Amplified Fragment Length Polymorphism Profiles Research Articles

Banana somaclonal variation assessed by Amplified Fragment Length Polymorphism profiles at early cycles of in vitro culture

Banana micropropagation for obtaining free-virus plants frequently provokes somaclonal variation that could increase useful genetic variability in this asexually propagated crop. Both exploring the cycle of in vitro culture in which somaclonal variation occurs and the amount of generated polymorphism, are necessary. In this work, preliminary results of somaclonal variation during early cycles of banana in vitro culture are reported. Four randomly selected regenerated plants from the fifth cycle and two samples from the mother plant were analyzed. A total of 36 AFLP primer combinations were assayed, and 24 of them produced amplicons varying among 50- 500 bp. The mother plant presented a total of 125 different amplicons while the regenerated plants jointly showed 131 different amplicons with a mean of 119.75 ± 3.97 per individual. High level of DNA polymorphism (24.43 %) was found among micropropagated plants and, additionally, the occurrence of somaclonal variation at earlier cycles was suggested by multivariate analysis of Principal Coordinates. In this study, somaclonal variation at early cycles of banana micropropagation was validated and the adequacy of AFLP technique to assess it at the molecular level was verified. The phenotypic effects of the detected somaclonal variations remain to be evaluated.

Exploring genetic diversity, population structure, and phylogeography in Paracoccidioides species using AFLP markers.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii. DNA-sequencing of protein-coding loci (e.g., GP43, ARF, and TUB1) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides. We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67–87 fragments), the values of polymorphism information content (PIC = 0.3345–0.3456), marker index (MI = 0.0018), effective multiplex ratio (E = 44.6788–60.3818), resolving power (Rp = 22.3152–34.3152), discriminating power (D = 0.5183–0.5553), expected heterozygosity (H = 0.4247–0.4443), and mean heterozygosity (Havp = 0.00002–0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651–0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ2 = 1.025; P = 0.3113), P. venezuelensis (χ2 = 0.692; P = 0.4054), and P. lutzii (χ2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found for P. americana (χ2 = 8.909; P = 0.0028) and P. restrepiensis (χ2 = 4.571; P = 0.0325) with a preponderance of MAT1-1. Geographical distributions confirmed that P. americana, P. restrepiensis, and P. lutzii are more widespread than previously thought. P. brasiliensis s. str. is by far the most widely occurring lineage in Latin America countries, occurring in all regions of Brazil. Our new DNA fingerprint assay proved to be rapid, reproducible, and highly discriminatory, to give insights into the taxonomy, ecology, and epidemiology of Paracoccidioides species, guiding disease-control strategies to mitigate PCM.

Contribution to determination of extracellular DNA origin in the biofilm matrix.

This study is focused on the analysis of extracellular DNA (eDNA) from a biofilm matrix formed by Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica. The presence of eDNA in the biofilm of all the studied strains was confirmed by confocal laser scanning microscopy using fluorescent dyes with high affinity to nucleic acid. The protocol for eDNA isolation from the biofilm matrix was established, and subsequent characterization of the eDNA was performed. The purified eDNA obtained from the biofilm matrix of all three microorganisms was compared to the genomic DNA (gDNA) isolated from relevant planktonic grown cells. The process of eDNA isolation consisted of biofilm cultivation, its collection, sonication, membrane filtration, dialysis, lyophilisation, and extraction of DNA separated from the biofilm matrix with cetyltrimethylammonium bromide. An amplified fragment length polymorphism (AFLP) was used for comparing eDNA and gDNA. AFLP profiles showed a significant similarity between eDNA and gDNA at the strain level. The highest similarity, with a profile concordance rate of 94.7% per strain, was observed for S. aureus, L. monocytogenes, and S. enterica exhibited lower profiles similarity (78% and 60%, respectively). The obtained results support the hypothesis that the eDNA of studied bacterial species has its origin in the gDNA.

Molecular Subtyping of Salmonella enterica serovar Enteritidis isolated from Taiwan during 1992-1998 by amplified fragment length polymorphism and pulsed-field gel electrophoresis

Over the past 10 years, Salmonella enterica serovar Enteritidis has emerged as a new serovar and a frequent cause of food-borne diseases in Taiwan. We used two molecular subtyping methods to investigate the development of the infections by S. enterica Enteritidis in Taiwan. Twenty-eight strains of S. enterica Enteritidis isolated from Taiwan during the period of 1992 to 1998 were collected. The primer pairs, MseI+C/EcoRI+0 and MseI+G/PstI+C, were used in amplified fragment length polymorphism (AFLP) analyses, and the restriction enzyme, AvrII, was used in pulsed-field gel electrophoresis (PFGE). Twenty-two AFLP profiles with 53-59 fragments in each profile were found by using the primer pair, MseI+G/PstI+C. In the PFGE analysis, 7 PFGE types were identified. Some of the collected strains, especially the imported strains, had closer relationships with the SE 02 strain isolated from Taipei County. As our results indicate, AFLP analysis was time-saving, easy to perform, and highly discriminative. In this study, AFLP and PFGE were used to analyze the S. enterica Enteritidis isolated from patients in Taiwan. These results provide the epidemiological distribution of these isolated strains and additional evidence to illustrate the sources of the food-borne pathogens.

Molecular genetic variability of Juglans regia L. and Juglans sigillata D. as revealed by fluorescent amplified-fragment length polymorphism

Juglans regia L. and J. sigillata D. have a long cultivation history in China. With the purpose of detecting interspecific and intraspecific genetic variation, 35 clonal cultivars of J. regia, J. sigillata, J. sigillata×J. regia and J. hopeiensis Hu. (J. mandshurica Maxim. ×J. regia) were selected from a broad geographic range and applied in amplified-fragment length polymorphism (AFLP) analysis. Clear-cut AFLP profiles were produced using 9 EcoR I/Mse I primer combinations. As for J. regia, J. sigillata and their hybrids, the percentages of polymorphic bands (PPBs) averaged 93.86%, 93.94% and 60.46%; the observed number of alleles (Na) averaged 1.5370, 1.6389 and 1.2593; the effective number of alleles (Ne) averaged 1.2582, 1.2543 and 1.1646; Nei's gene diversity (H) scored 0.1560, 0.1556 and 0.0951; Shannon’s information index (I) averaged 0.2402, 0.2459 and 0.1414, respectively. These results implied a moderate level of genetic diversity within the investigated species. The total gene diversity (Ht = 0.1596) can be distributed into intra- (Hs = 0.1356) and inter- (Dst = 0.0240) species gene diversity. The proportion of the distributed genetic diversity among populations (Gst ) was 0.1505, indicating that only 15% of the total gene diversity was attributed to differences between species. The factorial correspondence analysis (FCA) and unweighted pair-group method of arithmetic averages (UPGMA) analysis revealed a close genetic distance between these two species.

Amplified fragment length polymorphism and whole genome sequencing: a comparison of methods in the investigation of a nosocomial outbreak with vancomycin resistant enterococci

BackgroundRecognition of nosocomial outbreaks with antimicrobial resistant (AMR) pathogens and appropriate infection prevention measures are essential to limit the consequences of AMR pathogens to patients in hospitals. Because unrelated, but genetically similar AMR pathogens may circulate simultaneously, rapid high-resolution molecular typing methods are needed for outbreak management. We compared amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) during a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VRE) that spanned 5 months.MethodsHierarchical clustering of AFLP profiles was performed using unweighted pair-grouping and similarity coefficients were calculated with Pearson correlation. For WGS-analysis, core single nucleotide polymorphisms (SNPs) were used to calculate the pairwise distance between isolates, construct a maximum likelihood phylogeny and establish a cut-off for relatedness of epidemiologically linked VRE isolates. SNP-variations in the vanB gene cluster were compared to increase the comparative resolution. Technical replicates of 2 isolates were sequenced to determine the number of core-SNPs derived from random sequencing errors.ResultsOf the 721 patients screened for VRE carriage, AFLP assigned isolates of 22 patients to the outbreak cluster. According to WGS, all 22 isolates belonged to ST117 but only 21 grouped in a tight phylogenetic cluster and carried vanB resistance gene clusters. Sequencing of technical replicates showed that 4–5 core-SNPs were derived by random sequencing errors. The cut-off for relatedness of epidemiologically linked VRE isolates was established at ≤7 core-SNPs. The discrepant isolate was separated from the index isolate by 61 core-SNPs and the vanB gene cluster was absent. In AFLP analysis this discrepant isolate was indistinguishable from the other outbreak isolates, forming a cluster with 92% similarity (cut-off for identical isolates ≥90%). The inclusion of the discrepant isolate in the outbreak resulted in the screening of 250 patients and quarantining of an entire ward.ConclusionAFLP was a rapid and affordable screening tool for characterising hospital VRE outbreaks. For in-depth understanding of the outbreak WGS was needed. Compared to AFLP, WGS provided higher resolution typing of VRE isolates with implications for outbreak management.

A68 Characterization and comparative genomic analysis of two Bacillus megaterium lytic bacteriophages

Next-generation sequencing technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. Many Bacillus bacteriophages have been isolated, but very few Bacillus megaterium bacteriophages have been characterized. Here, we describe biological characteristics and whole-genome sequences and their annotations for two new isolates of the B. megaterium bacteriophages (BM5 and BM10), isolated from Egyptian soil samples and representing two different groups according to their host range and amplified fragment length polymorphism profiles. Both phages have been displaying different thermal inactivation points (82 and 78 °C) and pH tolerance range (5–9.2 and 5–8.4 pH) while having the same longevity in vitro (192 h). Electron microscopy observation has proved that both phages belonged to the Myoviridiae family. Furthermore, growth analyses indicated that phages BM5 and BM10 have a shorter latent period (20 and 25 min) and smaller burst size (103 and 117 PFU) than is typical for Bacillus phages. The genome sizes of phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with a modular organization. Bioinformatic analyses of BM5 and BM10 genomes enabled assignments of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of BM5 and BM10 genome structures with other B. megaterium bacteriophages revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7) within L cluster and display different lifestyles (lysogenic and lytic). Sequenced phages encode proteins associated with Bacillus pathogenesis. BM5 does not contain any tRNA sequences, while BM10 genome codes for 17 tRNAs.

Application and limitation of molecular data and essential oil content in identification of Leutea elbursensis Mozaff in northern Iran

Abstract In this paper, the internal transcribed spacer (ITS) sequences, genetic structure and the chemical composition of essential oils of four populations belonging to Leutea elbursensis and Leutea petiolaris, two species endemic to northern Iran, are analyzed. Phylogenetic analysis based on the ITS data showed that all accessions of L. elbursensis formed a monophyletic clade, and L. elbursensis was a sister to the rest of Leutea species. Results of amplified fragment length polymorphism (AFLP) analysis performed on the total genome showed that all individuals presented in the study belonged to two different genetic clusters. The individuals belong to L. petiolaris had a different genetic structure and yielded no traceable amount of essential oils. The essential oil obtained from the ripe fruits of L. elbursensis yielded 0.5–0.6% of volatile essential oils. In total, 15-29 volatile natural components were identified on the basis of their mass spectra characteristics and retention indices, in which α-pinene (33.18-43.22%), β-pinene (32.4-40.9%) were the major constituents. Our results indicate that L. elbursensis is a distinct species, segregated from the other species based on morphology, ITS data and AFLP profile. In addition, despite the relatively uniform genetic structure of L. elbursensis, the chemical composition of essential oil could be highly affected by different factors.

Molecular Diagnostics of Arthroconidial Yeasts, Frequent Pulmonary Opportunists.

Magnusiomyces capitatus and Saprochaete clavata are members of the clade of arthroconidial yeasts that represent emerging opportunistic pulmonary pathogens in immunocompromised patients. Given that standard ribosomal DNA (rDNA) identification often provides confusing results, in this study, we analyzed 34 isolates with the goal of finding new genetic markers for classification using multilocus sequencing and amplified fragment length polymorphism (AFLP). The interspecific similarity obtained using rDNA markers (the internal transcribed spacer [ITS] and large subunit regions) was in the range of 96 to 99%, whereas that obtained using protein-coding loci (Rbp2, Act, and Tef1α) was lower at 89.4 to 95.2%. Ultimately, Rbp2 was selected as the best marker for species distinction. On the basis of cloned ITS data, some strains proved to be misidentified in comparison with the identities obtained with phenotypic characters, protein sequences, and AFLP profiles, indicating that different copies of the ribosomal operon were present in a single species. Antifungal susceptibility testing revealed that voriconazole had the lowest MIC against M. capitatus, while amphotericin B had the lowest MIC against S. clavata Both species exhibited in vitro resistance to fluconazole and micafungin.

Free-Ranging Synanthropic Birds (Ardea alba and Columba livia domestica) as Carriers of Salmonella spp. and Diarrheagenic Escherichia coli in the Vicinity of an Urban Zoo.

The presence of free-ranging urban birds is a risk factor for transmitting pathogens to captive animals and humans alike, including Salmonella spp. and diarrheagenic Escherichia coli. Cloacal swabs from 156 synanthropic Great egrets (Ardea alba) and feral pigeons (Columba livia domestica) that inhabit the surroundings of an urban zoo were processed for the identification of Salmonella spp. and diarrheagenic E. coli pathotypes. Bacterial species identification and genotypic characterization employed the matrix-assisted laser desorption ionization-time of flight mass spectrometry and PCR techniques, respectively, comparing their phylogenetic profiles through amplified fragment length polymorphism (AFLP) analysis. A total of 11 birds were positive for Salmonella Typhimurium (7%) and 9 individuals (5.8%) for diarrheagenic E. coli (enteropathogenic E. coli/Shiga-toxin producing E. coli [EPEC/STEC]) strains. S. Typhimurium strains presented highly similar AFLP profiles (85-100%), whereas EPEC/STEC strains showed more polymorphism. The results show free-ranging birds as carriers for both microorganisms in a zoo environment in Brazil for the first time and suggest these species as possible sources of infection to other animals as well as exposing personnel and visitors to potential zoonotic microorganisms. The presence of carriers highlights the importance of a surveillance system and the need for preventive measures to avoid attracting growing number of synanthropic avian species.

Amplified fragment length polymorphism analysis supports the valid separate species status of Lucilia caesar and L. illustris (Diptera: Calliphoridae)

ABSTRACTCommon DNA-based species determination methods fail to distinguish some blow flies in the forensically and medically important genus Lucilia Robineau-Desvoidy. This is a practical problem, and it has also been interpreted as casting doubt on the validity of some morphologically defined species. An example is Lucilia illustris and L. caesar, which co-occur in Europe whilst only L. illustris has been collected in North America. Reports that these species shared both mitochondrial and nuclear gene sequences, along with claims that diagnostic morphological characters are difficult to interpret, were used to question their separate species status. We report here that amplified fragment length polymorphism profiles strongly support the validity of both species based on both assignment and phylogenetic analysis, and that traditional identification criteria based on male and female genital morphology are more reliable than has been claimed.

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain.

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed. The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries.

Genotypic diversity in root‐endophytic fungi reflects efficient dispersal and environmental adaptation

Studying community structure and dynamics of plant-associated fungi is the basis for unravelling their interactions with hosts and ecosystem functions. A recent sampling revealed that only a few fungal groups, as defined by internal transcribed spacer region (ITS) sequence similarity, dominate culturable root endophytic communities of nonmycorrhizal Microthlaspi spp. plants across Europe. Strains of these fungi display a broad phenotypic and functional diversity, which suggests a genetic variability masked by ITS clustering into operational taxonomic units (OTUs). The aims of this study were to identify how genetic similarity patterns of these fungi change across environments and to evaluate their ability to disperse and adapt to ecological conditions. A first ITS-based haplotype analysis of ten widespread OTUs mostly showed a low to moderate genotypic differentiation, with the exception of a group identified as Cadophora sp. that was highly diverse. A multilocus phylogeny based on additional genetic loci (partial translation elongation factor 1α, beta-tubulin and actin) and amplified fragment length polymorphism profiling of 185 strains representative of the five dominant OTUs revealed a weak association of genetic differences with geography and environmental conditions, including bioclimatic and soil factors. Our findings suggest that dominant culturable root endophytic fungi have efficient dispersal capabilities, and that their distribution is little affected by environmental filtering. Other processes, such as inter- and intraspecific biotic interactions, may be more important for the local assembly of their communities.

Genetic variation of Sporothrix globosa isolates from diverse geographic and clinical origins in China

Sporothrix globosa is the main causative agent of sporotrichosis, a common mycosis that usually affects the skin, in China. Despite increasing efforts in the molecular identification of this fungal pathogen, its modes of transmission and epidemiology remain poorly understood. The goals of this study were to assess the genetic diversity of S. globosa using amplified fragment length polymorphism (AFLP) analysis and to assess the correlation of AFLP profiles with the geographic origins, growth rates, clinical forms, and antifungal susceptibilities of S. globosa isolates. AFLP analysis of 225 clinical S. globosa isolates from eight provinces or municipalities in China identified eight distinct clustering groups (I–VIII), with groups I, II and IV being the most common. The AFLP genotypes showed distinct distribution patterns among different regions within Jilin Province and between northern and southern China, but there was no obvious association between the AFLP genotypes and the growth rates, clinical forms or antifungal susceptibilities of the S. globosa isolates. These results expand our understanding of the genetic variation of S. globosa and suggest that AFLP analysis is a potentially useful tool for studying the epidemiology of this fungal pathogen.Emerging Microbes & Infections (2017) 6, e88; doi:10.1038/emi.2017.75; published online 11 October 2017

Genomic diversity of Sri Lankan New Improved Rice Varieties revealed by AFLP markers

Genetic relationships among 28 new improved rice varieties were established using Amplified Fragment Length Polymorphism (AFLP) markers. Cultivars were analyzed with 10 EcoR1 and MseI primer combinations. A total of 517 fluorescent AFLP markers were generated and analyzed. Of these 480 fragments were polymorphic (92.84%) and 37 (7.16%) fragments were monomorphic. The Jaccard’s similarity indices (J) based on the AFLP profiles of the 28 varieties were computed and Unweighted Pair Group Method with Arithmetic mean (UPGMA) based dendrogram was constructed. The dendrogram separated varieties into three major clusters. Outliers used in the study were uniquely separated from the rest confirming the reliability of data and analysis. The Cophenetic correlation with 0.862 strongly supported the clustering pattern of UPGMA dendrogram. Principal Coordinate analysis and the unrooted tree also confirmed the clustering pattern of the UPGMA dendrogram. Rice varieties in the same cluster showed similar characteristic features (Eg. Grain colour, life span etc). Therefore this genetic diversity data at molecular level will provide detailed estimates of the genetic variation among Sri Lankan new improved rice varieties and also useful in ex situ and in situ genetic conservation, utilization and exchange of genetic material. Int J Appl Sci Biotechnol, Vol 4(1): 32-38

The Widespread Presence of a Multidrug-Resistant Escherichia coli ST131 Clade among Community-Associated and Hospitalized Patients.

Background & AimsThe extent of entry of multidrug-resistant Escherichia coli from the community into the hospital and subsequent clonal spread amongst patients is unclear. To investigate the extent and direction of clonal spread of these bacteria within a large teaching hospital, we prospectively genotyped multidrug-resistant E. coli obtained from community- and hospital associated patient groups and compared the distribution of diverse genetic markers.MethodsA total of 222 E. coli, classified as multi-drug resistant according to national guidelines, were retrieved from both screening (n = 184) and non-screening clinical cultures (n = 38) from outpatients and patients hospitalized for various periods. All isolates were routinely genotyped using an amplified fragment length polymorphism (AFLP) assay and real-time PCR for CTX-M genes. Multi-locus sequence typing was additionally performed to confirm clusters. Based on demographics, patients were categorized into two groups: patients that were not hospitalized or less than 72 hours at time of strain isolation (group I) and patients that were hospitalized for at least 72 hours (group II).ResultsGenotyping showed that most multi-drug resistant E. coli either had unique AFLP profiles or grouped in small clusters of maximally 8 isolates. We identified one large ST131 clade comprising 31% of all isolates, containing several AFLP clusters with similar profiles. Although different AFLP clusters were found in the two patient groups, overall genetic heterogeneity was similar (35% vs 28% of isolates containing unique AFLP profiles, respectively). In addition, similar distributions of CTX-M groups, including CTX-M 15 (40% and 44% of isolates in group I and II, respectively) and ST131 (32% and 30% of isolates, respectively) were found.ConclusionWe conclude that multi-drug resistant E. coli from the CTX-M 15 associated lineage ST131 are widespread amongst both community- and hospital associated patient groups, with similar genetic diversity and similar distributions of genetic markers.

Genetic diversity and specialisation of Eudarluca caricis on some graminaceous Puccinia species

Eudarluca caricis is a common hyperparasite of rusts. A total of 100 cultures were isolated from six Puccinia species or forms growing on 10 species of British grasses at two sites approximately 3 km apart. 82 isolates collected in 2005 were partially sequenced at the ITS locus, and amplified fragment length polymorphism profiles generated for 86 isolates from 2005 and 12 from 2007. Partial ITS sequences of most isolates grouped closely, in a clade with previously reported graminaceous Puccinia isolates and a number of Melampsora isolates. A second clade was very distinct and contained mostly isolates from Puccinia poarum on Poa trivialis . All isolates had distinct AFLP haplotypes. The P. poarum isolates were very distinct from isolates collected from other rusts at the same site. Isolates from P. brachypodii f. sp. arrhenatheri growing on Arrhenatherum elatius in 2005 and 2007 at the same location were distinct (P < 0.001). Isolates from each rust or grass in one year and site were more similar than expected from overall variation between isolates (P < 0.001). Isolates from P. coronata on different grasses clustered together (with isolates from P. brachypodii f. sp. poae-nemoralis ), suggesting partial host rust specialisation in E. caricis .

The phenotypic and genomic diversity of Aspergillus strains producing glucose dehydrogenase

Twelve Aspergillus sp. strains producing glucose dehydrogenase were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship of the analyzed strains was investigated. Moreover, partial gdh gene sequences were determined and aligned. The amplified fragment length polymorphism (AFLP) method was applied for genomic fingerprinting of twelve Aspergillus isolates. Using one PstI restriction endonuclease and five selective primers in an AFLP assay, 556 DNA fragments were generated, including 532 polymorphic bands. The AFLP profiles were found to be highly specific for each strain and they unambiguously distinguished twelve Aspergilli fungi. The AFLP-based dendrogram generated by the UPGMA method grouped all the Aspergillus fungi studied into two major clusters. All the Aspergillus strains were also characterized using Biolog FF MicroPlates to obtain data on C-substrate utilization and mitochondrial activity. The ability to decompose various substrates differed among the analyzed strains up to three folds. All of the studied strains mainly decomposed carbohydrates.

Clonal Structure of Two Wild Lotus (Nelumbo nucifera Gaertn.) Populations Inferred from Amplified Fragment Length Polymorphism Fingerprints

The lotus Nelumbo nucifera often forms dense populations at the margins of lakes and ponds. Many aquatic plant species such as N. nucifera are characterized by the ability to reproduce both sexually and asexually, which can affect the genetic diversity of populations growing in different types of environments. We compared the clonal diversities of two natural lotus populations in Japan. Diversity was quantified by DNA fingerprinting of amplified fragment length polymorphisms (AFLPs) in leaves sampled across each population. The DNA fingerprints revealed different amounts of diversity in each population. In 162 samples from Lake Kasumigaura, where lotus has grown continuously for many years, 35 AFLP patterns were detected. One particular clone was distributed widely, suggesting that growth of seed-derived plants was inhibited because of the superior competitive ability of rhizomes, resulting in low genetic diversity within the population. In 214 samples from Uchi-numa Pond in Miyagi Prefecture, where the lotus population was totally destroyed by heavy rains in 1998 and is now recovering, 213 different AFLP profiles were detected. The spatial distance between samples from Uchi-numa Pond was correlated with the genetic distance. These results suggest that, in lotus, sexual reproduction is favored when rapidly varying water level damages the population, because small seedlings are better able to survive in a low-density population. In contrast, vegetative reproduction is favored when environmental conditions are stable.

Genetic structure of Italian populations ofPyrenochaeta lycopersici, the causal agent of corky root rot of tomato

Pyrenochaeta lycopersici is the causal agent of corky root rot, which is a serious disease worldwide that attacks the roots of tomato. A total of 139 isolates were sampled from eight locations in Italy and Israel and assigned to two molecular types (type 1 and type 2) based on internal transcribed spacer (ITS) sequences. These isolates were genotyped using amplified fragment length polymorphisms (AFLPs) to decipher the population structure. Based on this population structure analysis, three groups of P. lycopersici were identified. One group correlated to ITS type 1, while the other two correlated to ITS type 2. AMOVA indicated high genetic divergence (FST = 0� 40) between the Italian types 1 and 2. These data support the view that the two ITS types represent significant evolutionary entities, although there might be incomplete lineage sorting present. Some isolates of different ITS type were observed to have very similar multilocus AFLP profiles, and some genotypes were intermediate between the two ITS types. This suggests that parasexual hybridization between the two types has had a significant role in shaping the population structure of P. lycopersici. Finally, the average divergence among the populations within the ITS types was very high (FSC = 0� 710, P < 10 � 5 ), probably