Thymus zygis subsp. Gracilis: watering level effect on phytomass production and essential oil quality

Thymus zygis subsp. gracilis (chemotype thymol) was evaluated on the basis of its phytomass production and essential oil quality. Three different watering levels were assayed to achieve 63, 44, and 30% of the local potential evapotranspiration (Eto). According to the statistical analysis, a water supplement equivalent to 44% Eto in this cultivation area was optimal for maximum plant dry matter production and essential oil yield. Capillary GC-MS analysis of the essential oil allowed the identification of 86 volatile components. Among them, 30 are described for the first time as volatile constituents of the essential oil in this thyme subspecies and chemotype. The watering level effect on essential oil composition was noticeable, because the application of a water supplement equivalent to the 63% Eto favored the production of an essential oil richer in low molecular weight components. However, the greatest thymol concentrations were obtained under the 30 and 44% Eto watering levels.     

Comparative Analysis of Genetic Diversity in Two Tunisian Collections of Fig Cultivars Based on Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats Fingerprints

This study characterized the genetic diversity of 18 Tunisian fig cultivars using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR). Both random and ISSR primers tested generated a total of 116 RAPD and 47 ISSR markers. Considerable genetic variation was observed among fig cultivars sampled from two regional Tunisian collections with an average diversity of 4.57. RAPD and ISSR banding patterns and genetic distances values reflected the high level of diversity among the collections and lower variability between the two collections. The correlation between the RAPD and ISSR similarity matrices computed for the 153 pairwise comparisons among the 18 varieties was lower and significant. An analysis of molecular variance showed that 92% of the total genetic diversity resided within collections, whereas only 8% between collections. The results indicated that in the local fig germ plasm the information provided by RAPD and ISSR is not analogous, most likely as a consequence of the fact that the two classes of markers explore, at least in part, different portions of the genome.     

Characterization of Some Italian Common Bean (<Emphasis Type="Italic">Phaseolus Vulgaris</Emphasis> L.) Landraces by RAPD,Semi-random and ISSR Molecular Markers

Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and a semi-random PCR system were used to analyze the genetic diversity of 16 Italian common bean landraces and their relationship to four commercial cultivars. Of the primers tested, 8 ISSR, 6 RAPD and 7 semi-random primers produced polymorphic and reproducible DNA fragments. A higher proportion of polymorphic bands were observed using ISSR (85%) and semi-random (90%) primers than RAPD (69%) method. The combination of any two semi-random markers allowed the identification of all 20 bean genotypes. In contrast ISSR (except for primer (CAC)₃GC) and RAPD markers appeared to be less informative as more than two markers were necessary to achieve the same diagnostic level. Moreover, 7 ISSR, 2 RAPD and 8 semi-random exclusive bands were identified as putative population-specific markers. Semi-random and ISSR derived dendrograms showed similar tendencies in terms of genetic relatedness, whereas clustering of genotypes within groups was not similar when compared with the RAPD technique. Despite the different ability to resolve genetic variation among the investigated landraces, two major clusters with less than 60% (ISSR) and 40% (RAPD and semi-random) genetic similarity were formed with all three marker systems. The two groups were correlated with the phaseolin patterns and seed size of the landraces. The analysis showed that the cultivar ȁ8Lingua di Fuocoȁ9 and most of the landraces (13 out of 16) collected in Italy belong to the Andean gene pool, whereas only the three populations from Pratomagno belong to the Middle American gene pool.     

An Assessment of the Genetic Diversity within a Collection of Wild Cranberry (<Emphasis Type="Italic">Vaccinium macrocarpon</Emphasis> Ait.) Clones with RAPD-PCR

Forty-three wild cranberry (Vaccinium macrocarpon Ait.) clones collected from four Canadian provinces and five cranberry cultivars were assessed for genetic variability by using random amplified polymorphic DNA (RAPD)-PCR. Fourteen primers generated 161 polymorphic RAPD-PCR bands. A substantial degree of genetic diversity was found among the wild cranberry collections. Cluster analysis by the unweighted pair-group method with arithmetic averages (UPGMA) separated the wild clones and three cultivars into five main clusters, and identified the two remaining cultivars as outliers. Furthermore, within four clusters, the genotypes tended to form sub-clusters that were in agreement with the principal coordinate (PCO) analysis. Geographical distribution explained 10% of total variation as revealed by analysis of molecular variance (AMOVA). The RAPD markers detected a sufficient degree of polymorphism to differentiate among cranberry clones and cultivars, making this technology valuable for germplasm management and the more efficient choice of parents in current cranberry breeding programs.     

Comparison of Growth,Pod Distribution and Canopy Structure of Old and New Cultivars of Oilseed Rape ( Brassica napus L.)

Growth, canopy structure and yield components of four selected spring-type cultivars of oilseed rape (Derby, Cyclone, Gulle and Christa) were studied in a field experiment conducted in 1993. Growth analysis was carried out by taking six samples at two-weekly intervals. Area indices of leaves, stems and pods as well as dry matter of plant components were measured. The cultivars showed two different patterns of development and were classified into two groups: old and new cultivars. The first group (Derby and Cyclone) was characterized by short plants, bearing more branches that started branching earlier and on a low position on the stem, while the second group (Gulle and Christa) were characterized by tall plants with thick stems and few branches, starting higher on the stem. Yield could not be directly related to differences in the canopy structure between old and new types. The highest yielding cultivar (Cyclone) belonged to the new category, but the second highest yielding cultivar (Gulle) was of the old type. The study of canopy structure revealed that the pods were distributed evenly on the uppermost branches of the cultivar Cyclone, while for the other cultivars most of the pods were on the main stem, especially in cultivars Gulle and Christa (old group). The cultivars differed significantly in most yield components. In addition to stem and pod development, number of branches and leaf area after anthesis were important characteristics for yield improvement. There were significant differences between cultivars in oil and protein content.     

Discriminating between cultivars and treatments of broccoli using mass spectral fingerprinting and analysis of variance-principal component analysis

Metabolite fingerprints, obtained with direct injection mass spectrometry (MS) with both positive and negative ionization, were used with analysis of variance-principal components analysis (ANOVA-PCA) to discriminate between cultivars and growing treatments of broccoli. The sample set consisted of two cultivars of broccoli, Majestic and Legacy, the first grown with four different levels of Se and the second grown organically and conventionally with two rates of irrigation. Chemical composition differences in the two cultivars and seven treatments produced patterns that were visually and statistically distinguishable using ANOVA-PCA. PCA loadings allowed identification of the molecular and fragment ions that provided the most significant chemical differences. A standardized profiling method for phenolic compounds showed that important discriminating ions were not phenolic compounds. The elution times of the discriminating ions and previous results suggest that they were common sugars and organic acids. ANOVA calculations of the positive and negative ionization MS fingerprints showed that 33% of the variance came from the cultivar, 59% from the growing treatment, and 8% from analytical uncertainty. Although the positive and negative ionization fingerprints differed significantly, there was no difference in the distribution of variance. High variance of individual masses with cultivars or growing treatment was correlated with high PCA loadings. The ANOVA data suggest that only variables with high variance for analytical uncertainty should be deleted. All other variables represent discriminating masses that allow separation of the samples with respect to cultivar and treatment.     

Tracing the origin of Spanish common bean cultivars using biochemical and molecular markers

Fifty-four accessions of Phaseolus vulgaris cultivated in Spain and representing a broad variability for this country, were studied together with 30 samples of wild forms of American origin. Two reference cultivars (from the Andes and Mesoamerica) plus two outgroups (P. coccineus and Vigna unguiculata) were also included. RAPD analysis of DNA leaf extracts were carried out with four selected primers. We also studied morphological characters of the seeds and the phaseolin electrophoretic patterns. Multivariate analysis with the UPGMA method using RAPD data clustered the samples in four groups and, comparing with morphological data and phaseolin types, showed that the Spanish cultivars were mainly of Andean origin. Nevertheless, occurrence of introgression in Spain and the consideration of the country as a second center of variability for beans can also explain the obtained results.     

应用RAPD技术区别芒果抗炭疽病品种的研究

应用RAPD技术在分子水平上对10个芒果品种的遗传变异程度研究分析结果表明,10个芒果品种被聚为3个RAPD组,RAPD组与品种对炭疽病的抗性有明显相关性。表明RAPD技术在筛选抗病品种时可作为辅助手段之一。     

草莓品种的亲缘关系及其不同继代次数的叶片组培苗的RAPD分析

从100个随机引物中筛选出10个引物,对16个草莓品种进行RAPD分析。结果显示,10个引物共扩增出73条DNA谱带,其中多态条带49条,多态性程度为67.13%;遗传距离D值在0.40水平上能将供试材料聚为3类,表明RAPD能很好地鉴别草莓品种之间的遗传关系。利用前面筛选的10个随机引物和其他20个随机引物对不同继代次数的草莓组培继代苗进行了RAPD分析,其特征带型表现一致,未发现变异,说明草莓叶盘离体继代培养30次以内能稳定遗传。     

Identification of some Benin Republic's Guinea yam (Dioscorea cayenensis/Dioscorea rotundata complex) cultivars using Randomly Amplified Polymorphic DNA

DNA from twenty-three late maturing cultivars of Guinea yams (D. cayenensis/D. rotundata complex) from the Benin Republic that could not be separated using isozyme markers, were examined using randomly amplified polymorphic DNA (RAPD) markers with decamer primers of arbitrary sequence. All the twelve primers tested were informative and yielded 63 amplified DNA bands from which 47 (75%) were polymorphic. Although no single primer produced polymorphic bands in all cultivars, the great majority of the cultivars were separated with the combinations of polymorphic bands generated by various primers. Putative duplicates and cultivar misclassifications were identified. Many morphologically distinct cultivars were close. The dwarf cultivar Tam-Sam considered as derived from Tabane, appeared more distant from the latter than was believed. RAPD analysis was found as a practical tool for the identification of duplicates toward establishment of an accurate core collection of Guinea yams in Benin Republic and in the other countries of the African yam belt.     

Antimicrobial and antioxidant activity of the essential oil and methanol extracts of Thymus pectinatus Fisch. et Mey. Var. pectinatus (Lamiaceae)

The essential oil, obtained by using a Clevenger distillation apparatus, and water-soluble (polar) and water-insoluble (nonpolar) subfractions of the methanol extract of Thymus pectinatus Fisch. et Mey. var. pectinatus were assayed for their antimicrobial and antioxidant properties. No (or slight) antimicrobial activity was observed when the subfractions were tested, whereas the essential oil showed strong antimicrobial activity against all microorganisms tested. Antioxidant activities of the polar subfraction and the essential oil were evaluated using 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radical, superoxide radical scavenging, and lipid peroxidation assays. The essential oil, in particular, and the polar subfraction of the methanol extract showed antioxidant activity. The essential oil was analyzed by GC/MS, and 24 compounds, representing 99.6% of the essential oil, were identified: thymol, gamma-terpinene, p-cymene, carvacrol, and borneol were the main components. An antimicrobial activity test carried out with fractions of the essential oil showed that the activity was mainly observed in those fractions containing thymol, in particular, and carvacrol. The activity was, therefore, attributed to the presence of these compounds. Other constituents of the essential oil, such as borneol, gamma-terpinene, and p-cymene, could be also taken into account for their possible synergistic or antagonistic effects. On the other hand, thymol and carvacrol were individually found to possess weaker antioxidant activity than the crude oil itself, indicating that other constituents of the essential oil may contribute to the antioxidant activity observed. In conclusion, the results presented here show that T. pectinatus essential oil could be considered as a natural antimicrobial and antioxidant source.     

Application of random amplified polymorphic DNA to study genetic diversity in Paspalum scrobiculatum L. (Kodo millet, Poaceae)

Summary Genetic diversity and patterns of geographic variation among collections of Paspalum scrobiculatum (kodo millet) and P. polystachyum were studied using molecular markers generated through the random amplified polymorphic DNA (RAPD) method. A high level of polymorphism in RAPD markers was observed among the individual accessions, demonstrating the high genetic diversity of the crop. The markers obtained from the RAPD method were analyzed with the cluster analysis, principal coordinates and minimum spanning tree methods. Three major groups were resolved, one representing the African accessions, and two for the Indian accessions. The accessions of the north African kodo millet and P. polystachyum (considered conspecific with P. scrobiculatum) were quite distinct. The Australian kodo millet showed higher affinity to the African types. The study demonstrated that the RAPD technique can be applied to resolving degrees and patterns of genetic variation at the population and species levels, identifying cultivars, and defining gene pools of this crop.     

Investigation of the Origanum onites L. essential oil using the chorioallantoic membrane (CAM) assay

The in vivo test on the chorioallantoic membrane of the fertilized hen's egg (CAM assay) is a current method to determine antiangiogenic, antiinflammatory activity and toxic effects of individual compounds or complex plant extracts. The method is used for testing natural compounds in small amounts for revealing various modes of action and the complex mechanisms related to angiogenesis and inflammation. Furthermore, possible side effects such as membrane irritation, toxic, and anticoagulant properties of the investigated material in question can be detected. For the evaluation, the essential oil obtained by hydrodistillation of the aerial parts of Origanum onites L., a common spice and medicinal plant, was tested for its effect in the chorioallantoic membrane (CAM) assay. The essential oil composition was revealed by means of gas chromatography-mass spectrometry (GC-MS). Eighty three components were identified, representing 99.1% of the total oil. Carvacrol, thymol, p-cymene, and gamma-terpinene were found as major components and were also individually tested in the CAM assay. Along with the monoterpenes carvacrol and thymol, their methyl ether derivatives were also examined for comparison of their physiological action. Neither the essential oil nor its components showed any pronounced antiinflammatory or antiangiogenic property in the CAM assay, at 10-250 microg/pellet. However, the irritant effect of the essential oil was linked to thymol in a dose-response fashion, up to 10 microg/pellet, where it was still showing irritation.     

Watering level effect on Thymus hyemalis Lange essential oil yield and composition

Thymus hyemalis Lange (chemotype thymol) was cultivated as an experimental crop under different watering level conditions in order to achieve 80, 60, 40, and 20% of the local potential evapotranspiration (Eto). Two harvesting periods were considered, winter and spring. As a consequence of the great variability among plants, essential oil yield percentages did not show statistically significant differences among seasons and different levels of water supply. Capillary GC-MS analysis of the essential oils permitted the detection of 84 volatile components. Among them, 54 are described for the first time as volatile constituents of the chromatographic profile of this Thymus species. Winter harvesting showed high concentrations in thymol percentage (25.92 +/- 4.39), the 40% Eto watering level being the best with respect to obtaining the optimum quality of this essential oil. However, in spring this thyme species needs a greater water supply (80% Eto) to achieve the same amount of thymol in the essential oil (29.20 +/- 2.83). From this, it was concluded that winter harvesting could be used for the extraction of the essential oil, with a low level of water supply, whereas spring harvesting could be employed for collection of leaves as a food condiment.     

RAPD and SSR markers for characterization and identification of ancient cultivars of <Emphasis Type="Italic">Olea europaea</Emphasis> L. in the Emilia region,Northern Italy

The Emilia region (Northern Italy) is characterised by the occurrence of microclimates that permit olive growing. The presence of the species, albeit sporadic, in these territories for several centuries as a fruit crop is well documented, by both archaeological and written testimony, and by a large number of plants well over a century old, located in particular sites, favourable for growth and development of the tree. Olive genetic diversity was studied using RAPD and SSR techniques, on plants growing in the Emilia territory (Reggio Emilia and Parma provinces). For genotype identification comparisons were made with 8 cultivars, some of which from Central Italy. Screening was obtained analysing patterns produced by 20 RAPD primers and 3 SSR primers, developed by other authors; the primers and we were able to discriminate olive cultivars with a sufficient degree of reliability. The dendrograms obtained from the analysis show the genetic relationship among accessions present in the Parma-Reggio Emilia district. Our results demonstrated the reliability of RAPDs and SSRs to identify all studied olive cultivars and to reveal the degree of their relatedness to each other. The analysis also reveals the presence of an interesting amount of genetic diversity among the studied individuals.     

RAPD and ISSR fingerprinting in cultivated chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and its wild progenitor <Emphasis Type="Italic">Cicer reticulatum</Emphasis> Ladizinsky

Detection of genetic relationships between 19 chickpea cultivars and five accessions of its wild progenitor Cicer reticulatum Ladizinsky were investigated by using RAPD and ISSR markers. On an average, six bands per primer were observed in RAPD analysis and 11 bands per primer in ISSR analysis. In RAPD, the wild accessions shared 77.8% polymorphic bands with chickpea cultivars, whereas they shared 79.6% polymorphic bands in ISSR analysis. In RAPD analysis 51.7% and 50.5% polymorphic bands were observed among wild accessions and chickpea cultivars, respectively. Similarly, 65.63% and 56.25% polymorphic bands were found in ISSR analysis. The dendrogram developed by pooling the data of RAPD and ISSR analysis revealed that the wild accessions and the ICCV lines showed similar pattern with the dendrogram of RAPD analysis. The ISSR analysis clearly indicated that even with six polymorphic primers, reliable estimation of genetic diversity could be obtained, while nearly 30 primers are required for RAPD. Moreover, RAPD can cause genotyping errors due to competition in the amplification of all RAPD fragments. The markers generated by ISSR and RAPD assays can provide practical information for the management of genetic resources. For the selection of good parental material in breeding programs the genetic data produced through ISSR can be used to correlate with the relationship measures based on pedigree data and morphological traits to minimize the individual inaccuracies in chickpea.     

Random amplified polymorphic DNA and genetic diversity in IndianMusa germplasm

Summary Fifty-seven accessions ofMusa including cultivated clones of 6 genomic groups (AA, AB, AAA, AAB, ABB, ABBB),M. balbisiana Colla (BB),M. acuminata Colla ssp.banksii F. Muell. (AA),M. acuminata Colla ssp.malaccensis Ridl. (AA) andM. velutina Wendl. & Drude were examined for random amplified polymorphic DNA (RAPD) genetic markers using PCR with sixty 10-mer random primers. Forty-nine of 60 tested primers gave reproducible DNA amplification patterns. The number of bands resolved per amplification was primer dependent and varied from 1 to a maximum of 24. The size range of the amplification products also differed with the selected primer sequence/genotype and ranged from 0.29 to 3.0 kb. RAPD data were used to generate Jaccard's similarity coefficients which were analyzed phenetically. Phenetic analysis separated clones into distinct groupings that were in agreement with clusterings revealed when data were subsequently analyzed by principal coordinate analysis (PCO). In both the phenetic and the PCO analyses, previously unclassified cultivars grouped with cultivars previously classified for their genomic group based on morphological keys. The implications of RAPD analysis forMusa germplasm classification, clonal identification, and management are discussed.     

RAPD and ISSR molecular markers in <Emphasis Type="Italic">Olea europaea</Emphasis> L.: Genetic variability and molecular cultivar identification

Thirty Portuguese and eight foreign olive (Olea europaea L.) cultivars were screened using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers. Twenty RAPD primers amplified 301 reproducible bands of which 262 were polymorphic; and 17 ISSR primers amplified 204 bands of which 180 were polymorphic. The percentage of polymorphic bands detected by ISSR and RAPD was similar (88 and 87%, respectively). The genetic variability observed was similar in the Portuguese and foreign olive cultivars. Seven ISSR and 12 RAPD primers were able to distinguish individually all 38 olive cultivars. Twenty specific molecular markers are now available to be converted into Sequence Characterised Amplified Region (SCAR) markers. Relationships among Portuguese and foreign cultivars is discussed.     

Bacterial susceptibility to and chemical composition of essential oils from Thymus kotschyanus and Thymus persicus

Susceptibility of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, and Pseudomonas aeroginosa to the essential oils extracted from two varieties of Thyme, i.e., Thymus kotschyanus Boiss. and Hohen. and Thymus persicus L. at preflowering and flowering stages were studied. The disk diffusion method was used to evaluate the zone of microbial growth inhibition at various concentrations of the oils. Minimal inhibitory concentration and minimal bactericidal concentration of the oils were determined and compared with each other. The oils from the above plants were found to be strongly bactericidal with that of T. kotschyanus being more effective. T. kotschyanusand T. persicus oils analyzed by gas chromatography (GC) and GC/mass spectrometry (MS) lead to identification of 33 and 26 components, respectively. The profile of the oil components from T. persicuswas similar to that of T. kotschyanus in almost all of the compounds but at different concentrations. The major components of T. kotschyanus oil before and at the flowering stages were carvacrol (35.06, 22.75%), thymol (26.60, 16.52%), gamma-terpinene (7.81, 0.34%), gamma-terpinene (4.34, 0%), borneol (2.29, 4.52%), myrcene (0.26, 12.65%), thymolquinone (0, 11.39%), nerol (0, 6.10%), and beta-caryophyllene (0, 5.54%), respectively, and those of T. persicus at the same stages were carvacrol (38.96, 27.07%), thymol (6.48, 11.86%), P-cymene (7.51, 10.16%), gamma-terpineol (0, 9.51%), nerol (15.66, 9.41%), gamma-terpinene (6.11, 6.51%), and thymol acetate (5.29, 5.30%), respectively. The contribution of oil components to its antibacterial property is discussed. High aromatic compound content of the phenol-rich oils seems to account for strong antibacterial activity.     

Discrimination of olive oils and fruits into cultivars and maturity stages based on phenolic and volatile compounds

Olive oil and fruit samples from six cultivars sampled at four different maturity stages were discriminated into cultivars and maturity stages. The variables-volatile and phenolic compounds-that significantly (p < 0.01) discriminated cultivars and maturity stage groups were identified. Separation by stepwise linear discriminant analysis revealed that Manzanilla olive cultivar was separated from cultivars Leccino, Barnea, Mission, Corregiola, and Paragon, whereas cultivars Corregiola and Paragon formed a cluster. The volatile compounds hexanol, hexanal, and 1-penten-3-ol were responsible for the discrimination of cultivars. All maturity stages were discriminated, with the separation of early stages attributed to oil phenolic compounds, tyrosol and oleuropein derivatives, whereas the volatile compounds (E)-2-hexenal, hexanol, 1-penten-3-ol, and (Z)-2-penten-3-ol characterized the separation of all maturity stages and in particular the late stages. Hexanol and 1-penten-3-ol characterized the separation of both cultivars and maturity stages.