., Gleditsia japonica Miq., Gleditsia japonica Miq. var. korainensis (= G. korainensis Nakai), Gleditsia triacanthos L., Gleditsia triacanthos L. (bulk), Gleditsia triacanthos f. inermis (L.) Zabel (bulk). Forty ISSR primers were tested and 18 were selected for their ability to produce clear and reproducible patterns of multiple bands.A total of 177 loci of 260-2600 bp were amplified, of which 89 (50%) were polymorphic, 14 (8%) monomorphic and 74 (42%) were accession-specific. Accession-specific ISSR loci were obtained for all of the seven accessions tested. A dendrogram generated using the UPGMA, based on a similarity measure of total character difference, showed that the Gleditsia accessions were clustered into two main groups (‘a’ and ‘b’). The first grup –‘a’– included: Gleditsia triacanthos L., Gleditsia triacanthos L. (bulk) and Gleditsia triacanthos f. inermis (L.) Zabel (similarity 0.61–0.75), the second –‘b’– included 2 species: Gleditsia japonica and Gleditsia japonica var. korainensis (similarity 0.43). Analysis of the phylogenetic similarity dendrogram has shown wide range of diversity between studied accessions. The clustering pattern obtained in our experiment was in agreement with the data based on morphological, allozyme and ITS analysis.
We expanded the basic ISSR-PCR protocol by an additional PCR reamplification round in order to detect whether increased PCR productivity would give new bands in ISSR patterns. We found that the reamplification step had a prominent impact on the quality of the inter-simple-sequence repeat (ISSR) PCR patterns of flax, depending on the particular primer used for PCR amplification. We could clearly distinguish between two types of reamplification effect. Most ISSR primers (16 out of 21) gave no reamplification effect as usual, but five primers (23.8%) provided a new ISSR fingerprinting pattern after the 2nd reamplification round, leaving the previous 1st round pattern completely blank. Therefore, we recommend the expansion of a basic ISSR-PCR protocol for another reamplification round in order to mine out full the fingerprinting potential from ISSR-PCR method.
A qualitative and quantitative analysis of anthocyanins in juices of three varieties of strawberry (Senga, Ducat, Marmolada), raspberry (Beskid, Canby, Malling Seedling), black currant (Ben Lomond, Titania, Ojebyn) and red currant (Rondom, Jonker, Holenderska) picked in three following years: 1998, 1999 and 2000, was presented in this paper. An HPLC technique was applied using a Gilson chromatograph and a DAD detector. Prior to the chromatographic analysis, anthocyanins were purified on a mini-column Sep-Pak C18 Waters. It was indicated that within species the juices examined differed in the quantitative and qualitative composition of anthocyanins. Pelargonidin-3-glucoside and cyanidin-3-xylorutinoside were the main anthocyanins in strawberry and red currant juices, respectively, independently of variety. Those anthocyanins were not detected in raspberry and black currant juices, in which cyanidin-3-sophoroside as well as delphinidin-3-rutinoside and cyanidin-3-rutinoside were the main anthocyanins, respectively. Differences of anthocyanin composition of juices obtained from different berry fruits create the possibility of detecting the adulterations of expensive raspberry and black currant juices with cheap strawberry and red currant juices on the basis of anthocyanin analysis.
The rep-PCR fingerprinting method, with the support of ERIC and REP primers, was used to analyse the genomic diversity of 93 E. coli strains isolated from lake water samples drawn at two different depths. The applied UPGMA for DNA analysis did not reveale any genomic similarities between the 48 E. coli strains derived from the subsurface-zone water and the 43 of the bottom-zone water. The considerable genomic diversity of the E. coli of the surface zone was expressed as a dendrogram in the form of 8 similarity groups comprising strains isolated from samples drawn over one month. The bottom-zone strains, which display a lesser degree of genomic diversity (5 similarity groups), showed distinct common features in their DNA fingerprints. In the similarity dendrogram for the bottom-zone, strains derived in different months of sampling were segregated into the same similarity groups. Applying REP primers in rep-PCR generates more complex fingerprints increasing the discriminatory power of the analysis, whereas the ERIC primer generates less complex fingerprint patterns, and is thus clearer to interpret.
The genetic integrity of four accessions of the cross-pollinating species rye (Secale cereale L.) was investigated. Seeds available from the first and most recent regeneration cycles, multiplied 8, 12 (twice) or 14 times were fingerprinted using microsatellite markers. In all four accessions the allele numbers and frequencies changed after regeneration. Alleles present in the original seed sample were not detectable in the regenerated populations, whereas on the other hand, alleles were found in the recent seed sample, which were not observed in the investigated plants of the original one.
In this study, the rep-PCR technique was used to differentiate isolates of bacteria belonging to genus Pseudomonas and phosphate-dissolving bacteria collected from the root vicinity of apple and sour cherry trees. DNA amplification was carried out with complementary primers for repetitive sequences: REP (repetitive extragenic palindromic sequence), ERIC (enterobacterial repetitive intergenic consensus) and the BOX element. The most differentiated DNA profiles were observed when using REP1R-I and REP2-I primers, in reactions with which 25 different DNA patterns were obtained for 28 isolates. In reactions with the primers ERIC1R and ERIC2 or BOXA1R, 24 and 22 patterns were obtained, respectively. Following the use of all the primers, no differences were found in the DNA profiles of two isolates of Pseudomonas bacteria and three isolates of phosphate-dissolving bacteria. This result suggests that the isolates in which no DNA polymorphism was observed belong to the same bacterial strain.
Methicillin resistant Staphylococcus aureus (MRSA), particularly strains with type III staphylococcal cassette chromosome mec (SCCmec), represent a serious human pathogen in Tehran, Iran. The disease-causing capability depends on their ability to produce a wide variety of virulent factors. The prevalence of exotoxin genes and multiple-locus variable number of tandem repeats fingerprinting (MLVF) profile among MRSA isolates, from patients in Tehran, was evaluated by PCR and Multiplex-PCR. The MLVF typing of 144 MRSA isolates with type III SCCmec produced 5 different MLVF types. Generally, 97.2% (140/144) of all the isolates were positive for at least one of the tested exotoxin genes. The most prevalent genes were hld, found in 87.5% (126/144) of the isolates followed by lukE-lukD and hla found in 72.9% (105/144) and 70.1% (101/144) of the isolates, respectively. The tst gene, belonging to MLVF types I, IV and V, was found among three of the isolates from blood and wound samples. The sea gene was detected in 58.3% (84/144) of the isolates and the sed and see genes were found in one isolate with MLVF type V. The coexistence of genes was observed in the 87.5% (126/144) of the isolates.The rate of coexistence of hld with lukE-lukD, hla with lukE-lukD and sea with lukE-lukD were 66.7% (96/144), 44.4% (64/144) and 44.4% (64/144), respectively. The present study demonstrated that MRSA strains with type III SCCmec show different MLVF patterns and exotoxin profiles.
W reakcji PCR - fingerprinting w układzie dwóch starterów ERIC 1 i ERIC 2 wśród 120 szczepów paciorkowców grupy В stwierdzono obecność 13 genotypów Metoda ta stwarza nowe możliwości różnicowania paciorkowców grupy В w badaniach epidemiologicznych.
The distinctness, uniformity and stability (DUS) requirements involve expensive, space- and time-consuming measurements of morphological traits. Moreover, for a majority of traits, interactions between genotype and environment complicate the evaluation. Molecular markers have a potential to facilitate this procedure, increase the reliability of decisions, and substantially save the time and space needed for experiments. We chose 25 varieties of pea (Pisum sativum L.) from the list of recommended varieties for cultivation in the Czech Republic, and made both a standard classification by 12 morphological descriptors and a classification by biochemical-molecular markers. Two isozyme systems, 10 microsatellite loci, 2 retrotransposons for multilocus inter-retrotransposon amplified polymorphism (IRAP), and 12 retrotransposon-based insertion polymorphism (RBIP) DNA markers were analysed. The main objective of the study was to examine the potential of each method for discrimination between pea varieties. The results demonstrate a high potential and resolving power of DNA-based methods. Superior in terms of high information content and discrimination power were SSR markers, owing to high allelic variation, which was the only biochemical-molecular method allowing clear identification of all varieties. Retrotransposon markers in RBIP format proved to be the most robust and easy to score method, while multilocus IRAP produced informative fingerprint already in a single analysis. Isozyme analysis offered a fast and less expensive alternative. The results showed that molecular identification could be used to assess distinctness and complement morphological assessment, especially in cases where the time frame plays an important role. Currently developed pea marker systems might serve also for germplasm management and genetic diversity studies.