A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

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

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.

A simple protocol for a low invasive DNA accessing in Stenella longirostris (Cetacea: Delphinidae)

The most significant studies about the spinner dolphin (Stenella longirostris) in the Southwestern Atlantic Ocean were conducted in Fernando de Noronha Archipelago, off Northeastern Brazil. The continuity of these studies depends upon the development of non-invasive methods. In this work, we present results from the skin swabbing sampling procedure for this species. We tested the performance of this method for nuclear and mitochondrial DNA analysis, unknown for this population. A total of skin 161 samples were collected during two expeditions. After the contacts the most of the dolphins remained close to the boat. Microsatellites markers and cytochrome b region primers were evaluated and the respective fragments were successfully amplified. Thus, skin swabbing may be considered an efficient strategy to obtain tissue samples for spinner dolphin genetic analysis in Fernando de Noronha Archipelago.

A single multiplex SNaPshot reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

SNaPshot minisequencing reaction is in increasing use because of its fast detection of many polymorphisms in a single assay. In this work we described a highly sensitive single nucleotide polymorphisms (SNPs) typing method with detection of 42 mitochondrial DNA (mtDNA) SNPs in a single PCR and SNaPshot multiplex reaction in order to allow haplogroup classification in Latin American admixture population. We validated the panel typing 160 Brazilian individuals. DNA was extracted from blood spotted on filter paper using Chelex protocol. Forty SNPs were selected targeting haplogroup-specific mutations in Europeans, Africans and Asians (only precursors of Native Americans haplogroups A2, B2, C1, and D1) and two non-coding SNPs were chosen to increase the power of discrimination between individuals (SNPs positions 16,519 and 16,362). It was done using a modified version of a previously published multiplex SNaPshot minisequencing reaction established to resolve European haplogroups, adding SNPs targeting Africans (L0, L1, L2, L3, and L*) and Asians (A, B, C, and D) haplogroups based on SNPs described at PhyloTree.org build 2. PCR primers were designed using PerlPrimer software and checked with the Autodimer program. Thirty-three primer-pairs were used to amplify 42 SNPs. Using this panel, we were able to successfully classify 160 individuals into their correct haplogroups. Complete SNP profiles were obtained from 10. pg of total DNA. We conclude that it is possible to build and genotype more than 40 mtDNA SNPs in a single multiplex PCR and SNaPshot reaction, with sensitivity and reliability, resolving haplogroup classification in admixture populations. © 2011.

Fast-Evolving Mitochondrial DNA in Ceriantharia: A Reflection of Hexacorallia Paraphyly?

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

Evolutionary Relationships of the Triatoma matogrossensis Subcomplex, the Endemic Triatoma in Central-Western Brazil, Based on Mitochondrial DNA Sequences

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Taxonomic status of the genus Sotalia: Species level ranking for tucuxi (Sotalia fluviatilis) and costero (Sotalia guianensis) dolphins

Dolphins of the genus Sotalia are found along the Caribbean and Atlantic coasts of Central and South America and in the Amazon River and most of its tributaries. At present, the taxonomy of these dolphins remains unresolved. Although five species were described in the late 1800s, only one species is recognized currently (Sotalia fluviatilis) with two ecotypes or subspecies, the coastal subspecies (Sotalia fluviatilis guianensis) and the riverine subspecies (Sotalia fluviatilis fluviatilis). Recent morphometric analyses, as well as mitochondrial DNA analysis, suggested recognition of each subspecies as separate species. Here we review the history of the classification of this genus and present new genetic evidence from ten nuclear and three mitochondrial genes supporting the elevation of each subspecies to the species level under the Genealogical/Lineage Concordance Species Concept and the criterion of irreversible divergence. We also review additional evidence for this taxonomic revision from previously published and unpublished genetic, morphological, and ecological studies. We propose the common name costero for the coastal species, Sotalia guianensis (Van Beneden 1864), and accept the previously proposed tucuxi dolphin, Sotalia fluviatilis (Gervais, 1853), for the riverine species.

Origem do suíno casco-de-burro e sua relação genética com populações ibéricas e americanas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Phylogenetic analysis of the order Pleuronectiformes (Teleostei) based on sequences of 12S and 16S mitochondrial genes

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

Populations analysis of the Brazilian Sharpnose Shark Rhizoprionodon lalandii (Chondrichthyes: Carcharhinidae) on the São Paulo coast, Southern Brazil: inferences from mt DNA sequences

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

Phylogenetic analysis of flatfish (order pleuronectiformes) based on mitochondrial 16s rDNA sequences

The phylogenetic relationships of the order Pleuronectiformes are controversial and at some crucial points remain unresolved. To date most phylogenetic studies on this order have been based on morpho-anatomical criteria, whereas only a few sequence comparisons based studies have been reported. In the present study, the phylogenetic relationships of 30 flatfish species pertaining to seven different families were examined by sequence analysis of the first half of the 16S mitochondrial DNA gene. The results obtained did not support percoids as the sister group of pleuronectiforms. The monophyletic origin of most families analyzed, Soleidae, Scophthalmidae, Achiridae, Pleuronectidae and Bothidae, was strongly supported, except for Paralichthyidae which was clearly subdivided into two groups, one of them associated with high confidence to Pleuronectidae. The analysis of the 16S rRNA gene also suggested the monophyly of Pleuronectiforms as the most probable hypothesis and consistently supported some major interfamily groupings.

Amplifiability of mitochondrial, microsatellite and amelogenin DNA loci from fecal samples of red brocket deer Mazama americana (Cetartiodactyla, Cervidae)

We tried to amplify mitochondrial, microsatellite and amelogenin loci in DNA from fecal samples of a wild Mazama americana population. Fifty-two deer fecal samples were collected from a 600-ha seasonal semideciduous forest fragment in a subtropical region of Brazil (21°20′, 47°17′W), with the help of a detection dog; then, stored in ethanol and georeferenced. Among these samples 16 were classified as fresh and 36 as non-fresh. DNA was extracted using the QIAamp® DNA Stool Mini Kit. Mitochondrial loci were amplified in 49 of the 52 samples. Five microsatellite loci were amplified by PCR; success in amplification varied according to locus size and sample age. Successful amplifications were achieved in 10/16 of the fresh and in 13/36 of the non-fresh samples; a negative correlation (R = -0.82) was found between successful amplification and locus size. Amplification of the amelogenin locus was successful in 22 of the 52 samples. The difficulty of amplifying nuclear loci in DNA samples extractedfrom feces collected in the field was evident. Some methodological improvements, including collecting fresh samples, selecting primers for shorter loci and quantifying the extracted DNA by real-time PCR, are suggested to increase amplification success in future studies. © FUNPEC-RP.

A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA

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

DNA barcoding reveals hidden diversity in the Neotropical freshwater fish Piabina argentea (Characiformes: Characidae) from the Upper Parana Basin of Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)