Jesús María Ortiz

The use of RAPD markers for identification of cultivated grapevine (Vitis vinifera L.)

Abstract Polymorphism among 31 grapevine accessions was studied utilizing random amplified polymorphic DNA (RAPD) markers. Fourteen decamer oligonucleotide primers were tested. All experiments were repeated three times. Primers could be grouped according to the polymorphism obtained as well as the constancy of results. Choosing the right primers, all varieties and some of the clones could be discriminated using reliable bands. Synonymies were confirmed, and a case of homonymy was detected. The use of RAPD markers as a technique for identification of grapevine varieties and clones can be encouraged.

Determination of relationships among autochthonous grapevine varieties (Vitis vinifera L.) in the Northwest of the Iberian Peninsula by using microsatellite markers

Fifty six grapevine varieties traditionally grown in the Northwest region of the Iberian Peninsula were analysed for six microsatellite loci, in order to determine the relationships among them as well as the plant material that should be collected and preserved in germplasm banks. Previous morphological and molecular results were taken into account for assessment of the existing synonymies among accessions from different European countries. Percent distribution of the main alleles was calculated. Multivariate analysis was carried out and similarities among the studied material were described and commented.

Morphological characterization of sweet and sour cherry cultivars in a germplasm bank at Portugal

Nine sweet cherry and eight sour cherry varieties located in a germplasm bank at Fundão, Portugal, were studied from the viewpoint of characterization. Most of them were autochthonous cultivars that have a high risk of extinction since at the present they are markedly minor varieties. Morphological characteristics were evaluated in different organs: crown and trunk of the trees, leaves, flowers and fruits, over a three consecutive years period. Statistical analyses were carried out in order to detect similarities between cultivars as well as the existence of synonymies. Qualitative characteristics of the fruits were scored in order to carry out the multivariate analysis. A dendrogram of the evaluated characters shows the marked differentiation between sour and sweet cherries and suggests the existing synonymies. Conservation of the autochthonous cultivars in the future is highly recommended.

Molecular and morphological characterization of a Vitis gene bank for the establishment of a base collection

A total of 621 accessions of Vitis vinifera L. from the Spanish gene bank at Alcalá de Henares (Madrid) were characterized using morphological descriptors, isoenzymes and microsatellites. Ampelographic evaluations included 27 morphometric plus 50 morphological characters. Of these twelve measurements or ratios were considered the most discriminant and 32 OIV descriptors were the most adequate for characterization because of their stability and objectiveness. Four isoenzyme systems provided 80 different patterns in these accessions. Analysis of six microsatellites in the studied samples resulted in 163 genotypes. The same microsatellite genotype was obtained for closely related varieties as in the case of mutations of the berry color, like Garnacha Blanca, Garnacha Gris and Garnacha Negra. Application of these characterization methods resulted in 177 accessions, which should be kept as a base collection, that represents less than 30% of the initial number.

Use of molecular markers in detection of synonymies and homonymies in grapevines (Vitis vinifera L.)

Abstract RAPD and microsatellites are used in the present study as molecular markers for characterisation of grapevine germplasm material. The studied varieties were located in two germplasm banks in Spain. Thirty nine accessions were included in the study, including some presumed synonymies and homonymies. Sixty six primers were used for the RAPD study. Twenty were selected for the multivariate analysis and grouping of the varieties. Seven varieties had a monotypic pattern. Twenty three accessions were also analysed with microsatellites with the VVMD7, VVS2, VVS5 and VVS29 loci, obtaining eight different patterns. The high discriminating ability of the RAPD analysis allowed all the studied varieties to be distinguished. A good concordance was obtained for both RAPD and microsatellites when the two analysis were carried out. The following synonymies were confirmed: Moristell and one of the accessions of Monastel; Maturana and Ribadavia; Concejon and one of the accessions of Monastrell; and most of the studied muscat varieties. Homonymies were detected for Miguel de Arco, Monastel, Monastrell and Turruntes. Both RAPD and microsatellites are considered as adequate molecular markers for characterisation of germplasm banks with the use of one or the other depending on the objectives of the study. When results are intended to compare with other laboratories or germplasm banks, microsatellites give simple and more comparable results.

Identification and relationships of accessions grown in the grapevine (Vitis vinifera L.) Germplasm Bank of Castilla y Leon (Spain) and the varieties authorized in the VQPRD areas of the region by SSR-marker analysis

A total of sixty-five accessions from the Spanish region of Castilla y León including those authorized in the VQPRD areas were characterized for six SSR loci. All the samples but one unknown were identified by comparison to other databases. Thirty synonymous samples and three cases of homonymy were found out, confirming in most cases ampelographic expectations. Five unique genotypes belonging to local varieties in risk of extinction were detected. Several parameters were calculated to assess the usefulness of the chosen loci in this work. A dendrogram representing the genetic similarities among the accessions was constructed using the neighbor-joining method to investigate possible parentage relationships in the sample, and to explain them from an historical and cultural point of view.

The use of isoenzymes in characterization of grapevines (Vitis vinifera, L.). Influence of the environment and time of sampling

Abstract Isoenzymes from grapevine woody stems and shoots were evaluated for their use in identification of varieties and clones. Plant extracts were separated by polyacrylamide gel electrophoresis. Isoenzyme analysis was carried out for esterases, peroxidases, catechol oxidase, glutamate oxalacetate transaminase and acid phosphatase. The plant material was grown and sampled at two localities in Spain, with different climatic conditions. Sampling was carried out bimonthly for two consecutive years in order to find out the influence of the environment and time of the year. Each isozyme system had a pattern defined by ‘fixed’ bands, that were always present at both localities and during the resting period of the plant (fall—winter). Esterases had a pattern with very few ‘fixed’ bands. Catechol oxidase was the most polymorphic system, thus being highly discriminating. The studied isoenzyme systems gave differences among varieties, although not among clones of the same variety. The use of this methodology for identification of grapevine plant material is discussed.

Potential of Cytisus and allied genera (Genisteae: Fabaceae) as forage shrubs

Abstract The chemical composition of the herbage produced by shrubs belonging to the genus Cytisus and allied genera (Cytisophyllum, Chamaecytisus, and Genista sect. Teline) was determined to appraise its potential value for forage. In their third year, eight accessions were assessed in a semi‐arid environment (400 mm annual rainfall) for seasonal variation of leaf/stem ratio, and contents of ashes, neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), and crude protein (CP). Fibre and crude protein concentration were similar to those of best‐quality shrubby legumes like Medicago sp. pi. Colutea sp. pi., or Chamaecytisus palmensis (ADF, 18–35%; CP, 14–24%). Genista monspessulana had low fibre and high protein contents, which were the most constant during the year. From the results obtained in this and the previous agronomic study, Part 1 of this series (Gonzalez‐Andres & Ortiz 1996), Genista monspessulana, Chamaecytisus podolicus, and C. supinus are recommended as potenti...

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

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

Effectiveness of AFLPs and Retrotransposon-Based Markers for the Identification of Portuguese Grapevine Cultivars and Clones

Grapevine germplasm, including 38 of the main Portuguese cultivars and three foreign cultivars, Pinot Noir, Pinot Blanc and Chasselas, used as a reference, and 37 true-to-type clones from the Alvarinho, Arinto, Loureiro, Moscatel Galego Branco, Trajadura and Vinhão cultivars were studied using AFLP and three retrotransposon-based molecular techniques, IRAP, REMAP and SSAP. To study the retrotransposon-based polymorphisms, 18 primers based on the LTR sequences of Tvv1, Gret1 and Vine-1 were used. In the analysis of 41 cultivars, 517 IRAP, REMAP, AFLP and SSAP fragments were obtained, 83% of which were polymorphic. For IRAP, only the Tvv1Fa primer amplified DNA fragments. In the REMAP analysis, the Tvv1Fa-Ms14 primer combination only produced polymorphic bands, and the Vine-1 primers produced mainly ISSR fragments. The highest number of polymorphic fragments was found for AFLP. Both AFLP and SSAP showed a greater capacity for identifying clones, resulting in 15 and 9 clones identified, respectively. Together, all of the techniques allowed for the identification of 54% of the studied clones, which is an important step in solving one of the challenges that viticulture currently faces.