Browsing by Author "Pereira, Ricardo"
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- A microsatellite sequence in the fifth intron provides a broad-spectrum SSR marker for multiple alleles of the er1/PsMLO1 powdery mildew resistance gene in Pisum sativum L.Publication . Cardoso, André; Pereira, Ricardo; Fonseca, Maria; M Leitão, JPowdery mildew caused by the biotrophic ascomycete fungus Erysiphe pisi Syd. is one the most devastating diseases of peas (Pisum sativum L.) with enormous impact in seed production. The most efficient genetic resistance to this disease, so far identified, is conferred by the naturally occurring or experimentally induced by chemical mutagenesis recessive state of the locus er1. Genetically mapped over 2 decades ago, this gene was recently identified as a homolog of the barley (Hordeum sativum L.) powdery mildew resistance gene MLO, and renamed as PsMLO1. The broad wide resistance conferred by the er1/PsMLO1 locus was found to be a consequence of the loss of function of the encoded PsMLO1 protein. After the publication of the expressed sequence of this gene by another research group, we published the genomic sequences of this gene which harbors a relatively long (TA) microsatellite sequence (SSR) in the fifth intron. SSR markers based on this highly polymorphic microsatellite can be used for marker-assisted selection in multiple pea powdery mildew resistance breeding programs involving the er1/PsMLO1 resistance, except in the rare circumstances where the progenitor lines are monomorphic for the microsatellite sequence.
- Assessment of Wild Rocket (Diplotaxis tenuifolia (L.) DC.) Germplasm accessions by NGS identified SSR and SNP markersPublication . Reis, João M.; Pereira, Ricardo; Coelho, Paula S.; Leitão, JoséRocket is the common designation for two baby-leaf salad crops of the Brassicaceae family: Eruca sativa (L.) Cav., usually referred to as annual garden rocket, and Diplotaxis tenuifolia (L.) DC. commonly named to as perennial wild rocket. E. sativa is used for human consumption since antiquity. However, the growing consumer preference for D. tenuifolia is being accompanied by the fast increase in its production area and commercialization of new cultivars. Nevertheless, the worldwide number of wild rocket accessions maintained in germplasm collections is very reduced, the solution for which situation the project “REMIRucula” intends to contribute, establishing a germplasm collection at the INIAV, Oeiras, Portugal. Herein, we report on the establishment via next generation sequencing (NGS) of the first genome assembly of D. tenuifolia and the identification of specific single sequence repeat (SSR) and single nucleotide polymorphisms (SNP) loci for the establishment of specific DNA-markers for this species. A representative set of 87 D. tenuifolia and 3 E. sativa accessions were assessed by 5 SSR and 9 SNP-CAPS markers, allowing a drastic discrimination between both species and the establishment of unequivocal molecular fingerprints for the analyzed accessions. The non-discrimination within six pairs and one trio of D. tenuifolia accessions is discussed.
- 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.
- 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.
- A Non-rogue mutant line induced by ENU mutagenesis in paramutated rogue peas (Pisum sativum L.) is still sensitive to the rogue ParamutationPublication . Pereira, Ricardo; Leitão, JoséThe spontaneously emerging rogue phenotype in peas (Pisum sativum L.), characterized by narrow and pointed leaf stipula and leaflets, was the first identified case of the epigenetic phenomenon paramutation. The crosses of homozygous or heterozygous (e.g., F1) rogue plants with non-rogue (wild type) plants, produce exclusively rogue plants in the first and all subsequent generations. The fact that the wild phenotype disappears forever, is in clear contradiction with the Mendelian rules of inheritance, a situation that impedes the positional cloning of genes involved in this epigenetic phenomenon. One way of overcoming this obstacle is the identification of plant genotypes harboring naturally occurring or artificially induced neutral alleles, non-sensitive to paramutation. So far, such alleles have never been described for the pea rogue paramutation. Here, we report the induction via 1-ethyl-1-nitrosourea (ENU) mutagenesis of a non-rogue revertant mutant in the rogue cv. Progreta, and the completely unusual fixation of the induced non-rogue phenotype through several generations. The reversion of the methylation status of two previously identified differentially methylated genomic sequences in the induced non-rogue mutant, confirms that the rogue paramutation is accompanied by alterations in DNA methylation. Nevertheless, unexpectedly, the induced non-rogue mutant showed to be still sensitive to paramutation.
- Vulnerable marine ecosystems survey pilot missions with EVA Hybrid AUV/ROVPublication . Almeida, Carlos; Martins, Alfredo; Soares, Eduardo; Santos, A. Miguel P.; Matias, Bruno; Silva, Pedro; Pereira, Ricardo; Sytnyk, Denys; Ferreira, António; Lima, Ana Paula; Cunha, Mariana R.; Ramalho, Sofia P.; Rodrigues, Clara F.; Figueiredo, Ivone; Rosa, Marcos; Almeida, JoséFishing for deep-sea species occurs on continental slopes, ridges, and seamounts. Fishing operations using fishing gears that contact the bottom (e.g., trawls and bottom longlines) may have significant impacts on Vulnerable Marine Ecosystems (VMEs). VMEs refer to marine ecosystems with a population or community of sensitive taxa or habitats that are likely to experience substantial alteration from short-term to chronic disturbance and that are unlikely to recover during the timeframe in which the disturbance occurs. The VME concept, introduced in the United Nations General Assembly Resolution 61/105, has been worldwide applied to the management of deep-sea fisheries. However, the effective identification and management of VMEs is highly constrained by the scarcity of data on VME indicator taxa. This data deficiency is usually surpassed by the use of VME predictive modelling. Video footage is a non-destructive method commonly used for exploring and investigating areas of seabed and for characterising and identifying habitat types. Remotely Operated Vehicles (ROVs) are one of the tools for seabed mapping. ROVs range in size from small observation-class to large work-class vehicles. Their sizes determine the payload, manoeuvrability, depth rating and ultimately uses of the vehicle. For epifaunal imaging, ROVs can be used in two modes: qualitative inspections and quantitative assessments. This paper presents the development of an innovative system composed of a compact support research vessel and a hybrid autonomous underwater vehicle capable of accurate georeferenced high-resolution imaging and profiling of the seabed for a detailed survey of the seabed for biodiversity studies. The experimental results obtained by the developed system in field work in real VME survey at 600m depth are presented.