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The common fig tree (Ficus carica L.) is a Mediterranean crop with problematic cultivar identification. The recovery and conservation of possible local varieties for ecological production requires the previous genetic characterization of the available germplasm. In this context, 42 lines corresponding to 12 local varieties and two caprifigs, in addition to 15 reference samples have been fingerprinted using 21 SSR markers. A total of 77 alleles were revealed, detecting a useful level of genetic variability within the local germplasm pools. UPGMA clustering analysis has revealed the genetic structure and relationships among the local and reference germplasm. Eleven of the local varieties could be identified and defined as obtained clusters, showing that SSR analysis is an efficient method to evaluate the Andalusian fig tree diversity for on-farm conservation.

Activity levels of oxidative stress-related enzymes in the root apoplast during the interaction of WR315 (resistant) and JG62 (susceptible) chickpeas (Cicer arietinum L.) with the highly virulent race 5 of Fusarium oxysporum f. sp. ciceris were compared. Because this fungus develops asymptomatic infections in the chickpea root cortex in both susceptible and resistant plants, but only intrudes into the root xylem in the susceptible variety, the interactions were compared at three specific stages during disease development in JG62: (i) before symptom development (10 days after inoculation); (ii) at the time of appearance of the first disease symptoms (15-17 days after inoculation) and (iii) when all plants had developed disease symptoms (20-22 days after inoculation). Diamine oxidase (DAO), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol-dependent peroxidase and superoxide dismutase (SOD), but not catalase (CAT), were found in the apoplast of chickpea roots. In terms of APX activity, infection by the pathogen caused a different response in the incompatible compared to the compatible plant. In the case of GR, SOD and DAO activities, the pathogen caused the same response, but it developed earlier (i.e. GR and SOD) or to higher levels (i.e. DAO) in the incompatible interaction. Expression of apx, cat, sod, lipoxygenase (lox) and actin genes was also analysed in infected roots. Infection by F. oxysporum f. sp. ciceris race 5 only caused a significant change in the root expression of lox and actin genes. This up-regulation was earlier (lox) or higher (actin) in the incompatible than in the compatible interaction. Thus, changes in oxidative metabolism differ in compatible and incompatible interactions in Fusarium wilt of chickpea.

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70-80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.

A selection of 51 barley Sequence-Tagged Sites (STSs) were studied for their utility in Hordeum chilense. They included four primer sets from wheat origin and six primer sets from oat origin. Forty-four primer pairs amplified H. chilense products consistently. Five primer pairs were suitable for studying the introgression of H. chilense in wheat because they amplified H. chilense products of distinct size. Six of the STSs showed polymorphism between different H. chilense accessions. The results showed that barley STSs could be useful for the genetic characterization of H. chilense, tritordeums and derived introgression lines.

Hordeum chilense is a wild relative of H. vulgare, cultivated barley, that has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. These amphiploids-named generically x Tritordeum-have been tested under field conditions, and one of them, the hexaploid tritordeum obtained following chromosome doubling of the hybrid H. chilense x T. turgidum, shows traits of interest inherited from the barley parent. Of great interest is the allelic variation observed in the endosperm storage proteins and their influence on the breadmaking and malting quality of tritordeum. We report here two mRNA (cDNA) sequences for a gamma-3 hordein from two accession lines of H. chilense, H1 and H7, and their characterization by quantitative real time (QRT)-PCR in the developing endosperm. Sequences were obtained by rapid amplification of cDNA ends and "edge-to-edge" amplification of open reading frames from cDNA of H. chilense. Eight putative single nucleotide polymorphisms and one codon insertion were identified in the sequences of the H1 and H7 gamma-3 hordeins. The deduced amino acid sequences showed similar features to that of the gamma-3 hordein and gamma-gliadins from barley and wheat, respectively. While the repetitive motif (PQQQPF) is similar to that of the gamma-3 hordein from H. vulgare, there are 19 motif repeats in H. vulgare, whereas H. chilense shows 15 tandem repeats. The transcription of the genes encoding for the gamma-3 hordein were monitored by QRT-PCR: in both lines maximum transcription occurred 12 days after flowering.

When developing SCARs by sequencing RAPD markers useful for olive variety identification, one RAPD sequence of 407 bp has been identified that shows significant DNA homology with more than 160 retrotransposon-like sequences. A generally coherent phylogenetic tree has been constructed based on the homologous retrotransposon-like sequences, reflecting genetic distances in 35 species belonging to eight kingdoms. The implications of this finding in the development of SCAR markers are discussed. In addition, specific oligonucleotide primers have been developed to amplify the DNA region and have a practical application as an internal amplification control in SCAR-based multiplexed PCRs. To our knowledge, this is the first report of a retrotransposon-like element in olive., Financial support from the CICYT andINIA, Spain (Projects OLI96–2184 and CAO98–001-C3–2), isalso acknowledged.

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described., Financial support from the Spanish ‘Ministerio de Ciencia y Tecnología’ ('Ramón y Cajal' Program and project AGL2002‐04079‐C02‐02 from DGI) is gratefully acknowledged.

The transferability of microsatellite markers developed for olive cultivars ( Olea europaea L.) has been tested and confirmed in the Olea complex. Thirty two genotypes, belonging to different taxa of the genus Olea, have been analyzed with four olive SSRs. Positive amplifications at all loci were obtained in 13 taxa (at least one accession per species). Sixty seven different alleles have been detected at the four loci analyzed. Polymorphic products have been observed at the inter- and intra-species level. Some SSR loci have shown multiple amplification products in some species. The high number of unique alleles has allowed the unambiguous discrimination of most accessions. Similarity coefficients and relationships among the Olea taxa have been calculated based on SSR amplification results. The reliability of SSRs as markers for intra-species variability evaluation has been confirmed while their use to explore relationships at the inter-species level is discussed, being dependent on the locus analyzed.

[EN] A range of single tagged site (STS), simple sequence repeat (SSR), and sequence-characterized amplified region (SCAR) markers were screened for their utility in detecting Hordeum vulgare and H. chilense chromosomes in a wheat background. PCR conditions were optimized for specific amplification of the targeted sequences and to avoid cross-species amplification. Two H. vulgare derived STSs, six H. vulgare derived SSRs, and nine H. chilense derived SCARs were usable for the detection of five H. vulgare and three H. chilense chromosomes by direct ethidium bromide staining of the PCR products in test tubes, avoiding the more costly and time-consuming DNA electrophoresis step. The practical application of the method is illustrated by the identification of a monotelosomic substitution of H. vulgare chromosome 6HS in tritordeum and a monosomic addition of H. chilense chromosome 6Hch in durum wheat.Key words: GISH, Hordeum chilense, Hordeum vulgare, marker-assisted selection, SSR, Tritordeum., [FR] Un ensemble de marquers de type STS (« single-tagged-site »), microsatellite (SSR) et SCAR (« sequence-characterized amplified region ») ont été examinés quant à leur utilité potentielle pour la détection de chromosomes duHordeum vulgareet duH. chilensedans un fond génétique du blé. Les conditions PCR ont été optimisées pourl’amplification spécifique des séquences ciblées et pour éviter l’amplification croisée entre espèces. Deux marqueursSTS dérivés duH. vulgare, six microsatellites dérivés duH. vulgareet neuf SCAR dérivés duH. chilensepermettaientde détecter cinq chromosomes duH. vulgareet trois chromosomes duH. chilensepar simple détection des ampliconsau bromure d’éthidium dans des tubes réactionnels, évitant ainsi l’étape plus longue et coûteuse de l’électrophorèse.L’intérêt pratique de cette méthode est illustré via l’identification d’une substitution monotélosomique du chromosome6HS duH. vulgarechez une lignée issue d’un croisement blé-orge (tritordeum) ainsi que par l’identification d’une ad-dition monosomique du chromosome 6HchduH. chilensechez le blé durum., Financial support from the Minesterio de Ciencia y Tecnologia, Spain, the Comisión Interministerial de Ciencia yTecnologia (CICYT), Spain (project numbers AGF98-0945-CO2-01 and AGF99-1036-CO2-02), and the Spanish–British Acciones Integradas program (concerted action HB1998-0199) is acknowledged.

RAPD markers generated by mixtures of two different 10-mer primers were developed for eight different olive cultivars used as parental lines in olive-breeding programs. Two RAPD bands were converted into dominant SCAR markers by direct sequencing of the RAPD products, avoiding the costly and time-consuming cloning step. The SCARs generated have maintained the original RAPD polymorphism among the cultivars and segregated according to Mendelian inheritance. Preliminary results suggest the use of the SCAR SCOeMS-2 for the marker-assisted selection of the high flesh/stone ratio. This strategy provides a rapid method for the characterization of RAPD markers and for the development of PCR-based markers with applications in olive mapping, paternal testing and germplasm characterization. The use of these markers in multiplexed PCRs, and the direct ethidium bromide detection of the PCR products in the test tube, facilitate their efficient and reliable breeding applications., Financial support from the CICYT andINIA, Spain (Projects OLI96-2184 and CAO98-001-C3-2), is acknowledged.

The first genetic map of the wild South Ameri- can barley species Hordeum chilense is presented. The map, based on an F2 population of 114 plants, contains 123 markers, including 82 RAPDs, 13 SSRs, 16 RFLPs, four SCARs, two seed storage proteins and two STS markers. The map spans 694 cM with an average distance of 5.7 cM between markers. Six additional SSRs and seven additional SCARs which were not polymorphic were assigned to chromosomes using wheat/H. chilense addition lines. Polymorphisms were revealed by 50% of the RAPD amplifications, 13% of wheat and barley SSR primers, and 78% of the Gramineae RFLP anchor probes. The utility of SSR and RFLP probes from other Gramineae species shows the usefulness of a comparative approach as a source of markers and for aligning the genetic map of H. chilense with other species. This also indicates that the overall structure of the H. chilense linkage groups is probably similar to that of the B and D genomes of wheat and the H genome of barley. Applications of the map for tritordeum and wheat breeding are discussed., Financial support from the Spanish-Britishconcerted action, HB1998–0199, and from the CICYT, Spain (Project number AGF98–0945-CO2–01), is gratefully acknowl-edged.

A survey of Gramineae markers was carried out with the aim of developing cost-effective methods for the molecular analysis of Miscanthus species. Ten out of twenty Gramineae RFLP probes from ”anchor” sets hybridized well to Miscanthus DNA while all 15 maize probes tested cross-hybridized successfully, showing similar patterns in both species. Cross-taxa amplification of maize microsatellite primers was then tested. This showed that 57 out of 76 (75%) give highly reproducible amplification with Miscanthus DNA. Amplification products differed in size from those in maize but there was no bias toward higher or lower molecular weights. Microsatellite polymorphism produced by 17 primer pairs was studied in detail in a panel of 11 Miscanthus clones belonging to the species Miscanthus sinensis, Miscanthus sacchariflorus, Miscanthus ×giganteus and Miscanthus condensatus. Intra- and inter-specific length polymorphisms were frequent between the tested Miscanthus clones with length polymorphisms being found for all primer pairs, detecting 3–22 alleles. Polymorphism information content (PIC) values for microsatellites ranged from 0.48 to 0.94 with an average of 0.83. Species-specific amplicons were produced by two microsatellites. Genetic similarity coefficients of the Miscanthus clones ranged from 0.35 to 0.92, with an average of 0.57. Five polymorphisms were studied in a segregating population, where they showed Mendelian inheritance. In addition, two microsatellite markers mapping 1.3-cM apart on maize chromosome 7 were linked in Miscanthus at an estimated distance of 8 cM, suggesting collinearity. The high transferability of microsatellite markers from maize will enhance the power and resolution of genome analysis in Miscanthus., Financial support from the EU (Project num-ber FAIR-CT98–3571) and from the Spanish-British concerted ac-tion, HB1998–0199, are gratefully acknowledged.

RAPD markers were developed for octoploid×Tritordeum (amphiploid Hordeum chilense×Triticum aestivum) and its parents. Addition lines were used to identify specific RAPD markers for the Hordeum chilense chromosomes detectable in a wheat background. Twelve RAPD fragments have been cloned, sequenced and converted into STS markers. Eleven of these STSs have maintained both the chromosome specificity and the possibility of detection in a wheat background. The use of these markers in multiplexed PCRs facilitates both the efficient and reliable screening of new addition lines as well as the monitoring of introgression of H. chilense in bread and durum wheat., Th first author acknowledges the Consejo Su-perior de Investigaciones Cientificas''for the award of a fellowship .Financial support from the CICYT, Madrid, Spain (Project num-bers AGF95-0964-C02-01 and AGF98-0945-C02-01) is also ac-knowledged.

We report the development of microsatellites or simple sequence repeats (SSRs) in the olive tree (Olea europaea L.). Forty three positive clones obtained by the screening of a GA-enriched genomic library were sequenced and primers were designed for 13 microsatellite loci. Five primer pairs amplified polymorphic products of the expected size range. SSR polymorphism was explored in a set of 46 olive cultivars. A total of 26 alleles were detected for the five loci. Heterozygosity ranged from 0.46 to 0.71. Ninety one per cent of the cultivars had unique multilocus genotypes. Microsatellite segregation was studied in a complex population from a cross between the commercial cultivars ’Leccino’ and ’Dolce Agogia’.