Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.

BackgroundA successful development of herbivorous insects into plant tissues depends on coordination of metabolic processes. Plants have evolved complex mechanisms to recognize such attacks, and to trigger a defense response. To understand the transcriptional basis of this response, we compare gene expression profiles of two coffee genotypes, susceptible and resistant to leaf miner (Leucoptera coffella). A total of 22000 EST sequences from the Coffee Genome Database were selected for a microarray analysis. Fluorescence probes were synthesized using mRNA from the infested and non-infested coffee plants. Array hybridization, scanning and data normalization were performed using Nimble Scan® e ArrayStar® platforms. Genes with foldchange values +/-2 were considered differentially expressed. A validation of 18 differentially expressed genes was performed in infected plants using qRT-PCR approach.ResultsThe microarray analysis indicated that resistant plants differ in gene expression profile. We identified relevant transcriptional changes in defense strategies before insect attack. Expression changes (>2.00-fold) were found in resistant plants for 2137 genes (1266 up-regulated and 873 down-regulated). Up-regulated genes include those responsible for defense mechanisms, hypersensitive response and genes involved with cellular function and maintenance. Also, our analyses indicated that differential expression profiles between resistant and susceptible genotypes are observed in the absence of leaf-miner, indicating that defense is already build up in resistant plants, as a priming mechanism. Validation of selected genes pointed to four selected genes as suitable candidates for markers in assisted-selection of novel cultivars.ConclusionsOur results show evidences that coffee defense responses against leaf-miner attack are balanced with other cellular functions. Also analyses suggest a major metabolic reconfiguration that highlights the complexity of this response.

No abstract

Puberty is a stage of sexual development determined by the interaction of environmental factors and genetic mechanisms. Among them, thyroid function plays a key role in sexual development and spermatogenic function and is under the control of several genes, including the well-described thyroglobulin gene (TG). Previous reports have shown genetic association between thyroid function and selected single nucleotide polymorphisms (SNPs) in taurine cattle. Therefore, the identification of genetic mechanisms involved in the regulation of this trait can assist with the selection for early pubertal bulls, thus improving genetic progress in livestock breeding. The aim of this study was to validate the association between TG SNPs and age at puberty in zebuine bulls. Three SNPs (rs110406764, rs109662686, rs109057985) were genotyped in 159 Guzerat animals using SEQUENOM technology. Results showed a significant association (p < .05) between the studied SNPs and puberty age, in agreement with our previous reports in a taurine breed. Interestingly, allele frequencies were different from those already reported, being GAT the most favourable allele for age at puberty in Guzerat (94.4 days lower). Overall, our findings corroborate previous reports and reinforce the importance of genetic influence in the regulation of sexual development and puberty through a thyroid pathway in zebuine cattle.

Evaluation of impacts of high CO 2 atmospheric concentration is strategically important for the development of adaptation measures and sustainable crop management. The objective of this study was to evaluate the effects of increased atmospheric CO 2 concentration on eucalypt rust (Puccinia psidii), C:N ratio, yield and chemical composition of essential oils and growth of eucalypt clonal plantlets. Two clones with different levels of rust resistance were studied: a Eucalyptus urophylla 9 E. camaldulensis hybrid (VM 01) and an E. urophylla (clone MN 463). The experiments were performed in open-top chambers (OTCs) with CO 2 mean concentrations (lmol mol À1 ) of 399 (unenclosed control), 412 (OTCs with ambient CO 2 concentration) and 508 (OTCs with high CO 2 concentration) and in closed chambers (CCs) with CO 2 mean concentrations of 390, 405, 520 and 700. Increased atmospheric CO 2 concentrations resulted in a decrease in rust pustules per leaf, uredinia per leaf area, spores per uredinia and area under the disease progress curve in VM 01 (hybrid) clonal plantlets. The disease did not occur in MN 463 clonal plantlets, which demonstrated that high CO 2 concentrations did not change the level of rust resistance. Plant growth of the two clones was stimulated by up to 23% in height and 26% in stem diameter in OTCs and by 18% for both clones in CCs. An increased C:N ratio in leaves, stems and roots was observed only for the VM 01 clonal plantlets. Essential oils produced by VM 01 (2.8 g 100 g À1 ) and MN 463 (1.4 g 100 g À1 ), as well as the major essential oil compounds (80% 1.8-cineole for VM 01; 50% 1.8-cineole and 32% a-pinene for MN 463), were not altered. In this study, increased concentrations of atmospheric CO 2 favourably impacted eucalypt growth and reduced rust severity, while not influencing the production of essential oils.