Juliana Pereira Bravo

Transferability and use of microsatellite markers for the genetic analysis of the germplasm of some Arachis section species of the genus Arachis

The Arachis section is the most important of the nine sections of the genus Arachis because it includes the cultivated peanut, Arachis hypogaea. The genetic improvement of A. hypogaea using wild relatives is at an early stage of development in spite of their potential as sources of genes, including those for disease and pests resistance, that are not found in the A. hypogaea primary gene pool. Section Arachis species germplasm has been collected and maintained in gene banks and its use and effective conservation depends on our knowledge of the genetic variability contained in this material. Microsatellites are routinely used for the analysis of genetic variability because they are highly polymorphic and codominant. The objective of this study was to evaluate the transferability of microsatellite primers and the assay of genetic variability between and within the germplasm of some species of the Arachis section. Fourteen microsatellite loci developed for three different species of Arachis were analyzed and 11 (78%) were found to be polymorphic. All loci had transferability to all the species analyzed. The polymorphic loci were very informative, with expected heterozygosity per locus ranging from 0.70 to 0.94. In general, the germplasm analyzed showed wide genetic variation.

Heterologous microsatellite primer pairs informative for the whole genus Arachis

The genus Arachis currently comprises 69 described species, some of which have potential and real value as human and animal foods. These Arachis species have been collected and maintained in germplasm banks to provide material that can be used as sources of genes in breeding programs and for the selection of new cultivars. One of the principal objectives of germplasm conservation is the evaluation of genetic variability, which is best conducted using molecular markers. We investigated the use of heterologous primers to amplify microsatellite loci that could be used to evaluate genetic variability in Arachis germplasm. Fifteen microsatellite primer pairs were tested in 76 accessions of 34 species from the nine Arachis sections. The data indicated that heterologous primers were very useful in Arachis since they had high transferability among the species (91%) and allowed the amplification of very polymorphic putative loci, which allowed both the characterization of most accessions and to make inferences about the mating systems of some species analyzed. Our data also revealed that the germplasm analyzed showed high variability, even when represented by few accessions.

Microsatellites as Tools for Genetic Diversity Analysis

Powerful tools for the analysis of genetic biodiversity are molecular markers, which are based on DNA sequence polymorphisms. Indeed, DNA sequences determine the diversity of organisms, and therefore, the techniques used to evaluate DNA polymorphisms directly measure the genetic diversity. Because molecular markers show Mendelian inheritance, it is possible to trace the fingerprint of each organism and determine the evolutionary history of the species by phylogenetic analysis, studies of genetic relationship, population genetic structures and genetic mapping.

The tonoplast intrinsic aquaporin (TIP) subfamily of Eucalyptus grandis: Characterization of EgTIP2, a root-specific and osmotic stress-responsive gene

Aquaporins have important roles in various physiological processes in plants, including growth, development and adaptation to stress. In this study, a gene encoding a root-specific tonoplast intrinsic aquaporin (TIP) from Eucalyptus grandis (named EgTIP2) was investigated. The root-specific expression of EgTIP2 was validated over a panel of five eucalyptus organ/tissues. In eucalyptus roots, EgTIP2 expression was significantly induced by osmotic stress imposed by PEG treatment. Histochemical analysis of transgenic tobacco lines (Nicotiana tabacum SR1) harboring an EgTIP2 promoter:GUS reporter cassette revealed major GUS staining in the vasculature and in root tips. Consistent with its osmotic-stress inducible expression in eucalyptus, EgTIP2 promoter activity was up-regulated by mannitol treatment, but was down-regulated by abscisic acid. Taken together, these results suggest that EgTIP2 might be involved in eucalyptus response to drought. Additional searches in the eucalyptus genome revealed the presence of four additional putative TIP coding genes, which could be individually assigned to the classical TIP1-5 groups.

UGP gene expression and UDP-glucose pyrophosphorylase enzymatic activity in grafting annonaceous plants

Grafting is commonly used to propagate commercial fruit species to ensure that the genetic characteristics of selected clones are maintained. However, the biochemical and molecular mechanisms involved in the graft incompatibility of woody trees are not well understood. We investigated the effect of grafting in vegetative growth, UDP-glucose pyrophosphorylase expression and activity of Annonaceous grafted plants: atemoya (Annona cherimola Mill. x Annona squamosa L.) ‘Thompson’ grafted onto different rootstocks, araticum-de-terra-fria (Annona emarginata Schltdl. H. Rainer “var. terra-fria”), araticum-mirim(Annona emarginata Schltdl. H. Rainer “var. mirim”) and biribá (Annona mucosa Schltdl. H. Rainer) at different post-grafting times. The growth of atemoya grafted onto araticum-mirim was lower than that of the rootstocks araticum-de terra-fria and biribá. The results also indicated that grafting alters UGPase gene expression; showing the combination atemoya grafted onto araticum-de-terra-fria (a compatible union) the higher levels of gene expression during the early stages of grafting development. However, no significant differences were detected in UGPase enzyme activity between the graft combinations. In addition, SDS-PAGE and MALDI-TOF analyses detected similar UGPase amino acid sequences in ungrafted atemoya samples to cherimoya (Annona cherimola Mill.), a female parent of the atemoya hybrid. These findings suggest that expression of the UGPase protein is related to graft compatibility in grafted Annona plants.

Expression Pattern and Promoter Analysis of a Eucalyptus grandis Germin-like Gene

Germin-like proteins (GLP) have been implicated in multiple physiological processes in plants, but our understanding of their functional roles, especially in trees, is far from complete. In the present study, an expressed sequence tag (EST) encoding a putative leaf-specific GLP was selected in silico using an expression-profile data from eucalyptus. Corroborating the in silico prediction, the expression of the corresponding gene (named EgGLP) in leaves of Eucalyptus grandis was validated using reverse transcription-PCR. Spatial EgGLP expression patterns were further investigated using a promoter-beta-glucuronidase (GUS) reporter gene fusion. In E. grandis seedlings, the EgGLP promoter was able to modulate transient GUS expression in the cotyledons, while in stably transformed tobacco plants, reporter expression was predominantly observed in the shoot apical meristem and in leaf vasculature. Phylogenetic analysis revealed that EgGLP belongs to subfamily III and is closely related to GLPs known to be regulated by the circadian rhythm. In this context, we show that the activity of the EgGLP promoter in tobacco is modulated by light/dark transition.

The receptor-like kinase SlSOBIR1 is differentially modulated by virus infection but its overexpression in tobacco has no significant impact on virus accumulation.

Key messageThe role of the tomato receptor-like kinase SlSOBIR1 in antiviral defense was investigated. SlSOBIR1 was transcriptionally modulated by unrelated viruses but its ectopic expression had no effect on virus accumulation.AbstractLeucine-rich repeat receptor-like kinases (LRR-RLK) constitute a diverse group of proteins allowing the cell to recognize and respond to the extracellular environment. In the present study we focused on a gene encoding a tomato LRR-RLK (named SlSOBIR1) involved in the host defense against fungal pathogens. Curiously, SlSOBIR1 has been previously reported to be down-regulated by Pepper yellow mosaic virus (PepYMV) infection. Here, we show that SlSOBIR1 is responsive to wounding and differentially modulated by unrelated virus infection, i.e., down-regulated by PepYMV and up-regulated by Tomato chlorotic spot virus (TCSV). Despite these divergent expression profiles, SlSOBIR1 overexpression in transgenic tobacco plants had no evident effect on TCSV and PepYMV accumulation. On the other hand, overexpression of SlSOBIR1 significantly increased the expression of selected defense genes (PR-1a and PR-6) and exacerbated superoxide production in wounded leaves. Our data indicate that the observed modulation of SlSOBIR1 expression is probably triggered by secondary effects of the virus infection process and suggest that SlSOBIR1 is not directly involved in antiviral signaling response.

The Eucalyptus Tonoplast Intrinsic Protein (TIP) Gene Subfamily: Genomic Organization, Structural Features, and Expression Profiles

Plant aquaporins are water channels implicated in various physiological processes, including growth, development and adaptation to stress. In this study, the Tonoplast Intrinsic Protein (TIP) gene subfamily of Eucalyptus, an economically important woody species, was investigated and characterized. A genome-wide survey of the Eucalyptus grandis genome revealed the presence of eleven putative TIP genes (referred as EgTIP), which were individually assigned by phylogeny to each of the classical TIP1–5 groups. Homology modeling confirmed the presence of the two highly conserved NPA (Asn-Pro-Ala) motifs in the identified EgTIPs. Residue variations in the corresponding selectivity filters, that might reflect differences in EgTIP substrate specificity, were observed. All EgTIP genes, except EgTIP5.1, were transcribed and the majority of them showed organ/tissue-enriched expression. Inspection of the EgTIP promoters revealed the presence of common cis-regulatory elements implicated in abiotic stress and hormone responses pointing to an involvement of the identified genes in abiotic stress responses. In line with these observations, additional gene expression profiling demonstrated increased expression under polyethylene glycol-imposed osmotic stress. Overall, the results obtained suggest that these novel EgTIPs might be functionally implicated in eucalyptus adaptation to stress.

Cloning and Functional characterization of the promoter of a high affinity potassium transporter gene from Eucalyptus grandis

The characterization of organ/tissue-specific promoters is of great interest to transgenic production. The construction of expression cassettes containing tissue-specific promoters is a viable alternative to limited transgene expression to specific organs and cell types. In this context, the purpose of this study was to functionally characterize the promoter of a Eucalyptus grandis gene encoding a high affinity potassium transporter (named EgHAK) shown to be specifically expressed in roots. For that, the 5’-flanking region of EgHAK (1,3 kb) was cloned and transcriptionally fused to the b-glucuronidase reporter gene (GUS), and then used to transform tobacco leaf discs. Histochemical analysis of GUS activity in transgenic plants showed that GUS staining was mainly detected in vascular tissues of leaf and root. To investigate the response of the studied promoter to potassium starvation, a hydroponic system was employed. In this case, enhanced GUS staining was observed in the roots of plants starved for 6 days when compared to control ones. Moreover, a weak induction of the promoter at low potassium conditions was observed using fluorimetric assays. Thus, our results indicate that, in a heterologous system, the studied promoter shows preferential expression in roots in the absence of potassium.