Systematics of Juniperus section Juniperus based on leaf essential oils and random amplified polymorphic DNAs (RAPDs)

The composition of the leaf essential oils of all the species of Juniperus in sect. Juniperus (=sect. Oxycedrus) are reported and compared (J. brevifolia, J. cedrus, J. communis, J. c. var. saxatilis, J. c. var. oblonga, J. formosana, J. oxycedrus, J. o. subsp. badia, J. o. subsp. macrocarpa, J. o. subsp. transtagana, J. rigida, J. r. subsp. conferta, J. sibirica, J. taxifolia and J. t. var. lutchuensis). In addition, DNA fingerprinting by RAPDs was utilized. Based on these data, several taxa remained at the same taxonomic level: J. brevifolia, J. cedrus, J. communis, J. c. var. saxatilis, J. formosana, J. oxycedrus, J. rigida, J. r. var. conferta, and J. taxifolia. However, several taxa exhibited considerable differentiation that warranted their recognition at the specific level: J. oblonga M.-Bieb. (=J. communis var. oblonga), J. badia H. Gay (=J. oxycedrus subsp. badia), J. macrocarpa Sibth. and Sm. (=J. oxycedrus subsp. macrocarpa), J. navicularis Gand. (=J. oxycedrus subsp. transtagana), J. sibirica Brugsd. (=J. communis var. saxatilis in part), and J. lutchuensis Koidz. (= J. taxifolia var. lutchuensis).     

Geographic variation in leaf essential oils and RAPDs of Juniperus polycarpos K. Koch in central Asia

Variations in the composition of the leaf essential oils and DNA fingerprints (RAPDs) of Juniperus excelsa, J. polycarpos, J. seravschanica, and J. turcomanica were examined. Juniperus procera was also included in the analyses to aid in determining the specific status of J. polycarpos. Based on these analyses, J. polycarpos is recognized as a distinct species from J. excelsa. The common, multi-seeded juniper of central Asia is J. polycarpos. Juniperus seravschanica and J. turcomanica are treated as part of the J. polycarpos complex but are not recognized as formal taxonomic groups at this time. The Balochistan, Pakistan juniper, usually called J. excelsa var. polycarpos or J. macropoda should be referred to as J. polycarpos in the future.     

Numerical taxonomy of Juniperus thurifera, J. excelsa and J. foetidissima (Cupressaceae) based on morphological characters

Juniperus excelsa and J. thurifera are considered to originate from the same ancestor. As a result of occurring in geographically isolated refuges – J. excelsa in SE Europe and SW Asia and J. thurifera in SW Europe and NW Africa – their divergence could have taken place at the end of the Tertiary, with the climate cooling. Juniperus foetidissima occurs in SE Europe and SW Asia in territories shared with J. excelsa and is similar to and sometimes misidentified with it. The occurrence of the latter two species over the same area suggests they should be more similar to each other than to the geographically distant J. thurifera . Four populations of J. excelsa ssp. excelsa , four of J. thurifera and two of Juniperus foetidissima were examined on the basis of features of 10 cones and 10 shoots of 18–36 specimens each. Results of discrimination analysis, Mahalanobis distances and cluster analysis showed great similarity to J. excelsa and J. thurifera , while J. foetidissima was more distant. The results support the hypothesis of a common ancestor of J. excelsa and J. thurifera , but suggest another origin of J. foetidissima .  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 483–495.     

Genetic Variability and Differentiation Among Populations of the Azorean Endemic Gymnosperm Juniperus brevifolia: Baseline Information for a Conservation and Restoration Perspective

The Azorean endemic gymnosperm Juniperus brevifolia (Seub.) Antoine is a top priority species for conservation in Macaronesia, based on its ecological significance in natural plant communities. To evaluate genetic variability and differentiation among J. brevifolia populations from the Azorean archipelago, we studied 15 ISSR and 15 RAPD markers in 178 individuals from 18 populations. The average number of polymorphic bands per population was 65 for both ISSR and RAPD. The majority of genetic variability was found within populations and among populations within islands, and this partitioning of variability was confirmed by AMOVA. The large majority of population pairwise F(ST) values were above 0.3 and below 0.6. The degree of population genetic differentiation in J. brevifolia was relatively high compared with other species, including Juniperus spp. The genetic differentiation among populations suggests that provenance should be considered when formulating augmentation or reintroduction strategies.     

Molecular characterization and identification of markers for toxic and non-toxic varieties of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. using RAPD,AFLP and SSR markers

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).     

RAPD-marking of genomes of representative Hordeum species

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum. High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgare sensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between the Hordeum species, between the H. spontaneum populations, and between regional H. vulgare cultivars and a dendrogram was constructed.     

Genetic differentiation of subspecies of the house mouse Mus musculus and their taxonomic relationships inferred from RAPD-PCR data

Genetic differentiation of six subspecies of the house mouse Mus musculus (Mus musculus musculus. M. m. domesticus, M. m. castaneus, M. m. gansuensis, M. m. wagneri, and M. m. ssp. (bactrianus?) was examined using RAPD-PCR analysis. In all, 373 loci of total length of about 530 kb were identified. Taxon-specific molecular markers were detected and the levels of genetic differences among the subspecies were estimated. Different degree of subspecific genetic differentiation was shown. The most similar subspecies pairs were M. m. castaneus--M. m. domesticus and M. m. musculus--M. m. gansuensis. In our phylogenetic reconstruction, M. m. wagnery proved to be most different from all the other subspecies. Genetic distances between it and other subspecies were two- to threefold higher than those between the "good"' species of the subgenus Mus (e.g., between M. m. musculus and M. spicilegus, M. musculus and M. abbottii). The estimates of genetic similarity and the taxonomic relationships between six house mouse subspecies inferred from RAPD partially conformed to the results based on cytogenetic and allozyme data. However, they were considerably different from phylogenetic reconstructions based on sequencing of the control mtDNA region, which reflects mutual inconsistency of different systems of inheritance.     

Molecular markers of nuclear DNA in the study of evolution and speciation process in an example of "Lacerta agilis complex" (Sauria: Lacertidae)

In order to elucidate the molecular-genetic relations of some Lacerta s. str. lizard populations, subspecies and species in comparison with some other genera we used methods revealing four types of nuclear DNA markers. Among these were taxonprint, RAPD, Inter-MIR-PCR markers and also satellite DNA monomer sequences. The aim was to compare the phylogeny and systematics of this reptilian group based on morphological and molecular criteria. This problem has a general importance for understanding a speciation process. Our results show a good correlation between both approaches when genera and species levels were studied. Systematic status of five subspecies of L. agilis were supported but not in all cases, some subspecies have no meaningful genetic differences by three types of molecular markers, but all of them were differed by RAPD markers. The data confirm the subdivision of L. agilis populations into west and east clades proposed by other authors earlier on the basis of mitochondrial DNA and morphology. The population structure of one of the subspecies--L. agilis exigua, was studied on a number of populations distributed from Ural region up to Kabardino-Balkaria by IMP method. There were no significant differences among these 14 populations investigated. The data testify the rapid distribution of the species after the end of Pleistocene glaciation.     

Cryptic speciation between Juniperus deltoides and Juniperus oxycedrus (Cupressaceae) in the Mediterranean

Analyses of individuals classically treated as Juniperus oxycedrus L. var. oxycedrus from Morocco, Portugal, Spain, France, Italy, Greece and Turkey, using DNA sequencing of nrDNA (ITS 1, 5.8S, ITS 2) plus RAPDs, leaf terpenoids and morphology revealed that two cryptic, genetically distinct but morphologically almost identical species are present. These species, J. oxycedrus L. var. oxycedrus and Juniperus deltoides R.P. Adams, are about as different from each other as Juniperus navicularis and Juniperus macrocarpa are from J. oxycedrus var. oxycedrus. Examination of herbarium specimens revealed that the two species are largely allopatric with J. deltoides occurring from Italy eastward through Turkey into the Caucasus Mts. and Iran. J. oxycedrus var. oxycedrus appears to be largely concentrated west of Italy (France, Spain, Portugal, Morocco). Cryptic speciation is discussed.     

Alkane composition diversity among populations of dwarf forms of Juniperus communis L.: comparison between western Europe and northern American populations

The cuticular wax composition of leaves has been analysed in three western European populations (Corsica, central Pyrenees, northern Alps) of Juniperus communis var. saxatilis Pall. (=  J. nana Willd., nom illeg.) and in one population of J. communis L. var. depressa Pursh. from North America (Sierra Nevada). Gas chromatography shows the presence of 13 alkanes in all samples ranging from C₂₃ to C₃₅ with important intraspecific polymorphism in alkane content. The dominant alkanes range from C₃₃ to C₃₅. Alkanes C₂₁ and C₂₂ were found only in Corsica and Sierra Nevada populations. Canonical discriminant analysis separated the J. communis L. var. depressa Pursh. of the population of Sierra Nevada from other populations of J. communis var. saxatilis Pall. on the basis of their higher C₃₁ content and the constant presence of C₂₁ and C₂₂ alkanes. J. communis var. saxatilis Pall. populations from the Pyrenees are close to northern Alps populations characterized by high concentrations of C₃₃, C₃₄ and C₃₅ alkanes. This paper confirms the existence of Juniperus var. saxatilis Pall. in the Pyrenees (France).  © The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 140 , 165–168.     

The use of RAPD markers in Hordeum phylogeny.

The phylogenetic relationships among 39 wild Hordeum species, subspecies, and cultivated barley were investigated using RAPD markers as discriminating characters. Seventy-six RAPD fragments were generated using 12 single decameric primers of arbitrary nucleotide sequences. Amplification reactions resulted in fragments ranging in length between 200 and 2000 bp. Clearly resolved bands were scored for their presence or absence in a binary matrix. Amplified products were treated as independent characters to generate a phenogram using the NTSYS-PC package. Tree topology was generally found to be consistent with those based on morphological treatments. However, a few species like H. erectifolium, H. jubatum and, to a lesser extent, H. bulbosum occupied a position different from previous classifications. The results demonstrated that RAPD technology represents a useful and reliable tool for detecting polymorphism for phylogenetic studies. Key words : RAPD analysis, molecular markers, phylogenetic studies, Hordeum species, barley.     

Isoenzyme diversity and cryptic speciation in Juniperus excelsa (Cupressaceae) complex in Iran

An isoenzyme analysis was performed on 109 individuals from 11 populations of Juniperus excelsa complex collected from Iran. A total of 61 allozymes from five enzyme systems were scored. The mean numbers of bands per presumed isoenzyme, ranged from 2.07 to 3.23. The value of Euclidean distance ranged from 1.920 to 4.411. The average value of Shannon diversity index (H′) estimated for each population ranged from 0.317 in the population sampled from ‘Fasa’ to 1.053 in the population of ‘Golestan’, and the average H′ value for each enzyme system ranged from 0.031 (SOD-3) to 1.670 (PPO-1). To reveal the relationships among these populations, a cluster analysis based on Euclidean distances was performed. The results of the analysis suggest recognizing three taxa in the complex in Iran: Juniperus polycarpos subsp. polycarpos, Juniperus polycarpos subsp. turcomanica, and one cryptic species not recorded from Iran until now. Moreover, the analysis presented, does not confirm the occurrence of J. excelsa in Iran.     

Molecular diversity of leuconostoc mesenteroides and leuconostoc citreum isolated from traditional french cheeses as revealed by RAPD fingerprinting, 16S rDNA sequencing and 16S rDNA fragment amplification

For a long time, the identification of the Leuconostoc species has been limited by a lack of accurate biochemical and physiological tests. Here, we use a combination of RAPD, 16S rDNA sequencing, and 16S rDNA fragment amplification with specific primers to classify different leuconostocs at the species and strain level. We analysed the molecular diversity of a collection of 221 strains mainly isolated from traditional French cheeses. The majority of the strains were classified as Leuconostoc mesenteroides (83.7%) or Leuconostoc citreum (14%) using molecular techniques. Despite their presence in French cheeses, the role of L. citreum in traditional technologies has not been determined, probably because of the lack of strain identification criteria. Only one strain of Leuconostoc lactis and Leuconostoc fallax were identified in this collection, and no Weissella paramesenteroides strain was found. However, dextran negative variants of L. mesenteroides, phenotypically misclassified as W. paramesenteroides, were present. The molecular techniques used did not allow us to separate strains of the three L. mesenteroides subspecies (mesenteroides, dextranicum and cremoris). In accordance with previously published results, our findings suggest that these subspecies may be classified as biovars. Correlation found between phenotypes dextranicum and mesenteroides of L. mesenteroides and cheese technology characteristics suggests that certain strains may be better adapted to particular technological environments.     

Genetic differentiation of subspecies of the house mouse Mus musculus and their taxonomic relationships inferred from RAPD-PCR data

Genetic differentiation of six subspecies of the house mouse Mus musculus (Mus musculus musculus, M. m. domesticus, M. m. castaneus, M. m. gansuensis, M. m. wagneri, and M. m. ssp. (bactrianus?) was examined using RAPD-PCR analysis. In all, 373 loci of total length of about 530 kb were identified. Taxonspecific molecular markers were detected and the levels of genetic differences among the subspecies were estimated. Different degree of subspecific genetic differentiation was shown. The most similar subspecies pairs were M. m. castaneus-M. m. domesticus and M. m. musculus-M. m. gansuensis. In our phylogenetic reconstruction, M. m. wagneri proved to be most different from all the other subspecies. Genetic distances between it and other subspecies were two-to threefold higher than those between the “good”species of the subgenus Mus (e.g., between M. m. musculus and M. spicilegus, M. musculus and M. abbotti). The estimates of genetic similarity and the phylogenetic relationships between six house mouse subspecies inferred from RAPD partially conformed to the results based on cytogenetic and allozyme data. However, they were considerably different from phylogenetic reconstructions based on sequencing of the control mtDNA region, which reflects mutual inconsistency of different systems of inheritance.     

Genetic variation detected with RAPD markers among upland and lowland rice cultivars (Oryza sativa L.)

Genetic variation of nine upland and four lowland rice cultivars (Oryza sativa L.) was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) method via the polymerase chain reaction (PCR). Forty-two random primers were used to amplify DNA segments and 260 PCR products were obtained. The results of agarosegel electrophoretic analysis of these PCR products indicated that 208 (80%) were polymorphic. All 42 primers used in this experiment were amplified and typically generated one-to-four major bands. Only two primers showed no polymorphisms. In general, a higher level of polymorphism was found between japonica and indica subspecies while fewer polymorphisms were found between upland and lowland cultivars within the indica subspecies. A dendrogram that shows the genetic distances of 13 rice cultivars was constructed based on their DNA polymorphisms. Classification of rice cultivars based on the results from the RAPD analysis was identical to the previous classification based on isozyme analysis. This study demonstrated that RAPD analysis is a useful tool in determining the genetic relationships among rice cultivars.     

Aspergillus fumigatus Fresenius, an endophytic fungus from Juniperus communis L. Horstmann as a novel source of the anticancer pro-drug deoxypodophyllotoxin

Aims:  Isolation, identification and characterization of an endophytic fungus from Juniperus communis L. Horstmann, as a novel producer of deoxypodophyllotoxin and its in vitro antimicrobial assay.
Methods and Results:  The methodology for the isolation, identification and characterization of a novel endophytic fungus from the twigs of the J. communis L. Horstmann plant, which specifically and consistently produces deoxypodophyllotoxin, was unequivocally established. The fungus was identified as Aspergillus fumigatus Fresenius by molecular, morphological and physiological methods. Deoxypodophyllotoxin was identified and quantified by high-resolution LC-MS, LC-MS² and LC-MS³. The antimicrobial efficacy of the fungal deoxypodophyllotoxin against a panel of pathogenic bacteria was established.
Conclusions:  The production of deoxypodophyllotoxin (found in the host) by the cultured endophyte is an enigmatic observation. It demonstrates the transfer of gene(s) for such accumulation by horizontal means from the host plant to its endophytic counterpart. It would be interesting to further study the deoxypodophyllotoxin production and regulation by the cultured endophyte in J.   communis and in axenic cultures.
Significance and Impact of the Study:  This endophyte is a potential handle for scientific and commercial exploitation. Although the current accumulation of deoxypodophyllotoxin by the endophyte is not very high, it could be scaled-up to provide adequate production to satisfy new drug development and clinical needs. However, further refined precursor-feeding and mass-balance studies are required to result in the consistent and dependable production.     

Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers

Jacaranda decurrens (Bignoniaceae) is an endemic species of the Cerrado with validated antitumoral activity. The genetic diversity of six populations of J. decurrens located in the State of São Paulo was determined in this study by using molecular markers for randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Following optimization of the amplification reaction, 10 selected primers generated 78 reproducible RAPD fragments that were mostly (69.2%) polymorphic. Two hundred and five reproducible AFLP fragments were generated by using four selected primer combinations; 46.3% of these fragments were polymorphic, indicating a considerable level of genetic diversity. Analysis of molecular variance (AMOVA) using these two groups of markers indicated that variability was strongly structured amongst populations. The unweighted pair group method with arithmatic mean (UPGMA) and Pearson''s correlation coefficient (RAPD -0.16, p = 0.2082; AFLP 0.37, p = 0.1006) between genetic matrices and geographic distances suggested that the population structure followed an island model in which a single population of infinite size gave rise to the current populations of J. decurrens, independently of their spatial position. The results of this study indicate that RAPD and AFLP markers were similarly efficient in measuring the genetic variability amongst natural populations of J. decurrens. These data may be useful for developing strategies for the preservation of this medicinal species in the Cerrado.     

Inter- and intraspecific genetic variation inHippophae (Elaeagnaceae) investigated by RAPD markers

Genetic diversity has been investigated by the application of molecular markers in, for the first time, all the taxa recognised in recent treatises of the genusHippophae. RAPD (random amplified polymorphic DNA) analyses were conducted with 9 decamer primers, which together yielded 219 polymorphic markers. We found 16 fixed RAPD markers, i.e. markers that either occurred in all plants of a population or were absent from all plants. Several of these markers were useful for analysis of interspecific relationships, whereas others can be considered as taxon-specific markers. Clustering of taxa and populations in our neighbour-joining based dendrogram was in good agreement with some recently suggested taxonomic treatises ofHippophae. Amount and distribution of genetic variability varied considerably between species. Partitioning of molecular variance withinH. rhamnoides supported earlier findings that a considerable part of the total variance resides among subspecies (59.6%) Within-population variability also differed considerably. Percentage polymorphic RAPD loci and Lynch and Milligan within-population gene diversity estimates showed relatively high values for some species close to the geographic centre of origin in Central Asia, e.g.H. tibetana and the putatively hybridogenousH. goniocarpa. Spatial autocorrelation analyses performed on 12 populations ofH. rhamnoides revealed positive autocorrelation of allele frequencies when geographic distances ranged from 0 to 700 km, and no or negative autocorrelation at higher distances. At distances between 700 and 1900 km, we observed deviations from the expected values with strongly negative autocorrelation of allele frequencies. A corresponding relationship between geographic and genetic distances could not be found when the analysis instead was based on one population from each of 8 species.     

Leaf essential oils and their application in systematics of Juniperus excelsa complex in Iran

The composition of the leaf essential oils of ten populations of Juniperus excelsa complex in Iran are reported and compared. Hydrodistillation yielded in 0.08%–3.78% dry weight (v/w) clear oils among populations studied. The oils are mostly dominated by α- Pinene, Limonene and β-Myrcene. The simplest oil was found in Qushchi (J. polycarpos var. polycarpos) with 25 components and the most complex oil was found in Lushan1 (J. polycarpos var. polycarpos) with 92 components. Quantitative data is presented for 164 components were found. The southern populations, Fasa and Khabr, show differences in composition of essential oils compared with other populations so that Borneol and isopinocamphone are diagnostic for them. These populations have been identified as Juniperus seravschanica in southeast of Iran and hybrid samples between J. polycarpos and J. seravschanica in southwest of Iran whereas other populations, as is confirmed in this study, are J. polycarpos. It is confirmed by essential oils data that there is no J. excelsa in Iran.     

Comprehensive genetic discrimination of Leonurus cardiaca populations by AFLP,ISSR, RAPD and IRAP molecular markers

Leonurus cardiaca is well known for its medicinal importance. In this investigation, genotypic characterization of this species from six eco-geographical regions of Iran was evaluated by four molecular techniques (AFLP, RAPD, ISSR and IRAP). A total of 899 polymorphic fragments were detected by used molecular markers (AFLP = 356, RAPD = 325, ISSR = 113 and IRAP = 105) with an overall average polymorphism of 81.24 %. Genetic variation calculated using Shannon’s Information index (I) and Nei’s gene diversity index (H) showed high genetic diversity in studied germplasm. Also, analysis of molecular variance showed high genetic variation among (55 %) and within populations (45 %). UPGMA dendrogram constructed from combined data of molecular markers distinguished studied populations in accordance with the results obtained by each marker which all individuals were clearly differentiated into two major clusters. The correlation coefficients were statistically significant for all marker systems with the highest correlation between similarity matrixes of RAPD and ISSR markers (r = 0.82). The present results have an important implication for L. cardiaca germplasm characterization, improvement, and conservation. Furthermore, the characterized individuals exhibited a great deal of molecular variation and they seem to have a rich gene pool for breeding programs.