Juan Pedro Martín

Genetic relationships among species of the genus Diplotaxis (Brassicaceae) using inter-simple sequence repeat markers

Abstract Inter-simple sequence repeat (ISSR) amplification was evaluated for its applicability as a genetic marker system to establish relationships among ten Diplotaxis species. ISSR amplification generated multiple banding profiles with the 12 primers from all DNA samples, with an average of 41.2 fragments per primer. This average was clearly higher for the 5´ triple-anchored primers than for other primers. The banding profiles were highly repeatable across separate PCR runs. DNA mixing procedures were found to be appropriate strategies to generate banding patterns representative of each species studied. Similarity values were calculated considering 494 ISSR bands, and a dendrogram was constructed based on the similarity matrix. The ten Diplotaxis species were clustered into two major groups. The first group consists of five species, Diplotaxis tenuifolia and Diplotaxis cretacea, and Diplotaxis muralis with their putative parents (D. tenuifolia and Diplotaxis viminea). In the second group three species are clustered that are closely related (Diplotaxis virgata, Diplotaxis catholica and Diplotaxis siettiana), in addition to Diplotaxis harra, and Diplotaxis erucoides, which has lowest similarity values with the rest of the species studied. The two groups defined in the present work may be concordant with the idea suggested by several authors of a biphyletic origin for Diplotaxis. The genetic relationships among the ten Diplotaxis species estimated by the polymorphism of ISSR markers are in agreement with those previously inferred by other morphological, biochemical and molecular data, indicating the reliability of the ISSR approach for this purpose.

A population genetic analysis of chloroplast DNA in wild populations of Prunus avium L. in Europe.

A population genetic study of chloroplast DNA was carried out in 23 wild populations of Prunus avium sampled from several European deciduous forests. An analysis of approx. 9% of the chloroplast genome detected mostly insertion–deletion mutations and one point mutation. In all, 16 haplotypes were detected. Six haplotypes were shared by two or more populations and 10 were unique. One haplotype was present in 21 of the 23 populations and 161 of 211 individuals, which probably indicates its ancient origin. The level of population subdivision, using unordered and ordered alleles, was low, GSTC=0.29 and NSTC=0.33, respectively. The difference between GSTC and NSTC is nonsignificant, indicating an absence of correlation between haplotype phylogeny and geographical distribution. The absence of phylogeographic structure in wild cherry may be attributed to long distance gene flow among populations by birds, animals and anthropogenic activities. The minimum-length spanning tree depicting the phylogenetic relationships between the haplotypes indicates the possible existence of two lineages represented by the haplotypes H3 and H4. The information about homogeneity or heterogeneity of populations in terms of haplotype constitution and detection of rare haplotypes in some populations will be useful for formulation of conservation and management strategies of wild cherry.

Chloroplast DNA diversity within and among populations of the allotetraploid Prunus spinosa L.

Abstract High chloroplast DNA (cpDNA) diversity was found within and among populations of Prunus spinosa sampled from seven European deciduous forests. A study of 12% of the total chloroplast genome detected 44 mutations, which were distributed over 24 haplotypes; four were common to two or more populations and the rest were unique haplotypes. The most-abundant and widely distributed haplotype was H2 (frequency = 41% approximately). Six of the seven populations were polymorphic. All of the six polymorphic populations had ”private” haplotypes (frequency < 5%) in addition to common haplotypes. The UPGMA dendrogram demonstrated a correlation between populations and their geographical locations. The total diversity was high (hT = 0.824) and a major portion of it was within populations (hs = 0.663). The level of population subdivision for unordered alleles was low (GST = 19.5%) and for ordered alleles was lower (NST = 13.6%). No phylogeographic structure could be demonstrated in the present geographical scale. High polymorphism in the cpDNA of P. spinosa has to be considered carefully when planning phylogenetic studies involving this species.

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.

Population genetic analysis of European Prunus spinosa (Rosaceae) using chloroplast DNA markers

Chloroplast DNA diversity in Prunus spinosa, a common shrub of European deciduous forests, was assessed using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Thirty-two haplotypes were detected in 25 populations spread across the European continent. Ten haplotypes were shared by two or more populations, and 22 were private. The major proportion of the total cpDNA diversity (H(T) = 0.73) was located within the populations (H(S) = 0.49), and differentiation between populations was low (G(ST) = 0.33) compared with other forest species. Haplotype diversity was higher in southern Europe than in northern Europe, indicating probable localization of glacial refugia in southern Europe. The minimum-length spanning tree of haplotypes showed incongruency between the phylogeny of haplotypes and their geographic locations. This might be the result of intensive seed movements following recolonization, which thereby erased the phylogeographic structure in P. spinosa.

Genetic variation in the endemic and endangered Rosmarinus tomentosus Huber-Morath & Maire (Labiatae) using RAPD markers

Rosmarinus tomentosus Huber-Morath & Maire, an endemic species of southern Spain, is critically endangered as a consequence of habitat destruction by anthropogenic activities. Random amplified polymorphic DNA (RAPD) markers were used for initial evaluation of genetic variation in this species; among zones, among populations (within zones and independently of zones), and among individuals (within populations and zones). The eight primers used to study five populations provided 126 reliable bands, of which 109 (86%) were polymorphic. Analyses of the RAPD markers conducted using different approaches (cluster analysis, Shannon’s diversity measure and analysis of molecular variance-AMOVA) showed similar results concerning genetic structure of populations of R. tomentosus. The three zones studied, and their corrresponding populations, were clearly defined through UPGMA analysis. AMOVA analyses indicated that 18.0% of the total genetic variation was attributable to differences among zones. When total variance was partitioned without considering the zonal distribution of the populations, 34.1% was attributable to differences among populations, and 65.9% to differences among individuals within populations. Similar results were obtained using Shannon’s information measure. Significant genetic distances (Phist values) were obtained between any two populations. This strong genetic differentiation among zones, populations and subpopulations may be explained by geographical distances and limited gene flow. This study will be of considerable help during subsequent formulations of conservation strategies for the endangered species R. tomentosus.

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.

Chloroplast and nuclear DNA studies in a few members of the Brassica oleracea L. group using PCR-RFLP and ISSR-PCR markers: a population genetic analysis

Abstract.A population genetic analysis of chloroplast and nuclear DNA was performed covering nine wild populations of Brassica oleracea. Three members of the n = 9 group, all close to B. oleracea, Brassica alboglabra Bailey, Brassica bourgeaui (Webb) O. Kuntze and Brassica montana Pourret, were also studied to better understand their relationship with B. oleracea. Chloroplast DNA was analysed using the PCR-RFLP (polymerase chain reaction – restriction fragment length polymorphism) method. The ISSR-PCR (inter-simple sequence repeat – polymerase chain reaction) technique was adopted to study nuclear DNA. Twelve primer pairs of chloroplast DNA showed very good amplification. The amplified product of each primer pair, digested by three restriction enzymes, revealed no variation of cpDNA among the taxa studied. This indicates they may have the same chloroplast genotype. Seven selected ISSR primers have detected genetic variation, both within and among the populations/taxa surveyed. The information obtained on the intra- and inter-populational genetic diversity of wild populations of B. oleracea neatly defined the individual plants. It could provide important guidelines for backing management and conservation strategies in this species. The study confirms a close relationship between B. alboglabra, B. bourgeaui and B. montana, which is parallel to their morphological similitude.

Chloroplast DNA study in wild populations and some cultivars of Prunus avium L.

Abstract The PCR-RFLP technique was used to detect chloroplast DNA diversity in wild populations of Prunus avium from five European deciduous forests and some cultivars. A study of 10.8% of the total chloroplast genome detected eight insertion-deletion (indel) mutations, distributed over 12 haplotypes. Six haplotypes (H1, H2, H3, H4, H5 and H6) were found in wild populations and eight (H2, H6, H7, H8, H9, H10, H11 and H12) in the cultivars. Only two haplotypes (H2 and H6) are shared by the wild populations and the cultivars. The most-abundant and frequent haplotype in wild populations is H2 (frequency=78%). The wider geographical distribution along with the high frequency reflects its ancient origin. Of the five populations, three are polymorphic. Populations GA (Scotland) and KE (Germany) have unique haplotypes. The total cpDNA diversity in wild populations is hT=0.40, and a major portion of it is within populations (hS=0.37). The genetic differentiation among populations was low (GSTC=0.08) and no genetic structure among wild populations was observed. A minimum-length spanning tree, demonstrating relationships among the haplotypes in wild populations, indicated two possible chloroplast lineages. The ten identified cultivars were represented by seven haplotypes; this result proposes the possible utilisation of the PCR-RFLP technique for the characterisation of sweet cherry cultivars. The cpDNA diversity in P. avium should be considered carefully for phylogenetic studies involving this species.

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.