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This study describes an efficient in vitro method using colchicinetreated triploid seedlings of Napiergrass and Pearl millet hybrids to produce hexaploid hybrids and their subsequent identification with flow cytometry. It also describes chromosome count and stomatal morphology and their use to ploidy analysis. Four-hundred and eighty triploid seeds, representing 12 different hybrids were sterilized and transferred to MS media to induce chromosome doubling. Surviving plants were analysed by flow cytometry. From six triploid (control) and hexaploid plants, the stomata sizes and frequency were analysed. Chromosome count was performed only in the plants identified as hexaploid. Seventeen plants were identified as hexaploid by flow cytometry analysis. Further confirmation of the hexaploid condition was performed with stomatal morphology (stomatal frequency reduction and stomatal length increase) and chromosome count (2n = 6x = 42). Chromosome doubling has numerous applications in Pennisetum breeding. It can be used to restore the fertility of interspecific hybrids and to improve seed size.The genus Pennisetum is one of the important genera of the family Poaceae. Pennisetum purpureum Schum. (Napiergrass) and Pennisetum glaucum (L.) R.Br. (Pearl millet) are important species widely used as forage. Pennisetum glaucum is a diploid species with 2n = 2x = 14 chromosomes and P. purpureum is tetraploid with 2n = 4x = 28 chromosomes (Martel et al. 2004). Napiergrass has been successfully crossed with Pearl millet to produce high quality and high yielding perennial interspecific forage hybrids (Hanna 1981). However, the sterility of this hybrid due to its triploid condition (2n = 3x = 21 chromosomes) has been pointed as a difficulty for its use in breeding programmes. The fertility of this hybrid can be restored with chromosome doubling. Colchicine is an alkaloid widely used for chromosome doubling and for the induction of polyploidy in plants (Pasakinskiene´2000). The technique of exposing explants to colchicine in vitro has been used in a number of cases (Kadota and Niimi 2002). Chromosome counts and stomatal morphology have been used routinely for polyploidy screening. However, these methods make the analysis of a great number of plants difficult and time-consuming. Flow cytometry represents a technology gain and has been increasingly used for highthroughput ploidy screening (Roy et al. 2001). This study describes an efficient in vitro method using colchicine-treated triploid seedlings of Napiergrass and Pearl millet hybrids to produce hexaploid hybrids and their subsequent identification with flow cytometry. It also describes chromosome count and stomatal morphology and their use for ploidy analysis. Plant materials and in vitro induction of chromosome doublingFour-hundred and eighty triploid seeds, representing 12 different hybrids (40 per hybrid), were sterilized and transferred to MS media to induce seedling development. The treatments (hybrids) have been arranged in a completely random design with four repetitions. Each...

BackgroundCytauxzoonosis is an emerging and life-threatening tick-borne feline disease caused by haemoprotozoan parasites of the genus Cytauxzoon. Information regarding epidemiological and clinical presentation of infections by species other than Cytauxzoon felis is scant. A case of Cytauxzoon sp. infection is described in a 2-year-old mixed breed male domestic cat from Portugal, presenting a history of acute lethargy, anorexia and pyrexia.ResultsComplete blood count revealed a severe anaemia, leucocytosis and thrombocytopenia. A pleural effusion was noticed on thoracic radiograph, and marked splenomegaly and free abdominal fluid were visualized by ultrasound. A molecular screening for the detection of causative agents of infectious anaemia was performed, and a positive result for Piroplasmorida was obtained. DNA sequencing of a 743 bp amplicon of the 18S rRNA gene (GenBank accession no. KU710344) revealed 99.9 % identity with Cytauxzoon manul.ConclusionsThis is the first report of Cytauxzoon sp. (clustering together with C. manul) in a felid from Portugal. Clinical manifestations along with molecular analysis suggest the hypothesis that domestic cats might be infected with and serve as a reservoir host for C. manul.

Like other eukaryotes, the nuclear genome of plants consists of DNA with a small proportion of low-copy DNA (genes and regulatory sequences) and very abundant DNA sequence motifs that are repeated thousands up to millions of times in the genomes including transposable elements (TEs) and satellite DNA. Retrotransposons, one class of TEs, are sequences that amplify via an RNA intermediate and reinsert into the genome, are often the major fraction of a genome. Here, we put research on retrotransposons into the larger context of plant repetitive DNA and genome behaviour, showing features of genome evolution in a grass genus, Brachiaria, in relation to other plant species. We show the contrasting amplification of different retroelement fractions across the genome with characteristics for various families and domains. The genus Brachiaria includes both diploid and polyploid species, with similar chromosome types and chromosome basic numbers x = 6, 7, 8 and 9. The polyploids reproduce asexually and are apomictic, but there are also sexual species. Cytogenetic studies and flow cytometry indicate a large variation in DNA content (C-value), chromosome sizes and genome organization. In order to evaluate the role of transposable elements in the genome and karyotype organization of species of Brachiaria, we searched for sequences similar to conserved regions of TEs in RNAseq reads library produced in Brachiaria decumbens. Of the 9649 TE-like contigs, 4454 corresponded to LTR-retrotransposons, and of these, 79.5 % were similar to members of the gypsy superfamily. Sequences of conserved protein domains of gypsy were used to design primers for producing the probes. The probes were used in FISH against chromosomes of accesses of B. decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola. Probes showed hybridization signals predominantly in proximal regions, especially those for retrotransposons of the clades CRM and Athila, while elements of Del and Tat exhibited dispersed signals, in addition to those proximal signals. These results show that the proximal region of Brachiaria chromosomes is a hotspot for retrotransposon insertion, particularly for the gypsy family. The combination of high-throughput sequencing and a chromosome-centric cytogenetic approach allows the abundance, organization and nature of transposable elements to be characterized in unprecedented detail. By their amplification and dispersal, retrotransposons can affect gene expression; they can lead to rapid diversification of chromosomes between species and, hence, are useful for studies of genome evolution and speciation in the Brachiaria genus. Centromeric regions can be identified and mapped, and retrotransposon markers can also assisting breeders in the developing and exploiting interspecific hybrids.

PCR is the best method for the detection of enteric viruses present at low concentrations in environmental samples. However, some organic and inorganic compounds present in these samples can interfere in the reaction. Many of these substances are cytotoxic, too. The ZP60S filter membranes used in addition to fluorpentane treatment are quite efficient for virus concentration and simultaneous elimination of cytotoxicity from environmental samples. In this study, both procedures were used to promote the elimination of reverse transcriptase PCR (RT-PCR) inhibitors from sewage and sewage-polluted creek water. Samples were subjected separately to each of the following procedures: filtration through electropositive filter membranes (ZP60S), organic extraction with Vertrel XF, and filtration through ZP60S followed by organic extraction. Afterwards, aliquots were experimentally inoculated with rotavirus SA-11 RNA and subjected to RT-seminested PCR for amplification of the VP7 gene. Results showed that the ZP60S membranes efficiently eliminated the RT-PCR inhibitors from water samples. The sample processing method was also applied to 31 in natura sewage and creek water samples for detection of naturally occurring rotavirus. A duplex seminested PCR was used for the quick detection of couples of the four rotavirus genotypes (G1 to G4). Eight samples (25.8%) were positive, and rotavirus sequences were not detected in 23 (74.2%). Results were confirmed by direct immunoperoxidase method. In summary, the use of electropositive filter membrane is appropriate for the elimination of substances that can interfere with RT-PCR, obviating additional sample purification methods.

RESUMOO presente trabalho foi realizado visando avaliar a viabilidade de grãos de pólen armazenados das cultivares copas cítricas Natal, Pêra e Valência. O pólen foi submetido a 3 temperaturas de armazenamento: -10 º C (freezer), 4 º C ( refrigerador) e temperatura ambiente; 2 ambientes (com e sem dessecador) e na presença e ausência de sílica-gel. A avaliação do índice de germinação foi feita com o pólen fresco e a cada 7 dias, durante 9 semanas. Para avaliar a germinação foi utilizado meio constituído de 1% de ágar e 10% de sacarose, 800 mg L -1 de nitrato de cálcio (Ca(NO 3 ) 2 4H 2 O), 200 mg L -1 de Ácido Bórico (H 3 BO 3 ) e pH corrigido para 6,5. Os grãos de pólen foram incubados à temperatura de 25 ± 2°C por 12 horas. As avaliações foram realizadas através da porcentagem de grãos de pólen germinados. Constatou-se que os grãos de pólen possuem redução na viabilidade com o aumento do tempo de armazenamento; o armazenamento em freezer (-10 o C) foi mais eficiente do que em refrigerador (4 o C) e à temperatura ambiente. Melhores resultados foram alcançados com os tratamentos em freezer com sílica-gel dentro de dessecador e em freezer sem sílica-gel dentro de dessecador. A cultivar Valência apresentou-se superior às demais em todos os tratamentos. Termos para indexação:Citrus sinensis , Palinologia, cultura de tecidos, melhoramento genético. ABSTRACTThe present work was accomplished to evaluate the effects of storage on the viability of pollen grains from Natal, Pêra and Valência cultivars of sweet oranges. The grains were stored at 3 temperatures: -10ºC (freezer), 4ºC (refrigerator), and room temperature; 2 environments (with or without desiccator) and in the presence or absence of silica-gel. The germination was evaluated every 7 days, during 9 weeks. To evaluate the germination, a medium consisting of 1% and sucrose 10%, 800 mg L -1 calcium nitrate (Ca(NO 3 ) 2 4H 2 O), 200 mg L -1 boric acid (H 3 BO 3 ) and pH 6,5 was used. The pollen was incubated at 25 ± 2°C temperature for 12 hours. The evaluation was performed using the percentage of pollen grains germinated. It was observed that pollen grains viability diminished as the storage time increased; the storage in freezer temperature (-10 º C) was much more efficient than in refrigerator (4 º C) and in room temperature. The best results were reached when freezer and desiccator were used. Valência cultivar was superior when compared whit the others, in all treatments.

It has been suggested that guidance, encouragement and opportunity are crucial factors for children's motor development. They are especially important to promote the mastery of fundamental movement skills. The present study investigated the association between two elements related to those factors -proposed curriculum of physical education and the most popular sport in a given country -and the incidence of mastery in the FMS in children from 4 to 10 years of age. In order to carry out a cross-cultural comparison, three studies from different countries (Portugal, China and USA) that used TGMD-II were selected. The results indicated association between proposed curriculum and the number of children mastering FMS. This association increases in older age groups of children from USA, the most structured curriculum related to FMS, presenting the highest average percentage of mastery. Additionally, we did not find association of the most popular sports in a given country with the mastery of the related skill. These results are interpreted as evidence for the importance of guidance on the mastery of FMS.

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