Analysis of DNA polymorphism in ancient barley herbarium material: validation of the KASP SNP genotyping platform

The availability of crop specimens archived in herbaria and old seed collections represent valuable resources for the analysis of plant genetic diversity and crop domestication. The ability to extract ancient DNA (aDNA) from such samples has recently allowed molecular genetic investigations to be undertaken in ancient materials. While analyses of aDNA initially focused on the use of markers which occur in multiple copies such as the internal transcribed spacer region (ITS) within ribosomal DNA and those requiring amplification of short DNA regions of variable length such as simple sequence repeats (SSRs), emphasis is now moving towards the genotyping of single nucleotide polymorphisms (SNPs), traditionally undertaken in aDNA by Sanger sequencing. Here, using a panel of barley aDNA samples previously surveyed by Sanger sequencing for putative causative SNPs within the flowering-time gene PPD-H1, we assess the utility of the Kompetitive Allele Specific PCR (KASP) genotyping platform for aDNA analysis. We find KASP to out-perform Sanger sequencing in the genotyping of aDNA samples (78% versus 61% success, respectively), as well as being robust to contamination. The small template size (≥46 bp) and one-step, closed-tube amplification/genotyping process make this platform ideally suited to the genotypic analysis of aDNA, a process which is often hampered by template DNA degradation and sample cross-contamination. Such attributes, as well as its flexibility of use and relatively low cost, make KASP particularly relevant to the genetic analysis of aDNA samples. Furthermore, KASP provides a common platform for the genotyping and analysis of corresponding SNPs in ancient, landrace and modern plant materials. The extended haplotype analysis of PPD-H1 undertaken here (allelic variation at which is thought to be important for the spread of domestication and local adaptation) provides further resolution to the previously identified geographic cline of flowering-time allele distribution, illustrating how KASP can be used to aid genetic analyses of aDNA from plant species. We further demonstrate the utility of KASP by genotyping ten additional genetic markers diagnostic for morphological traits in barley, shedding light on the phenotypic traits, alleles and allele combinations present in these unviable ancient specimens, as well as their geographic distributions.

Trypanosoma rangeli isolates of bats from Central Brazil: Genotyping and phylogenetic analysis enable description of a new lineage using spliced-leader gene sequences

Trypanosoma rangeli infects several mammalian orders but has never confidently been described in Chiroptera, which are commonly parasitized by many trypanosome species. Here, we described trypanosomes from bats captured in Central Brazil identified as T rangeli,.T. dionisii, T cruzimarinkellei and T cruzi. Two isolates, Tra643 from Platyrrhinus lineatus and Tra1719 from Artibeus plamirostris were identified as T rangeli by morphological, biological and molecular methods, and confirmed by phylogenetic analyses. Analysis using SSU rDNA sequences clustered these bat trypanosomes together with T rangeli from other hosts, and separated them from other trypanosomes from bats. Genotyping based on length and sequence polymorphism of PCR-amplified intergenic spliced-leader gene sequences assigned Tra1719 to the lineage A whereas Tra643 was shown to be a new genotype and was assigned to the new lineage E. To our knowledge, these two isolates are the earliest T rangeli from bats and the first isolates from Central Brazil molecularly characterized. Rhodnius stali captured for this study was found infected by T rangeli and T cruzi. (c) 2008 Elsevier B.V. All rights reserved.

Characterization and polymorphism screening of IGF-I and prolactin genes in Nelore heifers

Insulin growth factor I (IGF-I) and prolactin (PRL) are peptide hormones that exert complementary effects on reproductive traits by acting on folliculogenesis. In view of the lack of information about the IGF-I and PRL genes in Bos indicus, the objective of this study was to partially characterize the promoter regions of these genes and to screen animals of different ages at first pregnancy for the presence of polymorphisms in these regions. In addition, we determined whether polymorphisms influence the regulation of the two hormone genes, evaluating their association with sexual precocity.The animals were divided into three groups according to age at first pregnancy: 1) 100 heifers considered to be sexually precocious that became pregnant at 15-16 months of age, 2) 100 heifers that became pregnant during the normal breeding season at 24 months of age, and 3) 100 heifers that did not become pregnant until 24 months of age. For the IGF-I gene, PCR-RFLP-SnaBI analysis showed the presence of genotypes AB and BB at frequencies of 0.02 and 0.98, respectively. Sequencing of the IGF-I gene fragment revealed a single nitrogen base change from cytosine to thymine, corresponding to the restriction site of SnaBI. The polymorphisms identified in the 5'-flanking region of the IGF-I gene may serve as a basis for future studies of molecular markers in cattle. For the PRL gene, PCR-RFLP-HaeIII analysis showed the presence of only one migration pattern, a finding characterizing the region studied as monomorphic. The study of other regions in the IGF-I and PRL genes might provide molecular data that can be used in the future for the selection of sexually precocious animals.

Association between MUC1 gene polymorphism and expected progeny differences in Nelore cattle (Bos primigenius indicus)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synthesis and physical properties of lipid-based poly(ester-urethane)s, I: Effect of varying polyester segment length

Four aliphatic thermoplastic poly(ester-urethane)s (PEUs) with similar molecular weights but varying polyesters molecular weight (534-1488 g/mol) were prepared from polyester diols, obtained by melt condensation of Azelaic acid and 1,9-Nonanediol, and 1,7-heptamethylene di-isocyanate (HPMDI) all sourced from vegetable oil feedstock. The thermal, and mechanical properties, and crystal structure of PEUs were investigated using DSC, TGA, DMA, tensile analysis and WAXD. For sufficiently long polyester chain, WAXD data indicated no hydrogen bonds polyethylene (PE)-like crystalline packing and for short polyester chains, small crystal domains with significant H-bonded polyamide (PA)-like packing. Crystallinity decreased with decreasing polyester molecular weights. The polymorphism of PEUs and consequently their melting characteristics were found to be largely controlled by polyester segment length. TGA of the PEUs indicated improved thermal stability with decreasing polyester chain length, suggesting a stabilization effect by urethane groups. Mechanical properties investigated by DMA and tensile analysis were found to scale predictably with the overall crystallinity of PEUs. (C) 2012 Elsevier Ltd. All rights reserved.

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pbl 8) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pbl Ba by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

Shorter CAG repeat length in the AR gene is associated with poor outcome in head and neck cancer

Objective: Alterations in the size of the [CAG](n) repeats of the AR gene have been described in several types tumors. The purpose of this study was to evaluate if there is an association between the AR [CAG](n) repeat alleles and the relative risk for head and neck cancer and to analyse microsatellite instability (MSI) and loss of heterozygosity (LOH) in these tumors.Design: Matched samples of blood and head and neck tumors were evaluated using two methodologies, silver-stained gels to perform the analyses of MSI and LOH, and automated analysis to confirm these results and for genotyping of the AR [CAG](n), repeat length. Sixty-nine individuals without cancer were used as a control group for both procedures. The Log-rank test was used to compare overall survival and disease-free survival curves. The Cox proportional hazards regression models were performed to determine the [CAG], repeats as an independent prognostic factor.Results: Patients with alleles <= 20 in the male group showed a correlation with lower disease-free survival (P = 0.0325) and with recurrence or metastasis (RR 2.52, CI 95%). in the female group, the allele 2 (longer allele) showed a significant lower mean of [CAG](n), repeat when compared to the control group. Microsatellite instability was detected in nine cases in both procedures. In six out of these nine cases, we observed a reduction of the AR [CAG](n) repeat length. LOH was detected in one out of 17 women informative for oral cancer in both procedures.Conclusion: These results suggest that short [CAG](n) repeat length (: 20) polymorphism is associated with poor prognosis in a subset of male patients with head and neck cancer and that AR gene microsatellite instability is uncommon in these tumors. (C) 2007 Elsevier Ltd. All rights reserved.

The 434(G > C) polymorphism in the eosinophil cationic protein gene and its association with tissue eosinophilia in oral squamous cell carcinomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification and characterization of polymorphisms within the 5' flanking region, first exon and part of first intron of bovine GH gene

The aim of the present study was to identify and characterize polymorphisms within the 5' flanking region, first exon and part of first intron of the bovine growth hormone gene among different beef cattle breeds: Nelore (n = 25), Simmental (n = 39), Simbrasil (n = 24), Simmental x Nelore (n = 30), Canchim x Nelore (n = 30) and Angus x Nelore (n = 30). Two DNA fragments (GH1, 464 bp and GH2, 453 bp) were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. Within the GH1 fragment, five polymorphisms were identified, corresponding to three different alleles: GH1.1, GH1.2 and GH1.3 (GenBank: AY662648, AY662649 and AY662650, respectively). These allele sequences were aligned and compared with bovine GH gene nucleotide sequence (GenBank: M57764 and AF118837), resulting in the identification of five insertion/deletions (INDELs) and five single nucleotide polymorphisms (SNPs). In the GH2 fragment two alleles were identified, GH2.1 and GH2.2 (GenBank: AY662651 and AY662652, respectively). The allele sequences were compared with GenBank sequences (M57764, AF007750 and AH009106) and three INDELs and four SNPs were identified. In conclusion, we were able to identify six new polymorphisms of the bovine GH gene (one INDEL and five SNPs), which can be used as molecular markers in genetic studies.

Genetic polymorphism of Streptococcus mutans in Brazilian family members

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Association of apolipoprotein E polymorphism in late-onset Alzheimer's disease and vascular dementia in Brazilians

The genetic basis for dementias is complex. A common polymorphism in the apolipoprotein E (APOE) gene is considered to be the major risk factor in families with sporadic and late-onset Alzheimer's disease as well as in the general population. The distribution of alleles and genotypes of the APOE gene in late-onset Alzheimer's disease (N = 68), other late-life dementias (N = 39), and in cognitively normal controls (N = 58) was determined, as also was the risk for Alzheimer's disease associated with the epsilon4 allele. Peripheral blood samples were obtained from a total of 165 individuals living in Brazil aged 65-82 years. Genomic DNA was amplified by the polymerase chain reaction and the products were digested with HhaI restriction enzyme. APOE epsilon2 frequency was considerably lower in the Alzheimer's disease group (1%), and the epsilon3 allele and epsilon3/epsilon3 genotype frequencies were higher in the controls (84 and 72%, respectively) as were the epsilon4 allele and epsilon3/epsilon4 genotype frequencies in Alzheimer's disease (25 and 41%, respectively). The higher frequency of the epsilon4 allele in Alzheimer's disease confirmed its role as a risk factor, while epsilon2 provided a weak protection against development of the disease. However, in view of the unexpectedly low frequency of the epsilon4 allele, additional analyses in a more varied Brazilian sample are needed to clarify the real contribution of apolipoprotein E to the development of Alzheimer's disease in this population.

FISH using a gag-like fragment probe reveals a common Ty3-gypsy-like retrotransposon in genome of Coffea species

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Taenia solium and Taenia saginata: Identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies. © 2007 Elsevier Inc. All rights reserved.

Cytogenetic and random amplified polymorphic DNA analysis of Leptodactylus species from rural and urban environments (Anura, Amphibia)

Cytogenetic and random amplified polymorphic DNA analyses carried out in the species Leptodactylus podicipinus, L. ocellatus, L. labyrinthicus, and L. fuscus from rural and urban habitats of the northwest region of São Paulo State, Brazil, showed that the karyotypes (2n = 22), constitutive heterochromatin distribution and nucleolus organizer region (NOR) location did not differ between the populations from the two environments. The in situ hybridization with an rDNA probe confirmed the location of the NORs on chromosome 8 revealing an in tandem duplication of that region in one of the chromosomes of L. fuscus. DAPI showed that part of the C-band-positive heterochromatin is rich in AT, including that in the proximity the NORs in L. podicipinus and L. ocellatus. The molecular analyses showed that the two populations (urban and rural) of L. podicipinus and L. fuscus are similar from a genetic point of view. The urban and rural populations of species L. ocellatus and L. labyrinthicus showed differences in genetic structures, probably due to urbanization which interferes with the dispersion of those frogs. The marked differences observed between the two populations of L. ocellatus can be representing the cryptic condition of the species. Unweighted pair-group method of analysis and genetic distance analysis detected the genetic proximity between L. ocellatus and L. fuscus. The results indicate that there was no reduction in the genetic diversity in the populations from the urban environment; however, the survival of these frogs would not be guaranteed in the case of an increase in human impact especially for populations of L. labyrinthicus and L. ocellatus. ©FUNPEC-RP.