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Results for “Random Amplified Polymorphic DNA Technique”

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PubMed2004

Biomphalaria tenagophila: genetic variability within intermediate snail hosts susceptible and resistant to Schistosoma mansoni infection.

DNA analysis by molecular techniques has significantly expanded the perspectives of the study and understanding of genetic variability in molluscs that are vectors of schistosomiasis. In the present study, the genetic variability of susceptible and resistant B. lenagophila strains to S. mansoni infection was investigated using amplification of their genomic DNA by RAPD-PCR. The products were analyzed by PAGE and stained with silver. The results showed polymorphism between tested strains with four different primers. We found two bands of 1,900 and 3,420 bp that were characteristic of the susceptible strains with primer 2. The primers 9 and 10 identified a single polymorphic band that was also characteristic of (3,136 and 5,041 bp, respectively) susceptible snails. Two polymorphic bands were detected by primer 15: one with 1,800 bp was characteristic of the resistant strain and the other with approximately equal to 1,700 bp in the susceptible one. These results provide additional evidence showing that the RAPD-PCR technique is adequate for the study of polymorphisms in intermediate hosts snails of S. mansoni. The obtained results are expected to expand the knowledge about the genetic variability of the snails and to permit the future identification of genomic sequences specifically related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni.

Animals↗

PubMed2005

Patterns of AFLP variation in a core subset of cultivated hexaploid oat germplasm.

Many core collections have been developed from large collections of crop germplasm, but most of these have not been characterized, particularly using molecular techniques, for germplasm management and utilization. We have attempted to characterize a structured sample representing a world collection of 11,622 cultivated hexaploid oat accessions in the hope of understanding the genetic structure of the world collection. The amplified fragment length polymorphism (AFLP) technique was applied to screen 670 accessions representing 79 countries and one group of uncertain origin. For each accession, 170 AFLP polymorphic bands detected by five AFLP primer pairs were scored. Analyses of the AFLP data showed the effectiveness of the stratified sampling applied in capturing country-wise AFLP variation. The frequencies of polymorphic bands ranged from 0.11 to 0.99, with an average of 0.72. The majority (89.9%) of the AFLP variation resided within accessions of each country, and only 6.2% of the AFLP differences existed among accessions of major geographic regions. Accessions from the Mediterranean region were the most distinct, while those from Russia and the USA were the most diverse. The two distinct groups that were observed were separated largely on the basis of common oat and red oat. Red oat was genetically more diverse than its common and hull-less counterparts, and hull-less oat was more related to common oat than red oat. Landrace and non-landrace accessions displayed similar AFLP variation patterns. These patterns are significant for understanding the domestication of cultivated oat and are useful in classifying the intraspecific diversity of oat germplasm, developing specific core subsets of the oat collection, and exploring new sources of genes for oat improvement.

Avena↗

PubMed2001

Molecular analyses of the serotype of Cryptococcus neoformans.

Cryptococcus neoformans consists of two varieties and is divided into five serotypes: serotypes A, D and AD (C. neoformans var. neoformans) and serotypes B and C (C. neoformans var. gattii). This article deals with the investigation on the serotype of C. neoformans by molecular analysis technique in place of the immunological method with antisera against the capsule component of the yeast. For easier and more precise epidemiological surveillance, twenty-seven isolates of C. neoformans were molecularly analyzed by a RAPD method. This method differentiated these isolates of C. neoformans into 4 groups corresponding to the serotypes A, D, AD and complex of serotypes B and C. These results indicated that serotype A, D and AD could be differentiated by the molecular analysis technique described here. Furthermore, nucleotide sequences of CAP59 genes from five serotypes of C. neoformans were analyzed for their phylogenetic relationship. Approximately 600-bp genomic DNA fragments of the CAP59 gene were amplified from each isolate by PCR and sequenced. The CAP59 nucleotide sequences of C. neoformans showed more than 90% similarity among the five serotypes. The phylogenetic analysis of their sequences was divided into three clusters: serotype A and AD, serotype B and C, and serotype D. These results also indicated that serotype B and C isolates belonging to var. gattii were genetically homogeneous and closely related.

Base Sequence↗

PubMed2003

Detection of universal variable fragments as markers for genetic studies. A novel technology for DNA fingerprinting.

A novel DNA technology enables the detection of universal variable fragments (UVF), thus revealing genetic variation without a priori sequence information. The detection of UVF markers is based on two amplifications of genomic DNA with the polymerase chain reaction. In the first amplification, two short oligonucleotide primers produce a large number of fragments. One primer is based on a microsatellite sequence, whereas the second primer can have any sequence. In the second amplification, the length of the primers is increased in order to decrease the number of amplicons. This enables the selection of polymorphic fragments. Restriction digestion can be used to further increase the number of polymorphisms. Until now, we have demonstrated UVF in several different species. In addition, with the present study we have contributed to the linkage map of the rabbit by localizing 11 UVF markers on different linkage groups. Mendelian inheritance was shown in this linkage study through a backcross of two inbred rabbit strains. The power of the UVF technique is based on the selection for microsatellite variation in combination with the detection of single-nucleotide polymorphisms. UVF thus offers the possibility of increasing the clustering of markers and localizing genes in species for which sequence information is either not present or only scarcely present.

Animals↗

PubMed1998

Diversity of lactic acid bacteria associated with ducks.

The diversity of lactic acid bacteria occurring in the crop and intestinal content of ducks of different ages (5 to 34 days) was evaluated using the RAPD-PCR technique and comparative sequence analysis of 16S rRNA. Cluster analysis derived from isolates grown on Rogosa agar used for the enrichment of lactic acid bacteria, revealed at least twenty six different groups of organisms representing species, subspecies and strains belonging to Streptococcus, Enterococcus, Pediococcus, Weissella and Lactobacillus. Homofermentative and heterofermentative lactobacilli were identified, belonging to the phylogenetically defined Lb. acidophilus, Lb. salivarius and Lb. reuteri groups.

Age Factors↗

PubMed1997

Large deletions partially external to the human hprt gene result in chimeric transcripts.

We postulated that gene fusions sometimes occur in normal cells as a result of gene rearrangements as have been observed involving oncogene loci in tumours. To test this, we searched for fusion-gene transcripts in selected human T-lymphocyte large deletion mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene using the 3' rapid amplification of cDNA ends (RACE) technique. Aberrant hprt-containing transcripts were observed in seven out of 19 mutants (approximately 36%) indicating that a surprising number of these rearrangements code for processed mRNAs. RNA splicing and polyadenylation occurred downstream of the non-deleted hprt sequence in chimeric transcripts and the majority resulted from mutants with fusions of hprt into regions containing a repetitive element (Alu, LINE or microsatellite).

Base Sequence↗

PubMed2000

[Analysis of isozyme and RAPD for the germplasm lines from G. hirsutum x G. bickii].

To study the effectiveness of Gossypium interspecific hybrid breeding on molecular level, the six germplasm lines from interspecific hybrids between G. hirsutum and G. bickii were analysed by the techniques of RAPD and isozyme electrophoresis. The main results were summarized as follows: (1) The basic zymograms of peroxidase isozyme of the six germplasm lines were similar to their G. hirsutum parent, but two out of the germplasm lines showed one specific band of the wild parent of G. bickii (pI 4.85); (2) The RAPD fingerprint showed distinct difference of genomic DNA among the germplasm lines; (3) The four specific DNA fragments of G. bickii were observed in RAPD patters of four out of the six germplasm lines.

Gossypium↗

PubMed1999

Genetic diversity in Elymus caninus as revealed by isozyme, RAPD, and microsatellite markers.

Genetic diversity of 33 Elymus caninus accessions was investigated using isozyme, RAPD, and microsatellite markers. The three assays differed in the amount of polymorphism detected. Microsatellites detected the highest polymorphism. Six microsatellite primer pairs generated a total of 74 polymorphic bands (alleles), with an average of 15.7 bands per primer pair. Three genetic similarity matrices were estimated based on band presence or absence. Genetic diversity trees (dendrograms) were derived from each marker technique, and compared using Mantel's test. The correlation coefficients were 0.204, 0.267, and 0.164 between isozyme and RAPD distance matrices, RAPD and microsatellite distance matrices, and between isozyme and microsatellite distance matrices, respectively. The three methodologies gave differing views of the amount of variation present but all showed a high level of genetic variation in E. caninus. The following points may be drawn from this study whether based on RAPD, microsatellite, or isozyme data: (i) The Icelandic populations are consistently revealed by the three dendrograms. The congruence of the discrimination of this accession group by RAPD, microsatellite, and isozyme markers suggests that geographic isolation strongly influenced the evolution of the populations; (ii) The degree of genetic variation within accessions was notably great; and (iii) The DNA-based markers will be the more useful ones in detecting genetic diversity in closely related accessions. In addition, a dendrogram, which took into account all fragments produced by isozymes, RAPDs, and microsatellites, reflected better the relationships than did dendrograms based on only one type of marker.

Africa↗

PubMed2004

Diversity and molecular relationships of endophytic Colletotrichum isolates from the Iwokrama Forest Reserve, Guyana.

The diversity and host specificity were studied of a collection of Colletotrichum strains derived from endophytic colonies in leaves of 12 tree species in the Iwokrama Forest Reserve, Guyana. Analysis included ISSR-PCR and RAPD molecular fingerprinting techniques, rDNA ITS sequencing and morphological and cultural characterization. Most strains belonged to one of two species, C. gloeosporioides and a further taxon which is probably referable to C. boninense. Almost no strains were found to be genetically identical, indicating that clonal reproduction does not play a prominent role. No degree of host specificity could be detected even at molecular fingerprint level. The implications for estimation of fungal diversity in closed tropical forests may be profound.

Colletotrichum↗

PubMed2003

[Use of molecular markers for differentiation of cultivated strains of oyster and button mushrooms].

Analysis of commercial strains of two edible mushrooms, Pleurotus ostreatus and Agaricus bisporus, using PCR and isozyme electrophoresis techniques allowed us to differentiate groups of genetically similar and distant strains. Among the commercial strains of P. ostreatus, the level of genetic variation was higher suggesting a broader genetic basis employed in breeding of this mushroom. The cultivars and hybrids of, A. bisporus, showed a higher level of homology. The isozyme markers (nonspecific esterase, leucinaminopeptidase, and phosphoglucoisomerase) are recommended for identification of the commercial strains of edible mushrooms.

Agaricus↗

PubMed2005

[The application of DNA molecular markers in conservation of the rare and endangered medicinal plants].

In this paper, the advance in DNA molecular markers techniques in recent years was reviewed. The application of DNA markers in conservation of the rare and endangered medicinal plants was explicated, of which included identification of germ-plasm resource, determination of the habitats unite which should be protected in situ, sampling strategies of ex-situ conservation, evaluation of the conservation effects of the rare and endangered medicinal plants, as well as elucidation of their endangered mechanism etc. The information could help drawing up conservation strategies and conservation measures for references.

Conservation of Natural Resources↗

PubMed2000

Identification of RAPD markers tightly linked to wheat powdery mildew resistance gene Pm6.l.

Pm6 transferred from Triticum timopheevii L. to common wheat, is an effective resistance gene to powdery mildew disease caused by Erysiphe graminis f. sp. tritici. The RAPD technique, employing a total of 700 decamer primers, was used to identify polymorphic markers between resistant (IGVI463) and susceptible (Prins) near-isogenic lines. Primer OPV20 produced a 2,000 base pair (bp) reproducible fragment only in the resistant near-isogenic line. The 2,000-bp DNA fragment was present in all other introgression lines containing Pm6. Using the F2 mapping population from a cross of IGVI-463 (PI170914/7*Prins) x Prins, Pm6 was shown to be closely linked to the marker OPV20-2000 at a genetic distance of 3.0 +/- 2.2 cM. The marker was successfully used in detecting the presence of Pm6 in different genetic backgrounds.

Breeding↗

PubMed1996

Molecular analysis of the structure of the maize B-chromosome.

The overall composition of the maize B is similar to that of the standard chromosome complement (A-chromosomes). This has been demonstrated by the use of several methods including: (a) genomic in situ hybridization (GISH), (b) analysis of highly repetitive sequences by the comparison of restriction digests of 0B and +B DNA and (c) the characterization of middle-repetitive cloned sequences. By the use of the technique of random amplification of polymorphic DNA-polymerase chain reaction, we have identified a B-specific repetitive sequence family. Sequence analysis of the B-specific clone reveals a relationship to the PREM-1 family of maize retroelements, which are preferentially transcribed in pollen. These results suggest an internal origin of the B-chromosome from within the maize genome, but demonstrate also that specific sequences can evolve by rapid processes of genomic turnover. Models for the origin of the maize B are discussed in the context of the present data.

Base Sequence↗

PubMed1997

Molecular and phenotypic characterization of Aspergillus japonicus and Aspergillus aculeatus strains with special regard to their mitochondrial DNA polymorphisms.

Forty Aspergillus japonicus and A. aculeatus strains, most of them wild-type isolates, were examined using various molecular and phenotypic techniques. The rDNAs proved to be invariable (even strains of the species A. aculeatus exhibited the same restriction profile), while the strains could be classified into seven different mtDNA RFLP groups. Hybridisation data suggest that six of these mtDNA types have certain common restriction sites, while mtDNA type 7, which was exhibited by some A. aculeatus strains, probably has quite different mtDNA organisation and their size was smallest among the strains studied. The RAPD technique and isoenzyme analysis revealed some variabilities within these RFLP groups and strain specific features could also be recognised. Carbon source assimilation spectra were found to be very distinctive for strains of A. japonicus, A. aculeatus and A. niger, providing a useful tool for pre-characterising new wild-type isolates of black Aspergilli. Only a limited correlation was observed between the dendrograms based on genotypic and phenotypic characters.

Aspergillus↗

PubMed2000

[A decade of the RAPD method: possibilities and limitations for plant genetics relationship studies].

The RAPD method appeared a decade ago as an alternative in genetic relationship studies. The technique generates polymorphic band patterns, produced by PCR using arbitrary DNA sequence primers. If total DNA is used, RAPD yields abundant information about the analyzed genome in a rapid and inexpensive way. This information may be used in various types of plant genetic studies, such as hybrid detection, intra and interspecific genetic variation, genetic identity establishment, somaclonal variation analysis and, when combined with other methods, it helps in the elaboration of genetic maps. However, there are some requisites for its correct application. A strict control of working conditions is demanded. Furthermore, due to the anonymous character of polymorphic bands and the difficulties for establishing homologies, it is also recommended to confine RAPD uses to the specific or infra specific levels. Comparisons based on genetic distance calculations are accepted provided they do not require parsimony analysis methods.

Genetic Markers↗

PubMed2006

Genetic structure of Triatoma venosa (Hemiptera: Reduviidae): molecular and morphometric evidence.

Triatoma venosa presents a restricted geographical distribution in America and is considered as a secondary vector of Chagas disease in Colombia and Ecuador. A total of 120 adult insects were collected in domestic and peridomestic habitats in an endemic area of the department of Boyacá, Colombia, in order to determine their genetic structure through morphometric and molecular techniques. The head and wings of each specimen were used for the analyses of size, shape, and sexual dimorphism. A significant sexual dimorphism was found, although no differences in size among the studied groups were detected. Differences were found in the analyzed structures except for male heads. DNA was extracted from the legs in order to carry out the internal transcriber space-2 (ITS-2) amplification and the randon amplified polymorphic DNA (RAPD) analyses. Length polymorphisms were not detected in the ITS-2. Fst and Nm values were estimated (0.047 and 3.4, respectively). The high genetic flow found among the insects captured in the domicile and peridomiciliary environment does not permit a genetic differentiation, thus establishing the peridomicile as an important place for epidemiological surveillance.

Animals↗

PubMed1998

RAPD profile variation amongst provenances of neem.

Neem, described as a tree for solving global problems, is an evergreen, long-lived, multipurpose tree of the tropics with a wide distribution range in India. It is believed to be highly cross-pollinated. Inter-provenance variations have been reported in neem in case of morphological and physiological characters. Yet no reports about the genetic determinism for these variations are available to our knowledge. In order to have an idea about the extent and/or nature of genetic (DNA) variation in neem, the powerful RAPD technique has been employed. RAPD profiles of 34 accessions/provenances of neem were generated with 200 decamer random primers, of which the data from the 49 primers, that resulted in reproducible amplification products, were considered for analysis. Based on the presence/absence of bands, a similarity matrix was computed. Dendrogram was constructed by UPGMA method based on the pairwise similarities amongst the RAPD profiles. The similarities in RAPD profiles amongst the different DNAs was more than that expected due to the cross-pollinated nature of the tree and furthermore, these more-than-expected similarities were not due to random chance. These results suggest that neem may have a narrow genetic base.

DNA Primers↗

PubMed2000

[A study of genetic character of cultivar Shiyuan 321 from G. barbadense x G. thurberi x G. hirsutum using isozyme and RAPD techniques].

To study the genetic character of trispecific hybrid cultivar Shiyuan 321, we analyzed this cultivar and its parents by isozyme and RAPD techniques. The main results are as follows: (1) Esterase isozyme analysis showed that there was one specific band among cultivar Suiyuan 321 and its parents, G. thurberi and G. barbadense; (2) Six specific DNA fragments amplified by 4 primers were found among cultivar Shiyuan 321 and its parents G. barbadense, G. thurberi; (3) By comparing RAPD differences among Shiyuan 321 and its parents, it is indicated that the genetic heterogeneity of cultivar Shiyuan 321 is very high at its genomic DNA level. The above results proved that Shiyuan 321 is a trispecific cultivar from G. barbadense x G. thurberi x G. hirsutum as well as a new germplasm.

Esterases↗