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Research Project
Strategic Project - UI 115 - 2011-2012
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Publications
Unravelling wild carrot differentiation in Europe: preliminary data on a candidate gene approach
Publication . Nobre, T.; Ragonezi, C.; Arnholdt-Schmitt, B.
Carrot is an outcrossing species and levels of gene flow between populations,
and even between wild and domesticated relatives, are expected to be high. Cases of
natural hybridization and introgression of crops and wild relatives have been
reported. Have these events diluted any putative habitat-adapted genotypes? In other
words, can we still find a correlation between wild carrot genotypes and
regional/local environment? We have chosen to start addressing this question using a
member of the alternative oxidase (AOX) gene family. AOX genes seem to be linked to
all kinds of abiotic and biotic stress reactions. Wild carrots were sampled in an
environmental gradient across Western Europe. This gradient included sampling
points with more deviating conditions, such as Sierra de Guadarrama or the central
Pyrenees and the French Massif Central. Phylogenetic reconstruction on this
molecular marker is to be combined with geographic, climatic, and ecological
evidence. So far, the preliminary results suggest the existence of a biogeographical
barrier at the Pyrenees, and higher gene diversity than initially expected. From an
applied point of view, diversity of functional traits is much more relevant than species
diversity. Gene transfer from wild to cultivated plants has contributed to the evolution
of crop species. Providing that deterioration of genetic resources and biodiversity loss
have not been drastic, gene transfer from wild plants has the potential to further
contribute to a (targeted) improvement of cultivars.
Reference genes selection and normalization of oxidative stress responsive genes upon different temperature stress conditions in Hypericum perforatum L
Publication . Velada, Isabel; Ragonezi, Carla; Arnholdt-Schmitt, Birgit; Cardoso, Hélia
Reverse transcription-quantitative real-time PCR (RT-qPCR) is a widely used
technique for gene expression analysis. The reliability of this method depends
largely on the suitable selection of stable reference genes for accurate data
normalization. Hypericum perforatum L. (St. John’s wort) is a field growing plant that
is frequently exposed to a variety of adverse environmental stresses that can
negatively affect its productivity. This widely known medicinal plant with broad
pharmacological properties (anti-depressant, anti-tumor, anti-inflammatory,
antiviral, antioxidant, anti-cancer, and antibacterial) has been overlooked with
respect to the identification of reference genes suitable for RT-qPCR data
normalization. In this study, 11 candidate reference genes were analyzed in H.
perforatum plants subjected to cold and heat stresses. The expression stability of
these genes was assessed using GeNorm, NormFinder and BestKeeper
algorithms. The results revealed that the ranking of stability among the three
algorithms showed only minor differences within each treatment. The best-ranked
reference genes differed between cold- and heat-treated samples; nevertheless,
TUB was the most stable gene in both experimental conditions. GSA and GAPDH
were found to be reliable reference genes in cold-treated samples, while GAPDH
showed low expression stability in heat-treated samples. 26SrRNA and H2A had
the highest stabilities in the heat assay, whereas H2A was less stable in the cold
assay. Finally, AOX1, AOX2, CAT1 and CHS genes, associated with plant stress
responses and oxidative stress, were used as target genes to validate the reliability
of identified reference genes. These target genes showed differential expression profiles over time in treated samples. This study not only is the first systematic
analysis for the selection of suitable reference genes for RT-qPCR studies in H.
perforatum subjected to temperature stress conditions, but may also provide
valuable information about the roles of genes associated with temperature stress
responses.
Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in Portugal
Publication . Ragonezi, Carla; Caldeira, A. Teresa; Martins, M. Rosário; Salvador, Cátia; Santos-Silva, Celeste; Ganhão, Elsa; Klimaszewska, Krystyna; Zavattieri, Maria Amely
Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have benefi cial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of
stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root
growth cessation not only in vitro but also to improve root development during acclimation phase.
Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were col lected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P.
arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest
Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the
collected material, ITS amplification was applied using the pure cultures derived from the sporo carps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was
executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effective ness of the techniques used to amplify DNA polymorphic sequences, which enhances the characte rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity
at any stage of plant mycorrhization.
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Funders
Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
6820 - DCRRNI ID
Funding Award Number
PEst-C/AGR/UI0115/2011