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PubMed2005

Linkage map construction in allotetraploid creeping bentgrass (Agrostis stolonifera L.).

Creeping bentgrass (Agrostis stolonifera L.) is one of the most adapted bentgrass species for use on golf course fairways and putting greens because of its high tolerance to low mowing height. It is a highly outcrossing allotetraploid species (2n=4x=28, A(2) and A(3) subgenomes). The first linkage map in this species is reported herein, and it was constructed based on a population derived from a cross between two heterozygous clones using 169 RAPD, 180 AFLP, and 39 heterologous cereal and 36 homologous bentgrass cDNA RFLP markers. The linkage map consists of 424 mapped loci covering 1,110 cM in 14 linkage groups, of which seven pairs of homoeologous chromosomes were identified based on duplicated loci. The numbering of all seven linkage groups in the bentgrass map was assigned according to common markers mapped on syntenous chromosomes of ryegrass and wheat. The number of markers linked in coupling and repulsion phase was in a 1:1 ratio, indicating disomic inheritance. This supports a strict allotetraploid inheritance in creeping bentgrass, as suggested by previous work based on chromosomal pairing and isozymes. This linkage map will assist in the tagging and eventually in marker-assisted breeding of economically important quantitative traits like disease resistance to dollar spot (Sclerotinia homoeocarpa F.T. Bennett) and brown patch (Rhizoctonia solani Kuhn).

Agrostis↗

PubMed2003

RAPD method useful for distinguishing Leishmania species: design of specific primers for L. braziliensis.

The technique of Random Amplification Polymorphic DNA allows fragments of the genome to be amplified by means of polymerase chain reaction (PCR) without previous knowledge of their sequences. The protozoa of the genus Leishmania present great genetic variability, making it difficult to characterize the different species. A method is developed with a single 10-mers long primer, which allows the species L. braziliensis, L. mexicana, L. infantum, L. tropica, L. chagasi, L. amazonensis and L. major to be differentiated. These products amplified by RAPD have also facilitated the design of some primers that amplify L. braziliensis DNA exclusively.

Animals↗

PubMed2002

Typing of Saccharomyces cerevisiae and Kloeckera apiculata strains from Aglianico wine.

AIMS: Kloeckera apiculata and Saccharomyces cerevisiae yeast species are dominant, respectively, at the early and at the following stages of wine fermentation. In the present study, PCR fingerprinting and NTS region amplification and restriction were applied as techniques for monitoring yeast population performing Aglianico of Vulture grape must fermentation. METHODS AND RESULTS: Thirty S. cerevisiae and 30 K. apiculata strains were typed by PCR fingerprinting with (GAC)5 and (GTG)5 primers and by complete NTS region amplification followed by restriction with HaeIII and MspI enzymes. S. cerevisiae strains generated two patterns with (GAC)5 primer, while (GTG)5 primer yielded a higher genetic polymorphism. Conversely, in K. apiculata Aglianico wine strains (GAC)5 and (GTG)5 primers generated the same profile for all strains. Restriction analysis of the amplified NTS region gave the same profile for all strains within the same species, except for one strain of S. cerevisiae. CONCLUSIONS: The PCR fingerprinting technique was useful in discriminating at strain level S. cerevisiae, particularly with the primer (GTG)5. RFLP patterns generated from the NTS region of the two species can be more easily compared than the patterns resulting from PCR fingerprinting, thus RFLP is more suitable for the rapid monitoring of the species involved in different stages of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular techniques used allow discrimination of S. cerevisiae at strain level and monitoring of the ratio of S. cerevisiae/K. apiculata during the fermentation process. Thus, their application can assure technological adjustments in a suitable time.

Fermentation↗

PubMed2005

A single domestication for potato based on multilocus amplified fragment length polymorphism genotyping.

The cultivated potato, Solanum tuberosum, ultimately traces its origin to Andean and Chilean landraces developed by pre-Colombian cultivators. These Andean landraces exhibit tremendous morphological and genetic diversity, and are distributed throughout the Andes, from western Venezuela to northern Argentina, and in southern Chile. The wild species progenitors of these landraces have long been in dispute, but all hypotheses center on a group of approximately 20 morphologically very similar tuber-bearing (Solanum section Petota) wild taxa referred to as the S. brevicaule complex, distributed from central Peru to northern Argentina. We present phylogenetic analyses based on the representative cladistic diversity of 362 individual wild (261) and landrace (98) members of potato (all tuber-bearing) and three outgroup non-tuber-bearing members of Solanum section Etuberosum, genotyped with 438 robust amplified fragment length polymorphisms. Our analyses are consistent with a hypothesis of a "northern" (Peru) and "southern" (Bolivia and Argentina) cladistic split for members of the S. brevicaule complex, and with the need for considerable reduction of species in the complex. In contrast to all prior hypotheses, our data support a monophyletic origin of the landrace cultivars from the northern component of this complex in Peru, rather than from multiple independent origins from various northern and southern members.

Crops, Agricultural↗

PubMed1996

Comparison of three typing methods for Pseudomonas aeruginosa.

Fifty-seven independent isolates of Pseudomonas aeruginosa from blood specimens were typed with 3 different methods: ribotyping, random amplified polymorphic DNA (RAPD) typing, and pyocin typing. Ribotyping was performed by probing the rRNA genes of genomic DNA that was digested separately with 4 different restriction enzymes. Digestion of DNA from 57 P. aeruginosa isolates with BamHI, ClaI, EcoRI, and PstI produced 4, 4, 6, and 7 patterns, respectively. As a result, ribotyping classified the 57 isolates into 22 types. Six new ribotypes that had not been described previously were found. One BamHI, 1 ClaI, 2 EcoRI, and 2 PstI patterns were novel. RAPD typing was performed with two different polymerase chain reaction (PCR) primers (RAPD1 and RAPD2). Both primers classified the 57 isolates into 15 RAPD types and produced identical patterns. The pyocin typing method classified the 57 isolates into 10 types. According to the results obtained in this study, the ribotyping has a discriminatory index of 0.865, RAPD, 0.785, and pyocin typing, 0.676, respectively. The ribotyping method was the most effective among the 3 methods compared for typing P. aeruginosa isolates.

Bacterial Typing Techniques↗

PubMed1996

The novel untranslated first exon "exon 0N" of the rat estrogen receptor gene.

The 5'-untranslated region (UTR) of the estrogen receptor (ER) mRNA in the rat liver was analyzed by the use of the 5'-rapid amplification of the cDNA ends (5'-RACE) method. The nucleotide sequence of one of the positive RACE clones (clone 9) revealed that the existence of the novel untranslated first exon (termed "exon N") being spliced onto the exon 1 of the rat ER mRNA. We further analyzed the distribution of the ER mRNA containing the "exon 0N" (ER mRNA (0N-1)) and the ER mRNA containing the previously reported exon 0 (ER mRNA (0-1)) in the rat brain and peripheral tissues. In contrast to the wide distribution of the ER mRNA (0-1), the distribution of the ER mRNA (0N-1) was almost limited in the peripheral tissues. These results indicate that the "exon 0N" is the novel untranslated first exon of the rat ER gene, and the tissue specific expression of the ER is regulated, at least in part, by differential promoter usage in the rat.

Animals↗

PubMed2003

Evaluation and comparison of random amplification of polymorphic DNA, pulsed-field gel electrophoresis and ADSRRS-fingerprinting for typing Serratia marcescens outbreaks.

Amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) is a novel assay based on suppression of polymerase chain reaction (PCR). This phenomenon allows the amplification of only a limited subset of DNA fragments, since only those with two different oligonucleotides ligated at the ends of complementary DNA strands are amplified in the PCR. The DNA fragments can be easily analyzed on polyacrylamide gels, stained with ethidium bromide. We have implemented this method using a set of clinical Serratia marcescens isolates from three outbreaks ongoing in the Public Hospital in Gdańsk (Poland). Clustering of ADSRRS-fingerprinting data matched epidemiological, microbiological, random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) data. Based on this study, we found that there is at least a similar power of discrimination between the present 'gold-standard' PFGE and the novel method, ADSRRS-fingerprinting. Although the ADSRRS-fingerprinting method may appear to be more complex than the RAPD technique, we found it fast and reproducible.

Anti-Bacterial Agents↗

PubMed2001

Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates.

Trypanosomatids are widespread in several plant families and although most isolates have been classified as Phytomonas, other trypanosomatid genera can also infect plants. In order to assess the natural occurrence of non-Phytomonas trypanosomatids in plants we characterized 21 new trypanosomatid cultures, 18 from fruits and three from seeds of 17 plant species. The trypanosomatids from fruit and seeds were compared in terms of morphological, growth, biochemical and molecular features. The high diversity among the isolates permitted the classification of the new flagellates into the genera Crithidia and Leptomonas as well as Phytomonas. The data showed that natural fruit infection with non-Phytomonas trypanosomatids is more common than usually thought, being detected in 43% of the fruit isolates.

Animals↗

PubMed2001

Mycoplasmosis in evening and pine grosbeaks with conjunctivitis in Quebec.

An outbreak of conjunctivitis affected evening grosbeaks (Coccothraustes vespertinus) and pine grosbeaks (Pinicola enucleator) in Quebec (Canada) during the winter 1998-99. One to 30% of the individuals from these two species were sick at 13 feeding stations. Sick birds were thin and had unilateral or bilateral catarrhal and lymphoplasmacytic conjunctivitis and rhinitis, and mucopurulent infra-orbital sinusitis. Mycoplasmal organisms were isolated in cultures in an affected evening grosbeak and identified as Mycoplasma gallisepticum by direct immunofluorescence. Random amplified polymorphic DNA (RAPD) fingerprinting of this isolate resulted in a banding pattern that was identical to patterns of M. gallisepticum isolates made from similar lesions in house finches (Carpodacus mexicanus) and American gold finches (Carduelis tristis) throughout eastern North America. Mycoplasma gallisepticum was identified by polymerase chain reaction in another evening grosbeak and a pine grosbeak. These observations suggest that the same strain of M. gallisepticum is the likely etiology for the observed disease in evening and pine grosbeaks in Canada and represent an extension of the host-species range for the ongoing epidemic of M. gallisepticum conjunctivitis in eastern North America.

Animals↗

PubMed2001

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy.

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.

Animals↗

PubMed1998

RAPD technique for taxonomic studies of Pellia epiphylla-complex (Hepaticae, Metzgeriales).

The random amplified polymorphic DNA (RAPD) technique was used to study the genomic relationships of three liverworts from the genus Pellia: P.epiphylla, P. borealis and P. neesiana. Altogether 150 characters (150 DNA fragments obtained using PCR) were scored. These characters were used to create a matrix of pairwise distances between all the pairs of taxa. Both distance (UPGMA, Fitch-Margoliash and Neighbor-Joining) and binary character-state (Wagner and Camin-Sokal parsimony and compatibility) methods were applied for trees' construction. Our results strongly support distinction of the recently discovered sibling species of P.epiphylla - species N and P.epiphylla - species S, which have an allopatric distribution in Poland (N - North, S - South Poland). Moreover, our data also supports the hypothesis of a hybrid origin (alloploid) of the polyploid P. borealis from P. epiphylla-NxP.epiphylla-S. P.neesiana was excluded as a donor of either of the genomes of P. borealis.

Journal Article↗

PubMed1999

Species and genotypic diversities and similarities of pathogenic yeasts colonizing women.

We examined the patterns of strain relatedness among pathogenic yeasts from within and among groups of women to determine whether there were significant associations between genotype and host condition or body site. A total of 80 yeast strains were isolated, identified, and genotyped from 49 female volunteers, who were placed in three groups: (i) 19 women with AIDS, (ii) 11 pregnant women without human immunodeficiency virus (HIV) infection, and (iii) 19 women who were neither pregnant nor infected with HIV. Seven yeast species were recovered, including 59 isolates of Candida albicans, 9 isolates of Candida parapsilosis, 5 isolates of Candida krusei, 3 isolates of Candida glabrata, 2 isolates of Saccharomyces cerevisiae, and 1 isolate each of Candida tropicalis and Candida lusitaniae. Seventy unique genotypes were identified by PCR fingerprinting with the M13 core sequence and by random amplified polymorphic DNA analysis. Of the nine shared genotypes, isolates from three different hosts were of one genotype and pairs of strains from different body sites of the same host shared each of the other eight genotypes. Genetic similarities among groups of strains were calculated and compared. We found no significant difference in the patterns of relatedness of strains from the three body sites (oral cavity, vagina, and rectum), regardless of host conditions. The yeast microflora of all three host groups had similar species and genotypic diversities. Furthermore, a single host can be colonized with multiple species or multiple genotypes of the same species at the same or different body sites, indicating dynamic processes of yeast colonization on women.

AIDS-Related Opportunistic Infections↗

PubMed2004

Transfer of small chromosome fragments of Agropyron elongatum to wheat chromosome via asymmetric somatic hybridization.

The chromosome constitution of hybrids and chromatin patterns of Agropyron elongatum (Host)Neviski in F5 somatic hybrid lines II -1-3 and I-1-9 between Triticum aestivum L. and A. elongatum were analyzed. Based on the statistic data of pollen mother cells, F5 I-1-9 and II-1-3 had 20-21 bivalents with a frequency of 84.66% and 85.28%, of which, 89.83% and 89.57% were ring bivalents. The result indicated that both hybrid lines were basically stable in the chromosome constitution and behavior. RAPD analysis showed that the two hybrids contained biparental and integrated DNA. GISH (Genome in situ hybridization) revealed that in the form of small chromosome segments, A. elongatum chromatin was scattered on 4-6 wheat chromosomes near by the region of centromere and telomere in the two hybrid lines. SSR analysis indicated that A. elongatum DNA segments were distributed on the 2A, 5B, 6B and 2D wheat chromosomes in the hybrids, which was in accordance with the GISH results that small-segments intercalated poly-site.

Agropyron↗

PubMed2003

Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest.

We investigated the occurrence and genetic diversity of Trichoderma in the river Danube national park, a primeval, riparian forest area located south-east of Vienna (Austria) which represents one of the last cases of an original European river-floodplain landscape. Forty-six strains were isolated and identified at the species level by analysis of morphological characters, by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS 1 and 2) of the rDNA cluster and--in some cases--a fragment of the translation elongation factor 1alpha (tef1) gene, and RAPD-analysis. Twenty-one strains were positively identified as T. harzianum, thirteen as T. rossicum, four as T. cerinum, two as T. hamatum, and one each as T. atroviride and T. koningii: four strains yielded two different ITS1 and 2 as well as tef1 sequence types, which were not alignable with any known species. Our studies show that they represent two new taxa of Trichoderma.

Austria↗

PubMed2006

Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates.

In this study, we compared the potential of amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) analysis, restriction fragment length polymorphism (RFLP) of the gene encoding lipoprotein P146, and the variable number of tandem repeats (VNTR) of the P97 encoding gene, as possible methods for typing an international collection of Mycoplasma hyopneumoniae isolates. All techniques showed a typeability of 100% and high intraspecific diversity. However, the discriminatory power of the different techniques varied considerably. AFLP (>0.99) and PCR-RFLP of the P146 encoding gene (>0.98) were more discriminatory than RAPD (0.95) and estimation of the VNTR of P97 (<0.92). Other, preferentially well spread, tandem repeat regions should be included in order for this latter technique to become valuable for typing purposes. RAPD was also found to be a less interesting typing technique because of its low reproducibility between different runs. Nevertheless, all molecular techniques showed overall more resemblance between strains isolated from different pigs from the same herd. On the other hand, none of the techniques was able to show a clear relationship between the country of origin and the fingerprints obtained. We conclude that AFLP and an earlier described PFGE technique are highly reliable and discriminatory typing techniques for outlining the genomic diversity of M. hyopneumoniae isolates. Our data also show that RFLP of a highly variable gene encoding P146 may be an equally useful alternative for demonstrating intraspecific variability, although the generation of sequence variability of the gene remains unclear and must be further examined.

Adhesins, Bacterial↗

PubMed2003

Lactococcus garvieae in wild Red Sea wrasse Coris aygula (Labridae).

Lactococcus garvieae infection in wild wrasse Coris aygula is reported, and the serological and molecular characteristics of the isolate are described. This is the first evidence of the presence of this pathogen in the Red Sea, and it follows the recent diagnosis of Mycobacterium marinum and Streptococcus iniae in wild fish from the same region. Whether all 3 pathogens are strains endemic to the Red Sea, or recent introductions into the region, remains to be determined, but their appearance over a period of a few years in wild fish populations in the northern Red Sea is consistent with an emerging trend affecting marine organisms on a global level in areas subjected to intense anthropogenic impacts.

Animals↗

PubMed1996

Differentiation of Acinetobacter baumannii biotypes by amplification of 16S-23S rRNA intergenic spacer sequences.

Isolates of Acinetobacter baumannii (32 strains) from blood samples obtained from patients in five Chilean hospitals were identified and biotyped according to their phenotypic properties. They were also submitted to random amplified polymorphic DNA (RAPD) using eight randomly designed 10-mers and the core sequence of M13 phage (15-mers) as well as amplification of the spacer regions between 16S and 23S genes in the prokaryotic rRNA genetic loci. With some primers, RAPD discriminated between biotypes, whereas with others each isolate showed a particular profile. When amplification of spacer regions was performed, a clear correlation between patterns and biotypes was found. This last technique allowed correct biotyping of clinical isolates. Both genetic methods might be used for the identification of A. baumannii biotypes.

Acinetobacter↗

PubMed2003

Molecular typing techniques as a tool to differentiate non-Saccharomyces wine species.

A total of 32 yeast strains belonging to four non-Saccharomyces species associated with winemaking was characterized by different molecular techniques. The PCR amplification of 18S rRNA-coding DNA and nontranscribed spacer, followed by restriction analysis with the endonucleases HaeIII and MspI, and PCR fingerprinting with microsatellite primers (GAC)(5) and (GTG)(5) were used. The methods used provided species-specific profiles and proved to be fast and reliable for monitoring the evolution of the four non-Saccharomyces yeast populations throughout wine fermentation.

DNA Fingerprinting↗