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- Identification and validation of microsatellite markers in strawberry tree (Arbutusunedo L.)Publication . Fazenda, Pedro; Pereira, Ricardo; Fonseca, Maria; Carlier, Jorge; Leitão, JoséStrawberry tree (Arbutus unedo L.), an evergreen shrub/small tree of the family Ericaceae, is a main constituent of the Mediterranean basin flora; although it is also found in southwestern Prance, Macaronesia, and Ireland. The small fruits are edible but mostly used for preparation of preserves and jams, and for liquors such as the Portuguese traditional "aguardente de medronho". Traditionally cultivated by small farmers, often in consociation with Quercus sp., strawberry tree is presently emerging as a new important fruit crop cultivated in large orchards by modern export-oriented enterprises. This change of paradigm requires a growing role of plant breeding, upstream of the production process. Genomic tools for this species are mostly limited to the chloroplast genome sequence and to genomic data described in this work. In order to identify strawberry tree microsatellite (SSR) loci we performed partial genome next-generation sequencing using the Ion Torrent technology. The sequenced similar to 24.6M nucleotides resulted in the identification of 1185 microsatellite markers mostly constituted by dinucleotide motifs. The relative amount of microsatellite dinucleotide motifs (AG/CT - 71.7%, AC/GT - 20.5%, AT/AT - 2.9%, and CG/CG - 0.3%) is similar to the one observed in other Ericaceae species. Among a tested sample of 40 SSR primer pairs, 20 amplified well-defined PCR products, 12 (30%) were validated as polymorphic. Used in our collaborative project for molecular identification of selected and improved clones, the identified SSR loci constitute a strong tool for a large panoply of applied and fundamental studies of this emerging fruit crop.
- Identification of mandarin hybrids by isozyme and RAPD analysisPublication . Elisiário, P. J.; Justo, E. M.; Leitão, J. M.A cross between the monoembryonic Clementine MA3 SRA 649 used as seed parent and the polyembryonic Carvalhais mandarin used as pollen progenitor was carried out in the SRA INRA-CIRAD, S. Giuliano, Corsica. Cuttings of 61 plants putatively resulting from this cross were sent to the Centro de Citricultura, Fare, Portugal for further agronomic evaluation under field trials. Isozyme analysis allowed the identification of 59 plants as having sexual origin, however, two plants that showed isozyme patterns identical to their seed parent were impossible to identify as being either of zygotic or of somatic origin. Isozymes confirmed Carvalhais mandarin as the pollen parent of 42 progeny plants, while RAPD analysis confirmed Carvalhais paternity over the remaining 19 plants. A fast and low pollutant method for extraction of Citrus total genomic DNA suitable for RAPD analysis is reported. (C) 1999 Elsevier Science B.V. All rights reserved.
- Purification and characterization of a pore-forming protein from the marine sponge Tethya lyncuriumPublication . Mangel, A.; Leitão, J. M.; Batel, R.; Zimmermann, H.; Muller, W. E. G.; Schroder, H. C.A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.
- SCAR and CAPS markers flanking the Brassica oleracea L. Pp523 downy mildew resistance locus demarcate a genomic region syntenic to the top arm end of Arabidopsis thaliana L. chromosome 1Publication . Farinhó, M.; Coelho, P.; Monteiro, A. A.; Leitão, J. M.We recently mapped the Pp523 locus that includes a single, dominant gene conferring resistance to downy mildew expressed in adult plants to a 75.1 cm long linkage group on a genetic linkage map of Brassica oleracea L. More recently, we identified a new AFLP marker 2.8 cm downstream from the resistance gene. The five DNA markers within an 8.5 cm region encompassing the Pp523 gene were cloned and sequenced. Three of these markers were transformed into SCARs (sequence characterised amplified regions), however, two among them were monomorphic and were analysed as CAPS (cleaved amplified polymorphic sequence) markers among the mapping population. Searched against genomic databases, the five B. oleracea DNA-marker sequences matched Arabidopsis thaliana L. gene sequences that delimit a conserved syntenic region in the top arm end of chromosome 1 of this last species. Considering the close genetic relatedness between both species, the information on this specific genomic region in A. thaliana is particularly useful for the construction of a fine-scale map of the corresponding genomic region in B. oleracea. The identified SCAR and CAPS markers can be used for marker assisted selection (MAS) in breeding programs aimed at the introgression of the Pp523 resistance locus, allowing the reliable indirect identification of plants harbouring the resistance gene with a margin of error of approximately six in ten-thousand selected plants.
- Next-Generation Sequencing (NGS) identified Species-Specific SSR and SNP markers, allow the unequivocal identification of Strawberry Tree (Arbutus unedo L.) germplasm accessions and contribute to assess their genetic relationshipsPublication . Pereira, Ricardo; Anjos, Isabela Vera Dos; M. Reis, João; Dias, Carolina; M Leitão, JThe strawberry tree (Arbutus unedo L.), an evergreen bush to small tree of the Ericaceae family, is a main component of the natural flora of the Mediterranean basin that also grows profusely through the Iberian Peninsula, southwestern France, and Ireland. The small edible red fruits are usually used to produce preserves, jams, and liquors, as the Portuguese “aguardente de medronho”. The leaves and fruits have been used for a long time in traditional medicine, and their bioactive compounds are presently the subject of intense research. A strawberry tree germplasm collection was recently established by the company Corte Velada (Odiáxere, Portugal). A set of 50 germplasm accessions was selected for a breeding program. A next-generation sequencing project was performed, resulting in the establishment of the first strawberry tree genome assembly and further identification of 500 SSR and 500 SNP loci. Individual molecular fingerprints for the unequivocal identification of the selected 50 accessions were established based on 71 markers alleles amplified by 4 SSR and 9 SNP markers. The same species-specific markers alleles combined with 61 random amplified markers amplified by 5 RAPD and 5 ISSR primers were used to assess the genetic variability and genetic relationships among the selected accessions.
- Isozyme analysis revealed that the Portuguese mandarin 'Carvalhais' originated as a single clonePublication . Elisiário, P. J.; Santos, G. G.; Guerreiro, A. R.; Ollitrault, P.; Luro, F.; Leitão, J. M.'Carvalhais' mandarin is a well-known Portuguese mandarin used in several Citrus breeding programmes throughout the world. The first step in the implementation of a breeding programme of this variety was to screen for its natural genetic variability. A significant number of adult trees and young seedlings were submitted to isozyme analysis. For better interpretation of the isozyme patterns of 'Carvalhais', several plants resulting from crosses between this Portuguese mandarin cultivar and the monoembryonic mandarin cultivar Clementine MA3 SRA 649, were also analysed. All the analysed trees of 'Carvalhais' showed similar monomorphic patterns for each isozyme system, indicating that this mandarin cultivar originated as a single initial clone. Young seedlings exhibited monomorphic isozyme patterns identical to the adult plants revealing a high level of apomixis in this cultivar. (C) 1999 Elsevier Science B.V. All rights reserved.
- Two powdery mildew resistance mutations induced by ENU in Pisum sativum L. affect the locus er1Publication . Pereira, G.; Leitão, J. M.The alkylating compounds and strong mutagens methylnitrosourea (MNU) and ethylnitrosourea (ENU) were used to widen the genetic variability of pea (Pisum sativum L.) via experimental mutagenesis. Amongst multiple mutations of agronomical interest, for the first time two mutations conferring broad-range resistance to powdery mildew (Erysiphe pisi Syd.) were induced in pea. Mutagenic treatments were carried out on seedlings using a technique that ensures very high mutagenic efficiency. Two-hour exposure of cv. Solara seedlings to chemical mutagens resulted in almost non noticeable lethality and sterility in the M1 generation and very high mutation rates: similar to 9% families with visible mutations in the M2 generation. The influence of the cell cycle phase on the process of mutagenesis was studied in cv. Frilene using a previously developed technique for synchronization of shoot apical meristem cells. The cell cycle phase at which cells were treated apparently did not influence the lethality and sterility in the M1 generation, while the visible mutation rate, assessed in the M2 generation, showed a clear cell cycle dependency. Seedlings treated at the G2 and M phases gave rise to progenies exhibiting the highest mutation rate, over 50% of M2 families with visible mutations. The powdery mildew resistant (PMR) mutant S(er1mut1) was induced by treatment of cv. Solara seedlings for 2 h with ENU, while the PMR mutant F(er1mut2) was induced by treatment of cv. Frilene seedlings with the same chemical mutagen for 1 h during the G2 phase of the cell cycle. The genetic analysis of the novel PMR mutant lines showed that both resistances are inherited as monogenic recessive traits. The performed genetic complementation analyses revealed that both mutations affect the same locus-er1, which determines most of the natural sources of PMR in pea. A project aiming at the isolation of the powdery mildew resistance mutated gene via map based cloning is currently under way.
- Correction of the misclassification of species in the Portuguese collection of Cucurbita pepo L. using DNA markersPublication . Rodrigues, R.; Veiga, I.; Marreiros, A.; Rocha, F.; Leitão, J. M.In this study, the genetic variability among 130 accessions of the Portuguese germplasm collection of Cucurbita pepo L. maintained at the Banco Portugues de Germoplasma Vegetal was assessed using AFLP (amplified fragment length polymorphism) and RAPD (random amplified polymorphic DNA) techniques for the identification of a genetically diverse core group of accessions for field phenotypic analysis. The surprisingly completely different molecular patterns exhibited by multiple accessions was later confirmed in the distribution of the putative C. pepo plants into two clusters drastically separated at a very low level of genetic similarity (DICE coefficient = 0.37). Additional analyses with RAPD and ISSR (inter single sequence repeat) markers and the introduction of standard genotypes of C. maxima L. and C. moschata L. into the analyses allowed the identification of multiple accessions of the last species wrongly included in the C. pepo collection. This study is a good example of the usefulness of DNA markers in the establishment and management of plant germplasm collections.
- Main results from the EU-funded project "evaluation and utilization of pineapple genetic resources from the amazon to breed resistant varieties"Publication . D'Eeckenbrugge, G. Coppens; Cabral, J. R. S.; Matos, A. P. de; Carlier, Jorge; Leitão, José; Duval, M. F.; Noyer, J. L.; Ferreira, F. R.; Leal, F.; Maggioni, L.; Suarez, Z.This project was aimed at obtaining knowledge and tools for the development of cultivars resistant to the main diseases in South America, by (i) characterizing and evaluating available genetic resources, (ii) studying the genetic structure of the genera Ananas and Pseudanas (iii) developing and applying resistance screening techniques for important pests and disease in the region, (iv) studying the heredity of agronomic traits, and (v) testing the potential of partial inbreeds in breeding. In addition, a standard database is being established in collaboration with IPGRI, to promote information on germplasm exchange. The project, which started in 1997 and ended in 2001, has produced an inventory of the germplasm held in its collections, allowing exchange and repatriation of lost germplasm. The list of descriptors has been revised. Multivariate analyses of the morphological data have produced consistent results. Molecular characterization, based on RFLP of genomic DNA and PCR-RFLP of cpDNA has confirmed the proximity of Ananas and Pseudananas, the absence of interspecific barriers in Ananas, the importance of a geographic component of variation, and the existence of a higher genetic diversity in the North of South America. Pineapple taxonomy has been revised. Genetic mapping was based on isozyme, RAPD, AFLP and ISSR markers and was carried out on a hybrid progeny between A. bracteatus and A. comosus, following the pseudotestcross approach. It resulted in a first map of 390 and 174 DNA markers distributed in 46 and 31 linkage groups for these two species. New sources of resistance to fusariosis have been identified, some of which show other interesting traits for the Brazilian producers. According to the first inheritance studies, this resistance seems monogenic and dominant, a result still to be confirmed. Selfprogenies obtained from the cultivars Perolera, Primavera, and Roxo de Tefé, showed inbreeding depression, but normal segregation for major leaf traits (spininess, colour). Subsequent hybridization of the inbreeds restored vigour.
- Genetic diversity assessment of the almond (Prunus dulcis (Mill.) DA Webb) traditional germplasm of Algarve, Portugal, using molecular markersPublication . Cabrita, L.; Apostolova, E.; Neves, Alcinda; Marreiros, A.; Leitão, JoséIn this study, 123 almond (Prunus dulcis (Mill.) D. A. Webb) trees identified among traditional orchards in the Algarve region and 53 trees of the local field collection managed by the regional office of the Portuguese Ministry of Agriculture (DRAALG) were assessed using isozyme, inter- single sequence repeat and simple sequence repeat or microsatellite techniques for the evaluation of genetic diversity and genetic relatedness and identification of new accessions for the field collection. The isozyme analysis allowed the distribution of the 176 plants into 13 different classes of enzyme similarity, while the use of DNA markers increased the distribution of the analysed trees among 140 discriminating DNA patterns. Multiple cases of homonymy and synonymy were identified in the local germplasm. Some traditional varieties, such as Lourencinha, appeared to be relatively homogeneous, while other local denominations, e.g. Galamba, included diverse genotypes. Of the 13 commercial varieties analysed in this study, 11 assembled in one major cluster clearly differentiated from the majority of the local genotypes. These results reinforced the perception that the Algarve traditional germplasm constitutes an important repository of genetic diversity, eventually carrying alleles of high agricultural interest such as the recently identified Phomopsis resistance in the traditional variety Barrinho Grado.