Genetic diversity and phylogenetic relationships in Vigna Savi germplasm revealed by DNA amplification fingerprinting.

The pantropical genus Vigna (Leguminosae) comprises 7 cultivated species that are adapted to a wide range of extreme agroclimatic conditions. Few data are available on the relationships among these cultivated species or on their importance as sources of resistance against biotic and abiotic stresses. Therefore, we optimized DNA amplification fingerprinting (DAF) to estimate the genetic diversity within, and genetic relationships among, a representative core collection of cowpea, as compared with 16 accessions representing cultivars from 6 Vigna species. A set of 26 primers was selected from 262 tested random primers and used for the characterization of 85 Vigna accessions (6 V. angularis, 4 each of V. mungo and V. radiata, 2 V. umbellata, 1 V. aconitifolia, and 68 V. unguiculata), with Phaseolus vulgaris subsp. vulgaris as outgroup. A total of 212 polymorphic bands were used for maximum parsimony analysis. Our results clearly distinguished Brazilian from African V. unguiculata genotypes. At the species level, V. angularis was the most related and V. radiata the most divergent species relative to V. unguiculata. DAF markers were also informative at the intraspecific level, detecting a large diversity between cowpea cultivars. The implications of the presented results for cowpea breeding programs are discussed.     

Genetic relationships of Japanese and Indian mulberry (Morus spp.) genotypes revealed by DNA fingerprinting

Mulberry (Morus spp.), a deciduous tree, originated at the foothills of the Himalayas and is used in sericulture for its leaf to feed the silkworm Bombyx mori L. Species differentiation among the genotypes of the genus Morus has never been out of debate as inter-specific hybridization events are often fertile. In the present study attempts were made to elucidate the genetic relationships among 18 mulberry genotypes collected from India and Japan using 15 Inter Simple Sequence Repeat (ISSR) and 15 Random Amplified Polymorphic DNA (RAPD) primers. The ISSR primers generated 81.13% polymorphism while the RAPDs generated 71.78% polymorphism. The polymorphic index of the primers identified UBC-812, UBC-826, UBC827, UBC-881, OPA-01, OPA-02, OPA-04 and OPH-17 as informative primers in mulberry. The genetic similarity coefficients and the dendrograms showed considerable genetic similarity among the genotypes. However, using the DNA markers, these genotypes were discriminated into two major groups in accordance with their geographic origin and species status. Distribution of the genotypes on a two-dimensional figure on the basis of the ALSCAL algorithm using Euclidean distance further confirmed the genetic divergence between these two groups. From the study it can be concluded that though morphologically Japanese and Indian mulberry genotypes show little divergence, genetic analysis using DNA markers could unravel significant genetic variation between these two groups. Similarly, while the species status of Japanese mulberry genotypes agrees with the genetic analysis, the same does not apply to Indian genotypes, in agreement with many earlier reports. The information generated in this study is of much use for taxonomical grouping and also for utilization in breeding and conservation programs.     

Diversity of DNA sequences among Vibrio cholerae O139 Bengal detected by PCR-based DNA fingerprinting

Abstract Vibrio cholerae O139, a causative agent of a large epidemic of cholera-like illness, has suddenly emerged and spread widely over several months. To investigate the characteristics unique to O139, traditional typing techniques for V. cholerae , such as biochemical characteristics, antibiotic susceptibility and detection of toxin production, were performed, with the result that 145 O139 strains, except for two O139 strains isolated from Argentina and Germany, were indistinguishable from O1 strains. Thus, in order to clarify the genetical relatedness among O139 strains, and between O139 and O1 strains, the RAPD (random amplified polymorphic DNA) DNA fingerprinting method was undertaken. Although the RAPD arrays in five O139 isolates from Vellore with one arbitrary primer were slightly different from the other O139 strains, the RAPD patterns of the 145 forty-five O139 strains except for two O139 strains from Argentina and Germany were quite similar to each other, but were different from those of O1 strains, indicating that those O139 epidemic strains are closely related to each other regardless of their place of isolation. Furthermore, the RAPD patterns of the O139 strains resembled those of E1 Tor strains rather than classical strain, and a small change in the RAPD pattern of O139 strains occurred during subculture for 200 generations. These results taken together suggested that O139 V. cholerae have emerged from a common origin associated with the E1 Tor strain.     

No evidence for extrapair fertilizations in the merlin revealed by DNA fingerprinting

Broods of young merlins were compared with the adults in attendance at their nest by DNA fingerprinting. No offspring were found that mismatched genetically suggesting that intraspecific brood parasitism and extrapair fertilization are very rare in this population. The results are discussed in the light of the Paternity Assurance Hypothesis.     

DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting.

The RAPD (or AP-PCR) DNA fingerprinting method was used to distinguish among clinical isolates of Helicobacter pylori, a bacterium whose long term carriage is associated with gastritis, peptic ulcers and gastric carcinomas. This method uses arbitrarily chosen oligonucleotides to prime DNA synthesis from genomic sites to which they are fortuitously matched, or almost matched. Most 10-nt primers with > or = 60% G + C yielded strain-specific arrays of up to 15 prominent fragments, as did most longer (> or = 17-nt) primers, whereas most 10-nt primers with 50% G+C did not. Each of 64 independent H. pylori isolates, 60 of which were from patients in the same hospital, was distinguishable with a single RAPD primer, which suggests a high level of DNA sequence diversity within this species. In contrast, isolates from initial and followup biopsies were indistinguishable in each of three cases tested.     

Genetic diversity in white clover and its progenitors as revealed by DNA fingerprinting

The genetic diversity and ancestral relationships of a number of Trifolium species was revealed by using the amplified fragment length polymorphism (AFLP) and the random amplified polymorphic DNA (RAPD) markers. Both markers produced few species-specific markers. Using distance and parsimony methods, in NTSYS-pc and PAUP software programs, we clearly differentiated the accessions of white clover from other closely related progenitors. The phylogenetic trees, produced by PAUP, also reinforced the close affinity of T. nigrescens and the allopolyploid white clover in support of former views that this diploid species could have been the donor of one of two genomes of the allotetraploid T. repens. In addition, the dendrograms, produced by NTSYS-pc, also indicated close affinity of T. nigrescens and T. occidentale to the accessions of T. repens. These data is congruent with karyological and phylogenetic affinities between the white clover and T. occidentale. The relationships between the examined accessions, in the T. repens gene pool, may be regarded to indicate the presence of shared alleles between T. repens, T. occidentale and T. uniflorum. Further, T. occidentale showed close phylogenetic relations to T. pallescens.     

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.     

Recognition of Yersinia enterocolitica multiple strain infection in twin infants using PCR-based DNA fingerprinting

AIMS: Yersinia enterocolitica causes several syndromes in humans. The most common presentation is enterocolitis in children, presenting as fever and diarrhoea. A Y. enterocolitica multiple strain infection in twin infants was investigated. METHODS AND RESULTS: One isolate was recovered from one patient and two morphologically-different isolates were recovered from the other infant. Biochemically, all isolates were identified as Y. enterocolitica group. The genomic DNA from each strain was purified and DNA fingerprinting was performed. The banding patterns observed for Y. enterocolitica isolates 2 and 3, from patients 1 and 2, respectively, were identical when comparing the presence or absence of major bands. However, Y. enterocolitica isolate 1, from patient 1, showed a distinctive banding pattern from isolates 2 and 3. CONCLUSION: The findings indicate that one infant was colonized by more than one strain of Y. enterocolitica, demonstrating that multiple strains can colonize and invade a patient. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of multiple strain infections can be important in diagnosis, treatment and prognosis of Y. enterocolitica infections, as well as in disease epidemiology. The technique described here offers a straightforward method for strain comparison.     

A PCR-based DNA fingerprinting technique: AFLP for molecular typing of bacteria.

Amplified restriction fragment polymorphism (AFLP) is a PCR-based DNA fingerprinting technique. In AFLP analysis, bacterial genomic DNA is digested with restriction enzymes, ligated to adapters, and a subset of DNA fragments are amplified using primers containing 16 adapter defined sequences with one additional arbitrary nucleotide. Polymorphisms of different Escherichia coli strains or Agrobacterium tumefaciens strains were demonstrated as distinct, unique bands in a denaturing sequencing gel using AFLP. The polymorphisms detected between BL21 and BL21F'IQ and between DH5 alpha and DH5 alpha F'IQ strains indicated that AFLP is able to resolve differences in F' episomal DNA (approximately 100 kb).     

Analysis of genotypic diversity and relationships among Pseudomonas stutzeri strains by PCR-based genomic fingerprinting and multilocus enzyme electrophoresis.

Molecular fingerprinting procedures including random amplified polymorphic DNA-PCR (RAPD), repetitive extragenic palindromic PCR (rep-PCR) with REP, ERIC, and BOX primers and multilocus enzyme electrophoresis (MLEE) were used for genotypic characterization of 16 P. stutzeri strains originally isolated from marine, waste water, clinical and soil samples. A distinct genotype of each strain and overall great genotypic diversity were found within P. stutzeri. Cluster analysis (UPGMA) of the electrophoretic patterns of all PCR-based methods used resulted in concordant grouping of 8 strains. With the other strains conflicting clustering was noticed. The variability of clustering in PCR-based analyses suggested the occurrence of chromosomal rearrangements. When RAPD-, rep-PCR and MLEE fingerprints were used in a cluster analysis of combined electrophoretic patterns, the P. stutzeri strains could be differentiated into seven distinct genotypic groups. These results supported the subdivision of the species in several genomovars and reproduced, with higher resolution, the strain grouping after 16S rRNA phylogenetic reconstruction. The combined use of several fingerprint-based genotypic analyses results in higher resolutive strain clustering by UPGMA than each of the single ones analyzed separately. Additionally, this combination of individual typings proved to be reliable of the determination of the great genotypic diversity and relationships among the P. stutzeri strains.     

Variation of mini- and microsatellite DNA repeats in parthenogenetic lizard Darevskia armeniaca as revealed by DNA fingerprinting analysis

Population and family samples of two morphological forms (mutant and normal with respect to dorsal color) of pathogenetic lizard Darevskia armeniaca were examined by means of DNA fingerprinting using M13 mini- and (GATA)n and (TCC)n microsatellite DNA markers. The morphological forms examined were characterized by clonally inherited, species-specific patterns of the DNA markers, which were different from the species-specific DNA fingerprints of the other parthenogenetic species of the genus Darevskia (D. dahli. D. unisexualis, and D. rostombekovi). The mean index of similarity (S) obtained for a sample of 36 individuals from three isolated populations using three types of DNA markers was 0.966. This was similar to the variability level observed in D. dahli (0.962) (P > 0.05), but higher than that in D. unisexualis (0.950) (P < 0.05) and D. rostombekovi (0.875) (P < 0.01). Inheritance of M13 minisatellite and (TCC)n microsatellite DNA markers in the F1 offspring of parthenogenetic lizards was examined. It was shown that variability and clonal diversity of the fingerprint phenotypes observed in the populations and families of D. armeniaca could be at least partly explained by RFLP mutations in microsatellite repeats.     

Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques

Strains of Xanthomonas axonopodis pv. manihotis (Xam) were characterized for pathogenicity and for DNA polymorphism using different PCR-based techniques. Using amplified restriction fragment length polymorphism (AFLP), strains were distinguished from each other and also from other Xanthomonas strains. Cluster analysis showed a high correlation between DNA polymorphism and pathogenicity. Four Xam strains were further analyzed using three PCR-based techniques, AFLP, AFLP-pthB and RAPD-pthB. Various primer combinations were used including primers specific to a Xam pathogenicity gene (pthB) along with RAPD or AFLP primers. The AFLP primer combinations EcoRI+T/MseI+A and EcoRI+T/MseI+T were the most efficient to discriminate among pathogenic and nonpathogenic Xam strains. Polymorphic bands were excised from the gel, amplified and cloned. Sequences analysis showed significant homology with bacterial pathogenicity island, genes involved in pathogenic fitness and regulators of virulence. Three cloned AFLP fragments were used as probes in DNA blot experiments and two of them showed significant polymorphism.     

Rubus vestervicensis (Rosaceae) — its hybrid origin revealed by DNA fingerprinting

Kraft, T., Nybom, H. & Werlemark, G. 199.5. Rubus vestervicensis (Rosaceae) — its hybrid origin revealed by DNA fingerprinting. — Nord. 3. Bot. 1.5: 237–242. Copenhagen. ISSN 0107-055X.
The large number of species in Rubus subgenus Rubus are thought to have arisen, at least in part, through hybridiation between facultatively apomictic species. R. vestervicensis is known only from a small island off the Swedish east coast. It has been suggested that it is a hybrid between R. grabowskii and R. pedemontanus . We have compared these species with DNA fingerprinting and our results support the hybridization hypothesis. All bands found in the fingerprint of R. grabowskii and about half of the bands found in R. pedemontanus were present in R. vestervicensis , suggesting that the latter species was derived from fertilization of an unreduced R. grabowskii egg cell with R. pedemontanus pollen.     

Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing

Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.     

Phylogenetic relationships among wild and cultivated Tartary buckwheat (Fagopyrum tataricum Gaert.) populations revealed by AFLP analyses

Phylogenetic relationships among cultivated landraces and natural populations of wild subspecies of Tartary buckwheat were investigated at the individual level by constructing a phylogenetic tree based on amplified fragment length polymorphism (AFLP) markers. Seven individuals from seven cultivated landraces and 35 individuals from 21 natural populations of wild subspecies were utilized for AFLP analyses. Three groups were recognized: (1) all cultivated landraces and wild subspecies from northern Pakistan, central and eastern Tibet, and northwestern Yunnan, (2) wild subspecies from central and southern Sichuan, (3) wild subspecies from northern Sichuan and eastern Tibet. It was concluded that cultivated Tartary buckwheat probably originated in eastern Tibet or northwestern Yunnan in China.     

Genetic variability in Metarhizium flavoviride revealed by telomeric fingerprinting

Previous studies using arbitrarily primed PCR (AP-PCR/RAPD) analysis have shown only little genetic variation among isolates of the entomopathogenic fungus Metarhizium flavoviride. In the current study, however, telomeric fingerprinting unambiguously differentiated several Brazilian strains of M. flavoviride as well as strains from Africa and Australia. Using this technique, similarity estimates of telomeric DNA among distinct strains were less than 50%, showing this locus to be highly mutable in this species.