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1.
Comp Immunol Microbiol Infect Dis ; 27(4): 247-53, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15177999

ABSTRACT

Staphylococcus aureus is a widespread pathogen causing infections in different animal species. The extensive use of antibiotics, particularly methicillin, causes the rise of antibiotic-resistant strains (MRSA). In order to verify the epidemiology and genetic relatedness among MRSA and sensible strains (MSSA), an accurate fingerprinting technique, the amplified fragment length polymorphism (AFLP), was carried out. The isolates were cultured, subdivided on MRSA and MSSA and submitted for the genomic DNA extraction that was utilized for AFLP. The data were analysed for genetic similarity using the Dice coefficient. The results of genomic analysis among MRSA and MSSA and within them revealed that the major component of variation was due to variation within strains (82.12%), while variance among strains was lower (17.88%). The low level of genomic similarity found among S. aureus strains implies high level of genetic diversity. Different similarity was found as well in all strains independently of the source.


Subject(s)
Methicillin Resistance/genetics , Staphylococcal Infections/microbiology , Staphylococcal Infections/veterinary , Staphylococcus aureus/genetics , Animals , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genetic Variation , Phylogeny , Polymerase Chain Reaction/veterinary , Polymorphism, Genetic , Staphylococcus aureus/classification , Staphylococcus aureus/drug effects , Staphylococcus aureus/isolation & purification
2.
Mol Genet Genomics ; 270(6): 539-48, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14648202

ABSTRACT

In plants, gametophytic apomixis is a form of asexual reproduction that leads to the formation of seed-derived offspring that are genetically identical to the mother plant. A common set of RFLP markers, including five rice anchor markers previously shown to be linked to apomixis in Paspalum simplex, were used to detect linkage with apomixis in P. notatum and P. malacophyllum. A comparative map of the region around the apomixis locus was constructed for the three Paspalum species, and compared to the rice map. The locus that controls apomixis in P. simplex was almost completely conserved in the closely related species P. malacophyllum, whereas it was only partially represented in the distantly related species P. notatum. Although strong synteny of markers was noted between this locus and a portion of rice chromosome 12 in both P. simplex and P. malacophyllum, the same locus in P. notatum was localized to a hybrid chromosome which carries markers that map to rice chromosomes 2 and 12. All three Paspalum species showed recombination suppression at the apomixis locus; in the case of P. notatum, this might be due to a heterozygosity for a translocation that most probably negatively interferes with chromosomal pairing near the locus. A common set of markers that show linkage with apomixis in all three Paspalum species define a portion of the apomixis-controlling locus that is likely to contain genes critical for apomictic reproduction.


Subject(s)
Genes, Plant , Paspalum/genetics , Chromosome Mapping/methods , Conserved Sequence , DNA, Plant/genetics , Genetic Markers , Genome, Plant , Oryza/genetics , Paspalum/classification , Seeds/genetics , Species Specificity
3.
Plant Cell Rep ; 22(5): 328-37, 2003 Dec.
Article in English | MEDLINE | ID: mdl-12937943

ABSTRACT

The aim of the experiments reported herein was to transiently test different gene constructs using green fluorescent protein (GFP) as a reporter gene for a future localization of the maize beta-zein in the chloroplast of alfalfa ( Medicago sativa L.). The transient expression of two GFP genes was compared in alfalfa leaves to determine which of these two mutants is the easier to detect. Based on the intensity of fluorescence emitted, the GFP S65C gene was used to assemble a chloroplast-targeted GFP to verify the efficiency of the transit peptide for chloroplast targeting. A chloroplast-targeted fusion protein between beta-zein and GFP was then assembled, and this protein was observed to accumulate in small aggregates into the chloroplasts of transiently transformed cells. To the best of our knowledge, this is the first report of the GFP S65C gene being used to obtain transformed alfalfa plants expressing GFP.


Subject(s)
Chloroplasts/metabolism , Medicago sativa/metabolism , Plants, Genetically Modified/metabolism , Zein/metabolism , Chloroplasts/genetics , Gene Expression Regulation, Plant , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Medicago sativa/cytology , Medicago sativa/genetics , Microscopy, Confocal , Microscopy, Fluorescence , Mutation , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/cytology , Plants, Genetically Modified/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Zein/genetics
4.
Plant Cell Rep ; 21(11): 1040-6, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12835996

ABSTRACT

Culture conditions have been established for the induction of callus from different explants of Paspalum simplex. Fast-growing calli were obtained from hypocotyls and roots excised from 5-day-old seedlings on culture medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Rapid plant regeneration from both apomictic and sexual lines was achieved when the medium was supplemented with alpha-naphthaleneacetic acid and benzylaminopurine. Restriction fragment length polymorphism analysis of the apomixis-controlling region of the regenerated plants showed an absence of restriction site variation for the loci analysed, whereas various degrees of variation were detected for the DNA methylation sites of the same loci.


Subject(s)
Paspalum/genetics , Paspalum/physiology , Polymorphism, Restriction Fragment Length , Regeneration/physiology , Adenine/analogs & derivatives , Adenine/pharmacology , Culture Media/chemistry , Culture Techniques , DNA Methylation , Genetic Variation , Naphthaleneacetic Acids/pharmacology , Paspalum/drug effects , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/physiology , Polymorphism, Genetic , Regeneration/drug effects , Restriction Mapping
5.
Mol Ecol ; 12(6): 1681-5, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12755896

ABSTRACT

Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment. Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants. In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. Plants of Lotus corniculatus L. transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor. Nontransgenic plants belonging to the species L. corniculatus L., L. tenuis Waldst. and Kit. ex Willd, and L. pedunculatus Cav., were utilized as recipients. Two experimental fields were established in two areas of central Italy. Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker. No transgene flow between L. corniculatus transformants and the nontransgenic L. tenuis and L. pedunculatus plants was detected. As regards nontransgenic L. corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot. In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.


Subject(s)
Genetics, Population , Lotus/genetics , Plants, Genetically Modified/physiology , Pollen/physiology , Transgenes/genetics , Aspartate-Ammonia Ligase/genetics , DNA Primers , Electrophoresis , Glucuronidase/genetics , Italy , Lotus/physiology , Plants, Genetically Modified/genetics , Pollen/genetics
6.
Sex Plant Reprod ; 14(4): 201-6, 2001 Dec.
Article in English | MEDLINE | ID: mdl-24573427

ABSTRACT

Segregating progenies of crosses between sexual and apomictic genotypes of Paspalum simplex were analysed for the formation of meiotic versus aposporous embryo sacs, zygotic versus parthenogenetic embryos, and autonomous versus pseudogamous endosperms by using cytoembryological and flow cytometric analyses. Reduced and unreduced 8-nucleated embryo sacs were the final product of female gametophyte development in sexual and aposporous genotypes, respectively. An incomplete penetrance of parthenogenesis was detected in aposporous genotypes. The relative DNA content of endosperm nuclei revealed the normal 2:1 maternal to paternal ratio in sexuals and a 4:1 ratio in apomicts, indicating insensitivity of the apomictic genotypes to endosperm imprinting. Apospory, parthenogenesis and pseudogamy are located on a relatively large linkage group and are inherited together with previously developed molecular markers as a single genetic unit in segregating progenies.

7.
FEMS Microbiol Ecol ; 34(2): 139-146, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11102691

ABSTRACT

Two repeated DNA sequences of European strains of the symbiotic fungus Tuber melanosporum were isolated and characterized. One of these, SS14, representing about 0.05% of the fungal genome, was shown to be a T. melanosporum-specific sequence by Southern and dot-blot hybridization. The second one, named SS15, is about 0.0025% of the entire genome, and it is specific not only to T. melanosporum but also to the Asian black truffle Tuber indicum. Neither of these two fragments hybridizes with any of the other European truffle species tested. By sequence analysis of these two fragments, PCR primers were designed and used to selectively amplify DNA from T. melanosporum ascocarps and ectomycorrhizae by simple and multiplex PCR. No amplification products were obtained with DNA from either mycorrhizal roots or fruit bodies of other ectosymbiotic fungi. The two identified genomic traits also provided useful information for a better understanding of the phylogenetic relationships among black truffle species and for testing T. melanosporum intraspecific variability.

9.
Plant Cell Rep ; 17(5): 339-344, 1998 Mar.
Article in English | MEDLINE | ID: mdl-30736569

ABSTRACT

Several dicotyledonous species were infected with an Agrobacterium rhizogenes binary vector harbouring the plasmid 121.Sn which contains the maize gene Sn under the constitutive promoter CaMV35S. In maize, Sn transactivates the anthocyanin pathway in different tissues. The aim of this work was to test the efficiency of this gene to regulate the anthocyanin pathway in heterologous systems and verify its suitability as a reporter gene. The pigmentation of the hairy roots was compared with hairy roots stained for ß-glucuronidase activity, which were used as a control. In two polymorphic genotypes of Lotus angustissimus, DNA integration and expression were assayed. The maize gene is competent to induce anthocyanin pigmentation in different species, but the complexity of the regulatory mechanisms of anthocyanin synthesis restricts the use of Sn as a reporter gene.

10.
FEMS Microbiol Lett ; 153(2): 255-60, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9271850

ABSTRACT

Morphologically similar species such as Chinese black truffles and Tuber melanosporum were typed by restriction length polymorphism of internal transcribed spacers. This analysis together with sequence comparison revealed the presence of high genetic variability among fruit bodies collected in China. Selection of primer pairs allowed the internal transcribed spacer region of both Chinese truffles and T. melanosporum to be specifically amplified.


Subject(s)
Ascomycota/classification , Mycological Typing Techniques , Polymorphism, Restriction Fragment Length , Ascomycota/genetics , Base Sequence , China , DNA, Fungal/genetics , DNA, Ribosomal/genetics , Genetic Markers , Genetic Variation , Molecular Sequence Data , Sequence Homology, Nucleic Acid
11.
Genome ; 40(6): 822-8, 1997 Dec.
Article in English | MEDLINE | ID: mdl-18464868

ABSTRACT

Apomictic tetraploid Paspalum simplex was crossed with colchicine-doubled diploid sexual plants belonging to the same species. Homologous genomic probes were selected from a partial PstI genomic library for their capacity to detect alleles specific to the apomictic parent, and their segregation was analyzed in the F1 progeny. High levels of polymorphism between apomictic and sexual genotypes were recorded. The heterozygosity was high in both tetraploid and diploid genotypes but the differences between them were not as great as expected. In the sexual parent, some markers segregated as either a monoallelic duplex or a diallelic duplex, while several allelic configurations were observed in the apomictic parent. The segregation of double-dose monoallelic fragments demonstrated the tetrasomic inheritance of apomictic P. simplex. The correlations between apomixis, ploidy level, and tetrasomic inheritance are discussed.

12.
Genome ; 39(5): 914-20, 1996 Oct.
Article in English | MEDLINE | ID: mdl-18469944

ABSTRACT

A cytological examination of the nucleolus organizing regions (NORs) of three species from the Medicago sativa complex was conducted to evaluate the structural and functional evolution of the ribosomal RNA (rRNA) loci that encode the 18S, 5.8S, and 26S rRNAs. Mitotic chromosomes in root-tip preparations from tetraploid M. sativa and diploids Medicago coerulea and Medicago falcata were visualized by four methods that provide new data. Fluorescent in situ hybridization using the M. sativa 18S gene as probe localized the structural rDNA to the constricted regions of the satellited chromosomes only. Chromomycin A3 (CMA3) staining and 4′,6-diamidino-2-phenylindole (DAPI) staining identified these chromosomal segments as the most GC-rich regions in the alfalfa karyotype. Medicago falcata exhibited fewer DAPI bands and chromocenters than did M. sativa and M. coerulea. Positive silver nitrate staining showed that all four rDNA regions in M. sativa (located in two chromosome pairs) and both rDNA sites in both diploid species remain transcriptionally active. Counts of nucleoli confirmed that all rDNA regions are independently capable of nucleolus organization. Thus, the number of active NORs in M. sativa is double the number found in M. coerulea or M. falcata. Consequently, if M. sativa originated from sexual hybridization of 2n gametes involving one or both diploid species, no major reorganization or loss of structural or functional rDNA loci has occurred. Key words : alfalfa evolution, CMA3 banding, DAPI banding, fluorescent in situ hybridization, silver nitrate staining.

13.
Theor Appl Genet ; 93(1-2): 183-9, 1996 Jul.
Article in English | MEDLINE | ID: mdl-24162216

ABSTRACT

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.

14.
Theor Appl Genet ; 93(5-6): 801-8, 1996 Oct.
Article in English | MEDLINE | ID: mdl-24162411

ABSTRACT

We have characterized the genetic consequences of somatic hybridization within the ribosomal DNA (rDNA) of three interspecific hybrids, each involving M. sativa as one of the parents. Restriction-fragment-length-polymorphisms (RFLPs) of rDNA spacers and fluorescent-in-situ-hybridization (FISH) of an 18S-gene probe to mitotic chromosomes were used to compare parental and hybrid species. The M. sativa-coerulea hybrid retained all six parental nucleolar-organizing regions (NORs) and all parental RFLPs representing a complete integration of rDNA. The M. sativa-arborea hybrid retained five of six parental NORs while losing half of the arborea-specific RFLPs, indicating that simple chromosome loss of one arborea NOR accounted for the RFLP losses. Dramatic alterations occurred within the M. sativa-falcata hybrid where five of six parental NORs were retained and new rDNA RFLPs were created and amplified differentially among somaclonal-variant plants. The molecular basis of the new RFLPs involved increased numbers of a 340-bp subrepeating element within the rDNA intergenic spacer (IGS), suggesting that recurrent cycles of unequal recombination occurred at high frequency within the rDNA in somatic lineages.

15.
Theor Appl Genet ; 90(3-4): 347-55, 1995 Mar.
Article in English | MEDLINE | ID: mdl-24173924

ABSTRACT

Somatic hybrid plants produced by protoplast fusion between tetraploid Medicago sativa (2n= 4x=32) and the diploid species Medicago coerulea (2n= 2x=16) have been RFLP fingerprinted to establish their nuclear composition. Although all of the chromosomes were present, molecular analysis revealed an incomplete incorporation of the alleles of the diploid parent in the fusion products. In the polycross progeny the alleles of both parents segregated in a Mendelian mode. Cytological observations indicated that in the somatic hybrid population minor abnormalities are present; these are restricted mainly to the formation of univalents and lagging chromosomes. Meiosis appeared to be more stable than has been previously reported in the hexaploids of alfalfa. The somatic hybrids grown in the field had a rather vigorous aspect, particularly with respect to the vegetative organs. Forage yield was comparable to that of thmore productive parent. The results are discussed with a view to utilizing the somatic hybrids as starting material for breeding alfalfa at the hexaploid level.

16.
Theor Appl Genet ; 84(7-8): 792-7, 1992 Sep.
Article in English | MEDLINE | ID: mdl-24201476

ABSTRACT

Symmetric somatic hybrid plants have been produced by electrofusion of leaf protoplasts of Medicago sativa and callus protoplasts of Medicago coerulea. The selection of hybrid individuals has been performed at the cellular level by recording the positions of single heterocaryons immobilized in a semisolid culture medium. The hybrid nature of the heterokaryons was assessed in fluorescent light on the basis of their color. Hybrid minicalli were picked up manually and grown first on propagating, and then on regenerating, media. Six putative hybrid calli were selected and two of them regenerated several plants. The hybrid nature of the regenerants was confirmed by cytological and isozyme analysis. Among the several morphological traits taken into account for the characterization of somatic hybrid plants, some were intermediate, some lower, and some higher, with respect to the parents. The somatic hybrid plants were fertile and set seed. The production of somatic hybrid plants in the genus Medicago is discussed in relation to the regenerating capability of parental protoplasts.

17.
Plant Cell Rep ; 10(6-7): 273-6, 1991 Sep.
Article in English | MEDLINE | ID: mdl-24221655

ABSTRACT

Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l(-1) maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.

18.
Plant Cell Rep ; 10(6-7): 300-3, 1991 Sep.
Article in English | MEDLINE | ID: mdl-24221661

ABSTRACT

Plants of Medicago arborea have been infected with Agrobacterium rhizogenes strain LBA9402 harbouring the plasmids Ri 1855 and AGS125 carrying a gene conferring resistance to the antibiotic hygromycin. About 7056 of the hairy roots showed callus formation on hygromycin-supplemented medium. Regeneration took place on antibiotic free medium only. Plantlets suitable for transfer to soil were obtained after the manual removal of most of the leaves. Plant morphology showed the usual alterations induced by the Ri plasmid; moreover, two years after soil-transfer, transformants have not flowered. Molecular analysis indicates the presence of T-DNA from both pAGS 125 and p1855. The expression of the hygromycin phosphotransferase gene allowed callus and protoplasts of transformed plants to grow on media supplemented with the antibiotic. This trait will be utilized as a marker in protoplast fusion between Medicago arborea and Medicago sativa (alfalfa).

19.
Theor Appl Genet ; 74(6): 700-5, 1987 Oct.
Article in English | MEDLINE | ID: mdl-24240327

ABSTRACT

From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.

20.
Theor Appl Genet ; 73(4): 523-30, 1987 Feb.
Article in English | MEDLINE | ID: mdl-24241108

ABSTRACT

The widely cultivated forage legume alfalfa (Medicago sativa L.) was transformed with the agropine type Agrobacterium rhizogenes NCPPB 1855. Sterile root and callus cultures were derived from tumorous hairy roots which were easily obtained independent of the plant variety or genotype. Plant regeneration, via somatic embryogenesis, was achieved only when a selected alfalfa line, characterized by high regenerative capability, was utilized. Genetic transformation was confirmed by the presence of agropine and T-DNA. Phenotypic alterations, mainly affecting the root system, were observed in transformed plants. The possibility that T-DNA-induced variations could be useful in the improvement of M. sativa is discussed.

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