Isaura Castro

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.

Chloroplast Genome Diversity in Portuguese Grapevine (Vitis vinifera L.) Cultivars

Grapevine chloroplast (cp) DNA diversity was analysed for the first time through amplification and digestion of fragments of the large single copy (LSC) region by polymerase chain reaction–restriction fragment length polymorphism methodology and also by amplification of three microsatellite loci, previously described as polymorphic in grapevine. Thirty-eight grapevine cultivars collected mainly in the North of Portugal, including some neglected cultivars, four international cultivars (Chasselas, Muscat of Alexandria, Muscat of Hamburg and Pinot) and Vitis riparia and Vitis rupestris, were used in this study with the main goal of finding out their cpDNA diversity and compare the obtained results with previously published data on cultivars from other regions to ascertain their possible origin. Two different alleles were found in each of the three cpSSR loci. Allele variants of the three loci combined in a total of three different haplotypes (A, B and D). The most frequent haplotype, A, was previously reported as the most frequent in Iberian Peninsula and Occidental Europe. Haplotype B was unique to Rabigato, Muscat of Alexandria, V. riparia and V. rupestris. This haplotype was previously proposed to be an ancestral haplotype. Twenty-seven fragments of the LSC region of Vitis vinifera cpDNA were amplified and then digested with HinfI and TaqI restriction enzymes. Polymorphisms were found in the trnT-psbC (TC) and orf184-petA (OA) fragments. In the TC fragment, the polymorphism corresponds to a point mutation in a restriction site of TaqI and is only present in all cultivars with cpSSR haplotype D. In the OA fragment, a short deletion exclusive to the Rabigato cultivar was found. In this case, one sequence tagged site-based marker was developed and will be very useful in future phylogenetic and fingerprinting studies in a broader number of cultivars and in wild grapevine populations. Inference about the progenitors of the Touriga Franca cultivar is done. The present work supports and completes its origin as a descendent of the female and male parents, Marufo and Touriga Nacional.

European cowpea landraces for a more sustainable agriculture system and novel foods

BACKGROUND Genetic diversity is fundamental to breeding programs and consequently has an important role in obtaining new varieties. To properly use the genetic diversity present in germplasm collections, a good knowledge of the agro-morphological traits of each accession is needed. The aim of this study was to explore the production capacity of 24 cowpea landraces from southern Europe, through phenotypic characterization and evaluation in three different locations in Greece and Portugal. RESULTS Most qualitative parameters tested showed a high stability among the three locations. A wide difference was observed among the three locations with respect to number of days to flowering, ranging from 55 to 99 days. Quantitative traits showed a higher genotype × environment than genetic variance component. In general, an inverse relationship between σ2ge /σ2g ratio (where σ2ge is genotype × genotype interaction and σ2g is genotype impact) and heritability value was observed. Principal component analysis was able to group accessions based on their origin. The first two principal components explained 97.52% of variation, being the number of seeds per plant, plant height and seed protein content, the traits which contributed most to variability. CONCLUSION The results show that sufficient variation exists in different traits within landraces in the studied cowpea germplasm to pursue a breeding program. However, the quantitative traits showed a higher genotype × environment component.

Spontaneous variation regarding grape berry skin color: A comprehensive study of berry development by means of biochemical and molecular markers

Understanding grape berry development and the metabolism of different classes of compounds responsible for traits like berrys color is imperative to control and improve quality aspects of grapes. A colorimetric, biochemical and molecular characterization allowed the comprehensive description of the pigment-related characteristics of nine berry skin color somatic variants, belonging to four different varieties. Although the observed berry skin color variability was not fully explained by MybA locus, the phenolic profiles allowed inferring about specific interferences among the biosynthetic pathways. Data were consistent concerning that grapes showing cyanidin-3-O-glucoside as the major anthocyanin and flavonols with two substituent groups in the lateral B-ring are generally originated by a white ancestor. After retro-mutation, these grapes seem to keep the dysfunction on flavonoid hydroxylases enzymes, which negatively affect the synthesis of both flavonols and anthocyanins with three substituent groups in the lateral B-ring. Overall, the obtained results indicate that the color differences observed between somatic variants are not solely the result of the total amount of compounds synthesized, but rather reflect a different dynamics of the phenolic pathway among the different color variants of the same variety. CHEMICAL COMPOUNDS Gallic acid (PubChem CID: 370); Caftaric acid (PubChem CID: 6,440,397); Catechin (PubChem CID: 73,160); Epigallocatechin gallate (PubChem CID: 65,064); Quercetin-3-O-galactoside (PubChem CID: 5,281,643); Quercetin-3-O-glucoside (PubChem CID: 25,203,368); Malvidin-3-O-glucoside (PubChem CID: 443,652); Peonidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,849); Malvidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,988); Resveratrol-3-O-glucoside (PubChem CID: 25,579,167).

Genetic diversity and structure of Iberian Peninsula cowpeas compared to world-wide cowpea accessions using high density SNP markers

BackgroundCowpea (Vigna unguiculata L. Walp) is an important legume crop due to its high protein content, adaptation to heat and drought and capacity to fix nitrogen. Europe has a deficit of cowpea production. Knowledge of genetic diversity among cowpea landraces is important for the preservation of local varieties and is the basis to obtain improved varieties. The aims of this study were to explore diversity and the genetic structure of a set of Iberian Peninsula cowpea accessions in comparison to a worldwide collection and to infer possible dispersion routes of cultivated cowpea.ResultsThe Illumina Cowpea iSelect Consortium Array containing 51,128 SNPs was used to genotype 96 cowpea accessions including 43 landraces and cultivars from the Iberian Peninsula, and 53 landraces collected worldwide. Four subpopulations were identified. Most Iberian Peninsula accessions clustered together with those from other southern European and northern African countries. Only one accession belonged to another subpopulation, while two accessions were ‘admixed’. A lower genetic diversity level was found in the Iberian Peninsula accessions compared to worldwide cowpeas.ConclusionsThe genetic analyses performed in this study brought some insights into worldwide genetic diversity and structure and possible dispersion routes of cultivated cowpea. Also, it provided an in-depth analysis of genetic diversity in Iberian Peninsula cowpeas that will help guide crossing strategies in breeding programs.

Caracterização agro-morfológica de acessos de feijão-frade (Vigna unguiculata): bases para o melhoramento

A caracterizacao morfologica dos recursos geneticos e essencial para o conhecimento da variacao fenotipica e das relacoes entre genotipos, sendo esta informacao fundamental no desenvolvimento de programas de melhoramento. O feijao-frade e uma leguminosa de grao cultivada um pouco por todo mundo devido ao seu elevado valor nutritivo, capacidade de fixar azoto e tolerância a seca. Com o objetivo de caracterizar uma colecao de 27 acessos portugueses de feijao-frade e 5 testemunhas procedeu-se a sua caracterizacao por parâmetros morfologicos e agronomicos com base em seis caracteristicas qualitativas e onze quantitativas. Os acessos apresentaram, na sua maioria, habito de crescimento ereto (96,87%), flores brancas (87,50%) e sementes de cor creme (84,38%). Os acessos mais precoces atingiram a floracao 77 dias apos a sementeira e o mais tardio apos 119 dias. O numero medio de vagens por planta variou entre 8,08 e 31,50, o peso medio de 100 sementes entre 11,80 g e 30,85 g e a producao total entre 40,96 g.m-2e 153,65 g.m-2. Em conclusao, foi observada uma elevada variabilidade entre os acessos de feijao-frade estudados, a qual podera ser utilizada em programas de melhoramento desta cultura. Visto que algumas das caracteristicas morfologicas sofrem influencia das condicoes ambientais, e importante complementar a caracterizacao destes acessos com estudos moleculares.

Berry color variation in grapevine as a source of diversity

Even though it is one of the oldest perennial domesticated fruit crops in the world, grapevine (Vitis vinifera L.) cultivation today is the result of both conventional breeding practices (i.e. hybridizations adopted during the last century) and vegetative propagation. Human-assisted asexual propagation has allowed the maintenance of desired traits but has largely impacted the frequency of spontaneous somatic mutations observed in the field. Consequently, many grapevine fruit attributes to date have been artificially selected, including: fruit yield, compactness, size and composition, the latter being greatly diversified in the pursuit of altering berry skin coloration. The present review provides an overview of various aspects related to grapevine diversity, with a special emphasis on grape berry skin color variation and will discuss the current knowledge of how grape skin color variation is affected by the synthesis of phenolic compounds, particularly anthocyanins and their underlying genetic factors. We hope this knowledge will be useful in supporting the importance of the berry color trait diversity in cultivated grapevines, which is used as basis for selection during breeding programs because of its application for vine growers, winemakers and consumers.

Speciation and periodic restricted environments. The case of genus Ononis L. (subsections Natrix and Viscosae)

Abstract The morpho–environmental similarity between subsections Natrix and Viscosae has been pointed out as the reason for the genetic complexity of these groups of taxa. Based on this characterization a question emerges: could a very recent ongoing evolutionary process explain that morpho–environmental similarity? ISSR and cpSSR amplifications for 45 specimens belonging to taxa of Natrix and Viscosae subsections were developed, along their biogeographic distribution areas. Twenty-nine haplotypes were detected in the biogeographic area of both subsections, 79% were exclusive haplotypes, but the rest is shared between subsections Natrix and Viscosae species. Could that haplotype sharing be the result of potential hybridization between these taxa? Do current environmental conditions restrict the gene flow among taxa? The combination of ancestral genetic polymorphism, introgression, coalescence processes and periodic restricted environments (PRE) by glacial–interglacial environmental dynamics were discussed to explain the relevant percentage of exclusive haplotypes detected, as well as the persistence of shared haplotypes. These results are in accordance with the morpho–environmental proximity previously described for both subsections.

Apple (Malus spp.) Breeding: Present and Future

Apple breeding has been extremely successful in providing a highly diverse fruit crop. Recent (>50 millions years ago) genome-wide duplication (GWD) resulted in the 17 chromosomes in the Pyreae and confirmed the origin of cultivated apple on Malus sieversii being the same species as M. × domestica. Malus, as many other species in the family Rosaceae, shows gametophytic self-incompatibility (GSI), which forces outcrossing. GSI at the pistil is regulated by extacellular ribonuclease, S-RNase, which is encoded by S locus. Growers and agronomists have provided multiple cultivars with different colors, shapes, resistances, climatic adaptation or industrial aptitudes. The aim in apple breeding was the combination of different kinds of resistance and good fruit quality to produce dessert cultivars and cultivars for processing. Some of the best of these cultivars display resistance to scab (Venturia inaequalis), mildew (Podosphaera leucotricha), fire blight (Erwinia amylovora), bacterial canker (Pseudomonas syringae), red spider mite (Panonychus ulmi), winterfrost and good fruit quality. Different scab resistance sources of wild species (Vf, Vr, VA) were combined in the new series of cultivars. Multiple efforts worldwide have conserved most of that variation, the pillar for the traditional and new techniques profiting from the analysis of the apple genome, the genome-wide association studies (GWAS), identifying SNPs and genes, the analysis of genes differentially expressed (GDE) identified by qRT-PCR and microarray analysis, and the recent molecular genetic tool CRISPR/Cas9 to edit and correct the genome.