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With the development of next-generation sequencing technology and bioinformatics tools, the process of assembling DNA sequences has become cheaper and easier, especially in the case of much shorter organelle genomes. The number of available DNA sequences of complete chloroplast genomes in public genetic databases is constantly increasing and the data are widely used in plant phylogenetic and biotechnological research. In this work, we investigated possible inconsistencies in the stored form of publicly available chloroplast genome sequence data. The impact of these inconsistencies on the results of the phylogenetic analysis was investigated and the bioinformatic solution to identify and correct inconsistencies was implemented. The whole procedure was demonstrated using five plant families (Apiaceae, Asteraceae, Campanulaceae, Lamiaceae and Rosaceae) as examples.

Landraces represent valuable genetic resources for breeding programmes to produce high-yielding varieties adapted to stressful environmental conditions. Although the common bean (Phaseolus vulgaris L.) is an economically important food legume for direct human consumption worldwide, common bean production in Croatia is based almost exclusively on landraces and there is no common bean breeding program. Information on phaseolin type and results of population structure and genetic diversity obtained by analysis of SSR and SNP markers, in combination with the morphological characterization of 174 accessions of 10 common bean landraces (morphotypes), enabled thorough classification of accessions. The accessions were classified into phaseolin type H1 (“S”) of Mesoamerican origin and phaseolin types H2 (“H” or “C”) and H3 (“T”) of Andean origin. By applying distance- and model-based clustering methods to SSR markers, the accessions were classified into two clusters at K = 2 separating the accessions according to the centres of origin, while at K = 3, the accessions of Andean origin were further classified into two clusters of accessions that differed in phaseolin type (H2 and H3). Using SNP markers, model-based analysis of population structure was performed, the results of which were consistent with those of SSR markers. In addition, 122 accessions were assigned to 14 newly formed true-type morphogenetic groups derived from three different domestication events: (1) Mesoamerican (H1A) (“Biser”, “Kukuruzar”, “Tetovac”, “Trešnjevac”), (2) Andean—indeterminate type (H2B1) (“Dan noć”, “Sivi”, “Puter”, ”Sivi prošarani”, “Trešnjevac”) and (3) Andean—determinate type (H3B2) (“Bijeli”, “Dan noć”, “Puter”, “Trešnjevac”, “Zelenčec”). The rest of the accessions could represent putative hybrids between morphogenetic groups. The differences between the true-type groups of accessions were further analysed based on nine quantitative traits, and the subsets of traits that best distinguish among centres of origin (A: Mesoamerican, B: Andean) and genetic groups (H1A, H2B1, H3B2) were proposed.

Ocimum basilicum L. exhibits a great variety of cultivars grown for various purposes. The aim of this research was to analyse the chemical composition of 85 O. basilicum accessions cultivated throughout the world using gas chromatography (GC/FID and GC/MS). All accessions were previously categorized into six morphotypes based on 24 morphological traits: True basil, Small-leaf, Lettuce-leaf, Purple basil A, Purple basil B and Purple basil C morphotype. A total of 77 volatiles were observed, with seven of them being in concentrations higher than 10% in at least one accession (1,8-cineole, linalool, linalool acetate, methyl chavicol, eugenol, trans-methyl cinnamate, trans-α-bergamotene). Based on the essential oil composition of 85 O. basilicum accessions, we propose the intraspecific characterization of O. basilicum into five chemotypes: (A) High-linalool, (B) Linalool/trans-α-bergamotene, (C) Linalool/methyl chavicol, (D) Linalool/trans-methyl cinnamate and (E) High-methyl chavicol chemotype. Groups based on morphological and biochemical traits do not necessarily coincide with one another. Due to great morphological and biochemical diversity, both morphological and chemical characterizations are crucial for the effective management of germplasm collections to facilitate the use of the accessions in future breeding programs.

By performing a high-resolution spatial-genetic analysis of a partially clonal Salvia brachyodon population, we elucidated its clonal architecture and seedling recruitment strategy. The sampling of the entire population was based on a 1 &times, 1 m grid and each sampled individual was genotyped. Population-genetic statistics were combined with geospatial analyses. On the population level, the presence of both sexual and clonal reproduction and repeated seedling recruitment as the prevailing strategy of new genets establishment were confirmed. On the patch level, a phalanx clonal architecture was detected. A significant negative correlation between patches&rsquo, sizes and genotypic richness was observed as young plants were not identified within existing patches of large genets but almost exclusively in surrounding areas. The erosion of the genetic variability of older patches is likely caused by the inter-genet competition and resulting selection or by a random die-off of individual genets accompanied by the absence of new seedlings establishment. This study contributes to our understanding of how clonal architecture and seedling recruitment strategies can shape the spatial-genetic structure of a partially clonal population and lays the foundation for the future research of the influence of the population&rsquo, s clonal organization on its sexual reproduction.

The essential oils of 25 populations of Dalmatian sage (Salvia officinalis L.) from nine Balkan countries, including 17 indigenous populations (representing almost the entire native distribution area) and eight non-indigenous (cultivated or naturalized) populations were analyzed. Their essential-oil yield ranged from 0.25 to 3.48%. Within the total of 80 detected compounds, ten (β-pinene, 1, 8-cineole, cis-thujone, trans-thujone, camphor, borneol, trans-caryophyllene, α-humulene, viridiflorol, and manool) represented 42.60 to 85.70% of the components in the analyzed essential oils. Strong positive correlations were observed between the contents of trans-caryophyllene and α-humulene, α-humulene and viridiflorol, and viridiflorol and manool. Principal component analysis (PCA) on the basis of the contents of the ten main compounds showed that four principal components had an eigenvalue greater than 1 and explained 79.87% of the total variation. Performing cluster analysis (CA), the sage populations could be grouped into four distinct chemotypes (A–D). The essential oils of 14 out of the 25 populations of Dalmatian sage belonged to Chemotype A and were rich in cis-thujone and camphor, with low contents of trans-thujone. The correlation between the essential-oil composition and geographic variables of the indigenous populations was not significant ; hence, the similarities in the essential-oil profile among populations could not be explained by the physical proximity of the populations. Additionally, the southeastern populations tended to have higher EO yields than the northwestern ones

The sweet chestnut (Castanea sativa Mill.) is a widely spread and important multipurpose tree species in the Mediterranean area, which has played an important role in human history. Natural events, such as glaciations, and human influence played significant roles in the distribution and genetic makeup of the sweet chestnut. In order to better understand how natural and human-mediated past events affected the current genetic diversity and structure of the sweet chestnut, we analysed populations from Central Europe and the western part of the Balkan Peninsula, utilizing ten polymorphic nuclear microsatellite markers. The study revealed the existence of three genetically and, to a large extent, geographically distinct and well-defined groups of sweet chestnut populations. Two not entirely separated groups of populations were detected in the northern part of the studied area and one in the southern. Our results indicate that the genetic structure of sweet chestnut populations in Central Europe and the western part of the Balkan Peninsula is the result of both natural colonization events and significant and lengthy human impact. Furthermore, it has been proven that the gene flow between cultivated/grafted trees’ and wild chestnut stands can influence their genetic structure. However, our results reveal that cultivated-to-wild introgression in the sweet chestnut is dependent on the close proximity of chestnut orchards and naturally occurring populations.

Helichrysum italicum (Roth) G. Don is a thermophilus perennial from family Asteraceae. Due to versatile biological activities and applications, the interest in Helichrysum italicum scientific research and use has increased in the last couple of decades. Consequently, due to the increased industrial demand its plantation farming experienced a huge expansion in Eastern European Mediterranean countries. The aim of this review article was to present the available data on Helichrysum italicum and to identify the gaps in the knowledge, and openings for future potential investigations and contributions to the topic. The review of the Scopus database was performed and scientific studies from 1990 to the most recent publications in 2019 were summarized. The article includes the last reviewed taxonomy, detailed description of morphological characteristics, geographical distribution, uses, chemical diversity, biological activity (antimicrobial, antiviral, antioxidant, anti-inflammatory, repellent, and insecticidal activity), and genetic diversity. Additionally, some other potential uses are described. The majority of the available scientific data is focused on the chemical composition of the essential oil, however, little is known about the cultivation management of Helichrysum italicum that includes sowing requirements, seedling production, plant density, fertilization and nutrition requirements, harvesting, diseases, insects, and weed control. Genetic information on Helichrysum italicum is also scarce, but needed for future breeding programs of Helichrysum italicum and selection of new cultivars.

Gutierrez, Natalia et al., Broomrapes are holoparasitic plants which infect faba bean (Vicia faba L.), among other legumes. Here, we aimed to identify and validate quantitative trait loci (QTLs) for broomrape resistance in the cross 29H × Vf136 and to investigate the existence of common and specific genomic regions against Orobanche crenata and O. foetida. A genetic map including 171 markers was constructed for QTL analyses. Field trials for O. crenata were conducted during three consecutive seasons at Córdoba (Spain) and in a single season at Kafr El-Sheikh (Egypt). QTL analysis for O. foetida was performed using data from a single season at Beja (Tunisia). Seven QTLs for O. crenata were identified. Oc7 on chromosome VI was detected over 3 years at Córdoba, explaining between 22 and 33 % of the phenotypic variation, which make it the most promising candidate for future marker-assisted breeding for broomrape resistance in faba bean. O. crenata QTLs identified at Kafr El-Sheikh did not co-localize with those identified in Córdoba. Environmental differences together with the diversity of parasitic populations between locations may account for the discrepancy. Three QTLs for O. foetida were detected. Co-localization of Oc8 and Of3 in chromosome V confirms a common resistance against both O. crenata and O. foetida, as previously reported. © 2013 Springer Science+Business Media Dordrecht., This work was co-financed by the EUFABA (QLK5-CT2002_02307), the GLIP-Grain Legumes Integrated Project (Food-CT-2004-506223) and by the Spanish Ministerio de Innovación y Ciencia (MICINN) projects: AGL2008-02305 and RTA2010-00059, co-financed with FEDER.

Olive breeding programs are only based on commercial cultivars as genitors. However, due to its wide variability, wild olive gremplasm could represent an interesting source of genes for the obtention of new cultivars. With purpose of evaluating the utility of wild olives in breeding programs, two open pollinated progenies (o.p.), originated from a wild olive (Alga05) and the main Spanish olive cultivar (Picual), were compared. Both progenies were analyzed by morphological descriptors, agronomical traits and SSR markers. The use of these 3 marker systems revealed great discrimination capacity, high level of morpho-agronomic and genetic diversity and their complementariness on the evaluation of these olive progenies. As expected, for most of morpho-agronomical traits, ‘Picual’ o.p. progeny showed superior values in comparison to the wild o.p. progeny. However, ‘Alga05′ wild olive progeny was more vigorous, with shorter juvenile period and more abundant flowering than ‘Picual’ o.p. For both progenies, PCA showed strong association between different agro-morphological traits (fruit and stone dimensions in wild olive progeny and fruit trait with oil content in ‘Picual’ progeny) which could facilitate the selection of the most appropriate traits for further evaluations, increasing thus, the efficiency of olive breeding programs. Our results indicate that the use of wild olive as genitors in breeding programs may be useful for generating new genotypes with interesting characters.

Dalmatian pyrethrum (Tanacetum cinerariifolium (TREVIR.) SCH.BIP.) is a plant species endemic to the east Adriatic coast. The bioactive substance of Dalmatian pyrethrum is a natural insecticide, pyrethrin, a mixture of six active components (pyrethrins I and II, cinerins I and II, and jasmolins I and II). The insecticidal potential of pyrethrin was recognized decades ago, and dried and ground flowers have traditionally been used in Croatian agriculture and households. A total of 25 Dalmatian pyrethrum populations from Croatia were studied to determine the pyrethrin content and composition, and to identify distinct chemotypes. The total pyrethrin content ranged from 0.36 to 1.30% (dry flower weight; DW) and the pyrethrin I/pyrethrin II ratio ranged from 0.64 to 3.33%. The statistical analyses revealed that the correlations between the percentage of pyrethrin I and of all the other components were significant and negative. The total pyrethrin content was positively correlated with the percentage of pyrethrin I and negatively correlated with cinerin II. The multivariate analysis of the chemical variability enabled the identification of five chemotypes among 25 Dalmatian pyrethrum populations. The chemical characterization of indigenous Dalmatian pyrethrum populations may serve as a good background for future breeding and agricultural exploitation.

This study presents the development of an enhanced map in faba bean. The map contains 258 loci, mostly gene-based markers, organized in 16 linkage groups that expand 1,875 cM, with an average inter-marker distance of 7.26 cM. The combination of EST-derived markers with a number of markers physically located or previously ascribed to chromosomes by trisomic segregation, allowed the allocation of eight linkage groups (229 markers), to specific chromosomes. Moreover, this approach provided anchor points to establish a global homology among the faba bean chromosomes and those of closely-related legumes species. The map was used to identify and validate, for the first time, QTLs controlling five flowering and reproductive traits: days to flowering, flowering length, pod length, number of seeds per pod and number of ovules per pod. Twelve QTLs stable in the 2 years of evaluation were identified in chromosomes II, V and VI. Comparative mapping suggested the conservation of one of the faba bean genomic regions controlling the character days to flowering in other five legume species (Medicago, Lotus, pea, lupine, chickpea). Additional syntenic co-localizations of QTLs controlling pod length and number of seeds per pod between faba bean and Lotus japonicus are likely. The new genetic map opens the way for further translational studies between faba bean and related legume species, and provides an efficient tool for breeding applications such as QTL analysis and marker-assisted selection. © 2012 Springer-Verlag., This research was developed at the Instituto de Investigación y For- mación Agraria y Pesquera (IFAPA) Córdoba, Spain belonging to the Campus de Excelencia Internacional Agroalimentario (ceiA3) and supported by the Spanish Ministerio de Ciencia e Innovación (MIC- INN) projects RTA-2007-00030, AGL-2008-02305 and RTA2010-00059, co-financed with FEDER.

Twenty-seven Ocimum basilicum cultivars were subjected to a chemical characterization of essential oil components by gas chromatography/mass spectrometry (GC/MS) and a genetic characterization using the amplified fragment-length polymorphism (AFLP) technique. Since the same 27 accessions had previously been classified into six morphotypes, these analyses allowed us to make detailed comparisons of chemistry, genetics, and morphology. The chemical composition and morphology of the studied cultivars appeared to have a strong genetic component. The AFLP analysis revealed a distinction between the green and purple morphotypes. The green morphotypes predominantly utilized the terpene biosynthetic pathway, while most purple morphotypes primarily utilized the phenylpropene biosynthetic pathway. The GC/MS analysis led to identification of 87 volatiles. Among the 27 cultivars, five chemotypes were identified. A detailed characterization of the essential oil constituents indicated the existence of both specific combinations of compounds and ‘private’ compounds with the potential to be used in many aspects of human life. The established relationship between a genetic profile, chemical composition, and morphology represents an important step in future breeding programs and in the cultivation of this species.

DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region (ITS) was performed to determine phylogenetic relationship between 49 isolates of rusts infecting grain and forage legumes. Isolates were collected from different hosts and distinct geographic origins and represent eight species of Uromyces: U. anthyllidis, U. appendiculatus, U. ciceris-arietini, U. minor, U. pisi, U. striatus, U. viciae-fabae and U. vignae. ITS sequences revealed length polymorphisms and variation in DNA sequence that were used to characterize phylogenetic relationships by maximum parsimony, maximum likelihood and Bayesian analyses which in general agreed revealing the presence of four clearly distinct clades. Clade one included the isolates causing rust on chickpea, fenugreek and alfalfa. Clade two was composed by rust isolates of field clover and pea plants, while the third clade was formed by bean and cowpea isolates. Clade four was the largest and included all the rust isolates infecting faba bean. Within this clade, the highly supported subclusters of U. viciae-fabae collected on Lens culinaris, U. viciae-fabae collected on Vicia sativa and U. viciae-fabae collected on Lathyrus palustris suggest an ongoing process of host specialization.

Genetic diversity and relationships between local cultivars and wild olive trees from three important Spanish olive-growing regions, Andalusia (South), Catalonia and Valencia (from Eastern Mediterranean Coastal area), were studied by means of eight SSR loci. Distinct allelic composition and heterozygosity levels were found in wild olive populations and cultivars. The observed patterns of genetic variation revealed: a) the independent clustering of Andalusian wild olives in a separate gene pool, b) the belonging of wild populations and most cultivars from Catalonia to another gene pool, c) the joined clustering of Andalusian and a set of Valencian cultivars in a third gene pool, and d) clustering of wild individuals from Valencia to the three different gene pools. These results suggest that wild populations of Andalusia may represent true oleasters, the ones from Catalonia may be feral forms derived from cultivar seed spreading, while the population of Valencia seems to be the most admixed one. The significant differentiation between Andalusian and most Catalonian cultivars is indicating an independent selection of olive cultivars in the two regions. The detection of a certain wild genetic background in some Catalonian and Valencian cultivars and the similarity found between wild and cultivated forms may suggest the use of local wild trees in olive domestication. The proposed scenario for the development of olive cultivars in Andalusia includes an empirical selection of outstanding local wild genotypes followed by various generations of crosses and various replanting campaigns, as well as possible introductions of ancestral cultivars. Therefore, our findings would lead us to support the hypothesis that the current diversity found in Spanish olive cultivars may be regionally differentiated and due to both, autochthonous and allochthonous origin. The information obtained in this work gives insights into the genetic resources of the main olive producing country, demonstrating that wild olive populations and local cultivars both represent potential sources of useful variability for olive breeding programs.

GC/MS was used to identify compounds of essential oils from seven Ocimum taxa (O. americanum L., O. basilicum L., O. campechianum Mill., O. x citriodorum Vis., O. kilimandscharicum Baker ex Gurke and three botanical varieties and cultivars of Ocimum basilicum L.: ‘Genovese’, var. difforme and var. purpurascens). Preliminary screening of their antibacterial activity was done against a number of common pathogens (Enterococcus faecalis, Enterococus faecium, Escherichia coli 0157:H7, Listeria monocytogenes, Listeria ivanovii, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermis) using the filter paper disc agar diffusion technique, while further analyses were done by modification of the disc diffusion method. A broad variation in the antibacterial properties of investigated essential oils was observed. E. coli 0157:H7 was inhibited by O. basilicum ‘Genovese’ essential oil, while Ocimum americanum and Ocimum x citriodorum essential oils were the most effective against Enterococcus faecalis, Enterococcus faecium, P. vulgaris, S. aureus and S. epidermis.

Genetic variation of 30 different Uromyces isolates collected on faba bean, lentil, common vetch, pea, chickpea, alfalfa, cowpea and lupin was studied. Random Amplified Polymorphic DNA markers were used showing clear differences among Uromyces species. Uromyces viciae-fabae isolates clustered according to the host, with a clear cluster including all U. viciae-fabae ex Vicia faba isolates. The U. viciae-fabae ex Lens culinaris isolate was the nearest to the cluster of U. viciae faba ex V. faba isolates, followed by U. pisi from Canada and U. viciae-fabae ex V. sativa. No association was found among molecular diversity and virulence or geographic origin within U. vicia-fabae ex V. faba isolates. Among the three U. pisi isolates considered, a great variability was observed and no grouping could be established. The most different isolate from the rest of species considered was U. striatus, followed by U. vignae. The two U. ciceris-arietini isolates clustered together and so did the two U. lupinicolus isolates., Financial support by the European Union project QLK5-CT2002-02307 and by the Spanish project AGL2005-01781 is acknowledged.

Orobanche foetida is widely distributed in western Mediterranean countries infecting wild legumes. Recently it has been reported in Morocco infecting common vetch, presenting a further constraint for legume production in this area. Comparative studies between Orobanche populations attacking wild species and Orobanche populations growing on crops from the same region may help to clarify whether a host specialisation process occurred. In the present study, the genetic variability of five O. foetida populations, four infecting wild plants and one infecting cultivated vetch in Morocco was characterised using amplified fragment-length polymorphism (AFLP) markers to elucidate the existing genetic relationship between populations and to suggest a potential origin for the recently detected vetch-infecting population. Analysis of molecular variance suggested the existence of genotypic differentiation among populations. The most genetic divergent population by cluster analysis was the population collected on Ornithopus sativus. The vetch-infecting O. foetida population was closer to the three populations infecting Scorpiurus muricatus. The possibility of the presence of a reservoir of diversity is balanced against direct genetic adaptation and completely new introduction. The potential for this Orobanche species to shift host and become a threat to agriculture exists and has to be taken into account for future legume breeding for these areas.

Aschochyta blight, caused by Mycosphaerella pinodes, is one of the most economically serious pea pathogens, particularly in winter sowings. The wild Pisum sativum subsp. syriacum accession P665 shows good levels of resistance to this pathogen. Knowledge of the genetic factors controlling resistance to M. pinodes in this wild accession would facilitate gene transfer to pea cultivars; however, previous studies mapping resistance to M. pinodes in pea have never included this wild species. The objective of this study was to identify quantitative trait loci (QTL) controlling resistance to M. pinodes in P. sativum subsp. syriacum and to compare these with QTLs previously described for the same trait in P. sativum. A population formed by 111 F6:7 recombinant inbred lines derived from a cross between accession P665 and a susceptible pea cultivar (Messire) was analysed using morphological, isozyme, RAPD, STS and EST markers. The map developed covered 1214 cM and contained 246 markers distributed in nine linkage groups, of which seven could be assigned to pea chromosomes. Six QTLs associated with resistance to M. pinodes were detected in linkage groups II, III, IV and V, which collectively explained between 31 and 75% of the phenotypic variation depending of the trait. While QTLs MpIII.1 and MpIII.2 were detected both for seedlings and field resistance, MpV.1 and MpII.1 were specific for growth chamber conditions and MpIII.3 and MpIV.1 for field resistance. Quantitative trait loci MpIII.1, MpII.1, MpIII.2 and MpIII.3 may coincide with other QTLs associated with resistance to M. pinodes previously described in P. sativum. Four QTLs associated with earliness of flowering were also identified. While dfIII.2 and dfVI.1, may correspond with other genes and QTLs controlling earliness in P. sativum, dfIII.1 and dfII.1 may be specific to P. sativum subsp. syriacum. Flowering date and growth habit were strongly associated with resistance to M. pinodes in the field evaluations. The relation observed between earliness, growth habit and resistance to M. pinodes is discussed.

Orobanche foetida Poir. is a parasitic plant widely distributed in the Western Mediterranean area. It typically parasitizes wild plants but has recently been described as an agricultural problem in legume crops in Tunisia. The pattern of genetic variation within and among O. foetida populations growing on chickpea and faba bean was analyzed by RAPD markers. The UPGMA cluster analysis based on Dice distance matrix showed a clear differentiation among O. foetida samples collected on chickpea and those on faba bean, suggesting a host-differentiation process. Although an AMOVA analysis revealed substantial internal variation among individuals within O. foetida populations (69.8%), there was a significant divergence between parasites on the two hosts considered (30.2%). Moreover, germination of O. foetida seeds collected on chickpea and faba bean in the presence of both host roots was studied. Germination percentages of O. foetida seeds varied depending on the host used both for collecting the seeds and evaluating the trait. According to these results, possible explanations for the origin of this new weedy parasite and the host differentiation process are discussed.

Some species of the genusOrobanche are among the most devastating parasitic weeds, causing extensive damage in agricultural fields. Considering the difficult control due to seed longevity in the soil, small seed size, high fecundity and a subterranean phase that allows them to parasitize the host before they emerge and become evident, the development of diagnostic markers is highly recommended. In our study we identified potential molecular diagnostic markers from the plastid genome in order to distinguish among the most importantOrobanche species attacking crops in Andalusia, the southern region of the Iberian Peninsula. The study has consideredO. crenata, O ramosa andO. cumana causing serious losses in legumes, solanaceous crops and sunflower fields, respectively, andO. minor that, although abundant in Andalusia, has to our knowledge not yet been found parasitizing agricultural hosts. We amplified a non-coding region from the plastid genome, studied sequence differences among the amplified fragments and digested those of the same length with selected restriction enzymes. Here, we propose a molecular protocol to distinguish the main parasitic plants in crop fields of southern Spain. Different applications such as identification ofOrobanche seeds in soil or crop seed lots are discussed in order to offer right crop recommendations or to prevent new infestation of parasite-free fields. Recommendations for further development of these diagnostic markers are also considered.

The Lilium carniolicum group consists of several taxonomically dubious taxa endemic to the European flora. Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA) were used to clarify both the delineation of, and relationships among, taxa in the group as well as to provide insight on the phylogenetic position of the group within the genus. Maximum parsimony, maximum likelihood and Bayesian analyses were in general agreement, with all taxa in the group being very closely related, and the entire group being monophyletic. L. pyrenaicum and L. pomponium are placed at the basal position in the group, while L. chalcedonicum is shown to be more closely related to L. carniolicum than previously thought. Our analyses suggested that L. albanicum and L. jankae are distinct from L. carniolicum, while no evidence was found to support the same separation for L. bosniacum.

Mullein (Verbascum) plants have extensive distribution and can grow in variable environmental conditions. Seed was collected from mullein plants grown at 4 locations, two metals contaminated and two metal uncontaminated areas. Genetic variability of progeny was examined. Populations collected from unpolluted areas were genetically more similar than those collected from polluted areas as revealed by RAPD and SSR markers and UPGMA analysis. The results indicate that there is genetic differentiation between examined populations and therefore they represent suitable material for further investigation of plant adaptation mechanisms to increased metal content. [Projekat Ministarstva nauke Republike Srbije, br. III43010]

A PCR approach with degenerate primers designed from conserved NBS–LRR (nucleotide binding site – leucine-rich repeat) regions of known disease-resistance (R) genes was used to amplify and clone homologous sequences from 5 faba bean (Vicia faba) lines and 2 chickpea (Cicer arietinum) accessions. Sixty-nine sequenced clones showed homologies to various R genes deposited in the GenBank database. The presence of internal kinase-2 and kinase-3a motifs in all the sequences isolated confirm that these clones correspond to NBS-containing genes. Using an amino-acid sequence identitiy of 70% as a threshold value, the clones were grouped into 10 classes of resistance-gene analogs (RGA01 to RGA10). The number of clones per class varied from 1 to 30. RGA classes 1, 6, 8, and 9 were comprised solely of clones isolated from faba bean, whereas classes 2, 3, 4, 5, and 7 included only chickpea clones. RGA10, showing a within-class identity of 99%, was the only class consisting of both faba bean and chickpea clones. A phylogenetic tree, based on the deduced amino-acid sequences of 12 representative clones from the 10 RGA classes and the NBS domains of 6 known R genes (I2 and Prf from tomato, RPP13 from Arabidopsis, Gro1–4 from potato, N from tobacco, L6 from flax), clearly indicated the separation between TIR (Toll/interleukin-1 receptor homology: Gro1–4, L6, N, RGA05 to RGA10)- and non-TIR (I2, Prf, RPP13, RGA01 to RGA04)-type NBS–LRR sequences. The development of suitable polymorphic markers based on cloned RGA sequences to be used in genetic mapping will facilitate the assessment of their potential linkage relationships with disease-resistance genes in faba bean and chickpea. This work is the first to report on faba bean RGAs.

The map locations and effects of quantitative trait loci (QTLs) were estimated for alpha-acid content in hop (Humulus lupulus L.) using amplified fragment length polymorphism (AFLP) and microsatellite marker (simple sequence repeat (SSR)) genetic linkage maps constructed from a double pseudotestcross. The mapping population consisted of 111 progeny from a cross between the German hop cultivar 'Magnum', which exhibits high levels of alpha-acids, and a wild Slovene male hop, 2/1. The progeny segregated quantitatively for alpha-acid content determined in 2002, 2003, and 2004. The maternal map consisted of 96 markers mapped on 14 linkage groups defining 661.90 cM of total map distance. The paternal map included 70 markers assigned to 12 linkage groups covering 445.90 cM of hop genome. QTL analysis indicated 4 putative QTLs (alpha1, alpha2, alpha3, and alpha4) on linkage groups (LGs) 03, 01, 09, and 03 of the female map, respectively. QTLs explained 11.9%–24.8% of the phenotypic variance. The most promising QTL to be used in marker-assisted selection is alpha2, the peak of which colocated exactly with the AFLP marker. Three chalcone synthase-like genes (chs2, chs3, and chs4) involved in hop bitter acid synthesis mapped together on LG04 of the female map. Saturation of the maps, particularly the putative QTL regions, will be carried out using SSR markers, and the stability of the QTLs will be tested in the coming years.Key words: Humulus lupulus L., genetic maps, alpha-acid content, QTLs.

Hop (Humulus lupulus L.) is a dioecious perennial plant native to the northern hemisphere cultivated for its use in the brewing industry. To investigate the genetic diversity present in wild hop accessions in comparison with cultivated hops, microsatellite marker variation was assessed at four loci in 124 accessions of wild (from Europe, Asia and from North America) and cultivated (varieties and breeding lines) hops. A total of 63 alleles were identified, with an average of 15.7 alleles per locus and an average PIC of 0.64 over four loci. The average number of alleles per locus in groups of accessions ranged from 5.75 to 8.30, with the highest number detected in groups of wild hops either of European (EU) or North American (NA) origin. Accessions from NA revealed the highest number of unique alleles indicating the high diversity present in this gene pool. Cluster analysis based on the DDor Dswdistance matrix divided accessions into 10 different clusters, which reflect the relationship among geographically diverse wild accessions and hop cultivars. The highest genetic differences were found between NA wild accessions, forming one distant cluster, and all the other accessions. The differentiation between European wild and cultivated accessions was revealed by PCoA based on the DDdistance matrix and by AMOVA results. Cultivated hops differ significantly from wild ones, although most of the variability was found within groups. The molecular variances within groups of cultivated and wild hops were homogeneous, suggesting that a similar level of molecular variability is found in both groups of accessions. The analysis of allele polymorphism and of allele sequences showed that hop germplasm can be differentiated to NA and EU geographic types according to the differences of allele sizes at three loci or by the specific microsatellite repeat type at one locus. The analysis also indicates the different evolutionary dynamics and complex mutations of microsatellite sequences within loci that can be followed in the two biogeographically separated germplasms.Key words: Humulus lupulus L., genetic diversity, germplasm, microsatellites, allele sequence variation.

Eighty-two Spanish olive cultivars from the World Germplasm Bank of the Centro de Investigación y Formación Agraria (CIFA) Alameda del Obispo in Cordoba (Spain) were analysed by RAPD markers to assess their genetic relatedness and to study patterns of genetic variation. The dendrogram based on unweighted pair group cluster analysis using Jaccard's index included two major groups that consisted mostly of cultivars from the southern and central part of Spain. Clustering together of cultivars from the Levante zone was also observed. The pattern of genetic variation among olive cultivars from three different Spanish zones (Levante, central and Andalusia) was analysed by means of the analysis of molecular variance (AMOVA). Although most of the genetic variability was attributable to differences of cultivars within each zone (95.88%), significant φ-values among zones (φst = 0.041; p < 0.001) suggested the existence of phenotypic differentiation. These results are consistent with the predominantly allogamous nature of Olea europaea L. species. Significant values of φst for the pair Andalusia/Levante indicate the presence of differentiation. The negative value of φst observed in the case of the Andalusia/central pair suggests that some varieties from central Spain are more similar to the Andalusian ones than to the varieties of their own geographic area, and vice versa.

The main objective of the present study was to locate the genomic regions responsible for ascochyta blight resistance in faba bean. Six QTLs were identified with the help of a linkage map constructed from a F(2) population from the cross between the inbred lines 29H (resistant) and VF136 (susceptible). Two pathogenically distinct Ascochyta isolates were used to study the genetic control against them and disease evaluations were performed separately on leaves and stems to investigate whether different genetic systems control resistance in each plant organ, as previously suggested. The six QTLs detected were named Af3 to Af8. Af3 and Af4 were effective against both Ascochyta isolates, Af5 was only effective against isolate CO99-01 while Af6, Af7 and Af8 were only effective against isolate LO98-01. Af3, Af4, Af5 and Af7 were revealed in both leaves and stems. By contrast, Af6 was only effective in leaves and Af8 only in stems. The validity and application of these results in a MAS program is discussed.

Chlorine and potassium content are important traits related to combustion quality of Miscanthus species. These traits were analysed in a cross between F(1.1) and F(1.7) entries of Miscanthus sinensis Anderss, both lines offspring of the cross between MS-90-2 and MS-88-110. Quantitative trait locus (QTL) analyses were performed on a previous linkage map constructed with the offspring cross mapping strategy. The mapqtl 4.0 package was used to perform QTL analyses. Six potential QTLs were detected with data collected over a 2-year period. Of these, four were associated with chlorine and two with potassium. These results could be used as an initial step to develop a marker-aided selection programme for biomass with low mineral content.

To identify how many chemotypes of Salvia officinalis exist in Montenegro, the chemical composition of the essential oils of 12 wild–growing populations was determined by GC–FID and GC/MS analyses. Among the 40 identified constituents, the most abundant were cis–thujone (16.98–40.35%), camphor (12.75–35.37%), 1, 8–cineol (6.40–12.06%), trans–thujone (1.5–10.35%), camphene (2.26–9.97%), borneol (0.97–8.81%), viridiflorol (3.46–7.8%), limonene (1.8–6.47%), alpha–pinene (1.59–5.46%), and alpha–humulene (1.77–5.02%). The composition of the essential oils under study did not meet the ISO 9909 requirements, while the oils of populations P02–P04, P09, and P10 complied with the German Drug Codex. A few of the main essential–oil constituents appeared to be highly intercorrelated. Strong positive correlations were observed between alpha–pinene and camphene, camphene and camphor, as well as between cis–thujone and trans–thujone. Strong negative correlations were evidenced between cis–thujone and alpha–pinene, cis–thujone and champhene, cis–thujone and camphor, as well as between trans–thujone and camphene. Multivariate analyses allowed the grouping of the populations into three distinct chemotypes, i.e., Chemotype A, rich in total thujones, Chemotype B, with intermediate contents of thujones, alpha–pinene, camphene, and camphor and high borneol contents, and Chemotype C, rich in camphor, camphene, and alpha–pinene. The chemotypes did not significantly differ in the total essential–oil content and the cis/trans–thujone ratio.

One of the most important postharvest diseases of peach and nectarine is brown rot caused by the fungus Monilinia laxa. Increasing concern related to the toxic effect of the pesticide residues on fruits has enhanced the interest for the control of postharvest pathogenic fungi using biologically active constituents of plants such as essential oils. Species from genus Ocimum are known to possess essential oils with antifungal and fungicidal properties. The present paper reports the results of essential oil composition of four basil taxa and preliminary screening of their antifungal activity against M. laxa. The greatest effectiveness was achieved by the essential oils from Ocimum basilicum var. purpurascens and Ocimum tenuiflorum, whose activity was high throughout the testing period of twenty-three days.

Numerous molecular systematic studies within Brassicaceae have resulted in a strongly improved classification of the family, as morphologically defined units at and above the generic level were often found to poorly reflect phylogenetic relationships. Here, we focus on tribe Alysseae, which despite its size (accounting for about 7% of all species) has only received limited coverage in previous phylogenetic studies. Specifically, we want to test phylogenetic hypotheses implied by current tribal and generic circumscriptions and to put diversification within tribe Alysseae into a temporal context. To this end, sequence data from the nrDNA ITS and two plastid regions (ndhF gene, trnL-F intergenic spacer) were obtained for 176 accessions, representing 16 out of 17 currently recognized genera of the tribe, and were phylogenetically analysed, among others, using a relaxed molecular clock. Due to large discrepancies with respect to published ages of Brassicaceae, age estimates concerning Alysseae are, however, burdened with considerable uncertainty. The tribe is monophyletic and contains four strongly supported major clades and Alyssum homalocarpum, whose relationships among each other remain uncertain due to incongruences between nuclear and plastid DNA markers. The largest genus of the tribe, Alyssum, is not monophyletic and contains, apart from A. homalocarpum, two distinct lineages, corresponding to sections Alyssum, Psilonema, Gamosepalum and to sections Odontarrhena and Meniocus, respectively. Clypeola, whose monophyly is supported only by the plastid data, is very closely related to and possibly nested within the second Alyssum lineage. Species of the genus Fibigia intermingle with those of Alyssoides, Clastopus, Degenia, and Physoptychis, rendering Fibigia polyphyletic. The monotypic genera Leptoplax and Physocardamum are embedded in Bornmuellera.

Pseudomonas syringae is the main pathogen responsible for bacterial blight disease in pea and can cause yield losses of 70%. P. syringae pv. pisi is prevalent in most countries but the importance of P. syringae pv. syringae (Psy) is increasing. Several sources of resistance to Psy have been identified but genetics of the resistance is unknown. In this study the inheritance of resistance to Psy was studied in the pea recombinant inbred line population P665 × 'Messire'. Results suggest a polygenic control of the resistance and two quantitative trait loci (QTL) associated with resistance, Psy1 and Psy2, were identified. The QTL explained individually 22.2 and 8.6% of the phenotypic variation, respectively. In addition 21 SSR markers were included in the P665 × 'Messire' map, of which six had not been mapped on the pea genome in previous studies. © 2011 Springer Science+Business Media B.V., Financial support by AGL2008-01239, Ministerio de Ciencia e Innovación GEN2006-27798-C6-3-E/VEG and Junta de Castilla y León ITACyL 2004/845 projects is acknowledged.

The present study reports the setting up of Diversity Arrays Technology (DArT) markers in olive (Olea europaea L.). Two genomic representations were generated using the PstI/. TaqI combination. A first one was aimed to cover the variability available at the World Olive Germplasm Bank (WOGB) and it was based on DNA from 87 olive cultivars carefully selected from the WOGB to represent the olive variability. The second one was obtained from DNA of the parents of a mapping population, 'Picual' and 'Arbequina', and was used to increase the number of markers segregating within them. A total of 2031 and 1630 markers were used for diversity and mapping analyses, respectively to test the utility of DArT markers in olive. A set of 62 cultivars was genotyped with olive-specific DArT markers. A dendrogram was constructed confirming the relationships among olive cultivars reported in previous works. Besides, DArT markers permitted the unambiguous discrimination of all 62 genotypes confirming thus their utility for identification studies, an important issue for management of germplasm collections. DArT markers also allowed the construction of a genetic map of olive using the population derived from the cross 'Picual'. ×. 'Arbequina' and the pseudo-testcross mapping strategy. This map will be used as a framework map to add new markers derived from olive sequencing originating from the project OLEAGEN. In conclusion, olive-specific DArT markers will enhance identification and genetic studies in olive given their high throughput and low cost per data. © 2011 Elsevier B.V.

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential-oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis-thujone, camphor, trans-thujone, 1,8-cineole, beta-pinene, camphene, borneol, and bornyl acetate) formed 78.1387.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and beta-pinene,beta-pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans-thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis-thujone from those rich in trans-thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis-thujone; trans-tujone, and camphor/beta-pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.

Most commercial basil cultivars belong to the species Ocimum basilicum L. Moreover, within O. basilicum species there are six main botanical varieties and cultivars (var. basilicum 'Genovese', var. basilicum 'Sweet Basil', var. difforme Benth., var. purpurascens Benth., 'Dark Opal' and var. thyrsiflorum /L./ Benth.) which are mainly found on the market. High levels of both morphological and chemical variability exist within the species due to the intra- and interspecific hybridization and long-term uses throughout the world. The aim of this work was to study the resolving power of morphological traits for reliable identification of basil accessions. Phenotypic dissimilarities between pairs of accessions were calculated and the UPGMA dendrogram was constructed. Six clearly defined clusters have been detected, giving a good representation of traditional taxonomic relationships. To compare the efficiency of the morphological traits in accession identification, confusion probability and discriminating power of each trait was calculated. Our results show that with a careful analysis and stringent selection of traits, morphological markers provide an inexpensive and reliable method for routine screening of a large number of accessions, in order to monitor and manage germplasm collections.

SummaryThe Croatian olive (Olea europaea L.) cultivar, ‘Lastovka’ is mainly used for oil production. While fruit yields are high and regular, the growing area is limited due to its sensitivity to low temperatures, with production concentrated on the South Dalmatian island of Korcula. Different morphological types have been observed during production. In order to determine the extent of intra-cultivar diversity, 47 individual ‘Lastovka’ trees were analysed for 11 morphological traits and assessed using AFLP markers, and compared to ten trees each of the cultivars ‘Oblica’ and ‘Drobnica’. Clear differentiation among cultivars was detected, and the results showed that both the morphological and molecular data were equally reliable for discriminating between cultivars. The existence of intra-cultivar diversity in ‘Lastovka’ was therefore confirmed at both the morphological and molecular levels, providing an opportunity for the selection of potentially superior clones.

In a previous study a genetic map had been developed using a RIL population derived from a cross between the Pisum sativum ssp. syriacum accession P665 and the P. sativum ssp. sativum cv. Messire. This population segregated for several agricultural important traits and was successfully used to identify QTLs (Quantitative Trait Loci) controlling resistance to Mycosphaerella pinodes and Orobanche crenata, earliness, root length and aerial biomass. However, this map contained only a few markers in common with the international pea consensus map, hampering comparison with other pea maps. The objective of this study was to incorporate a set of common transferable and reproducible markers into the P665 × Messire map to favour comparative mapping and QTL validation. Seventy-eight out of the 248 SSRs assayed resulted polymorphic in the parental lines. Thirty-eight of them, uniformly distributed all over the genome, were genotyped in the whole population and included in the map. This SSR enriched map allowed identification of six new QTLs (three for resistance to M. pinodes, two for resistance to broomrape and one for root length). Inclusion of the SSRs confirmed the homology of some of the QTLs identified in the population P665 × Messire with other QTLs associated with related traits in different pea genetic backgrounds. © 2011 Springer Science+Business Media B.V., Support by projects AGL2008-01239 and RTA2007-00030 is acknowledged. SF was funded by Juan de la Cierva Program of the Spanish MICINN.

DNA fingerprinting (AFLP) and chemical analyses of essential oils were utilized to define the extent of variation existing in the genus Ocimum. Research was carried out on 22 Ocimum accessions representing seven species. Concerning the essential oil composition of all investigated accessions, 115 compounds were identified. UPGMA cluster analysis, based on Euclidian distances of essential oil constituents between all pairs of accessions, showed four well-supported clusters (O. tenuiflorum, O. basilicum/O. africanum, O. basilicum, and O. americanum/O. africanum). Relating to the essential oil composition of all of the investigated accessions, 17 compounds were identified as the main ones, and according to them 13 chemotypes were determined. AFLP relationships were determined by neighbor-joining (NJ) cluster analysis based on Dice’s distance matrix and by maximum parsimony (MP) analysis. O. basilicum, O. americanum/O. africanum, O. tenuiflorum, and O. gratissimum represented four clusters supported with high bootstrap values. A neighbor-net diagram allowed the visualization of apparently conflicting data by revealing relationships between genotypes and chemotypes. Concerning the O. africanum species, two distinct chemotypes, geranial/neral (accession 11) and estragol (accession 10), have been established, while all the studied O. americanum accessions belong to the geranial/neral chemotype. This could be additional evidence that O. americanum is one of the parents of O. africanum. Furthermore, the fact that the O. africanum accession (10) as well as O. basilicum ‘Purpurascens’ and O. basilicum ‘Erevanskii’ accessions belong to the estragol chemotype supports the theory that O. africanum is one of the parents of these two O. basilicum accessions.