RESUMO

Complete mitochondrial sequences can be rapidly obtained and are widely available, providing a great source of species information and allowing for the discovery of new specific molecular markers. However, for some taxonomic groups, traditional approaches for species delimitation are impaired by the low genetic distance values. In these cases, other species-level markers are used. For Prochilodus, which includes important neotropical fish species, species-level delimitation usually results in poor phylogenetic resolution when using mitochondrial COI/cytB genes as barcoding markers because of low genetic variability and low species-level resolution. Thus, in this study, we developed an approach to design and validate new barcoding markers with high species-level resolution obtained from the D-loop region, using Prochilodus spp. as a model. For the new barcoding marker validation, the amplicon region was used to infer the phylogenetic relationships of Prochilodus spp. through three distinct methods: Bayesian inference (BI), Neighbor-Joining method (NJ), and Maximum Likelihood method (ML). The phylogenetic relationships of Prochilodus spp. revealed high resolution at species-level, nonoverlapping clades, and high branch support. The genetic distance results allied to two different clustering methods (Bayesian Poisson tree processes and automatic barcode gap discovery) revealed the existence of a barcoding gap, thus, validating the use of the barcoding markers designed in this study. The approach proposed here may, therefore, be expanded to other taxa to access and validate new barcoding markers with higher resolution at the species level.

Assuntos

Caraciformes/classificação , Marcadores Genéticos , Mitocôndrias/genética , Animais , Teorema de Bayes , Caraciformes/genética , Código de Barras de DNA Taxonômico , Genoma Mitocondrial , Filogenia , Especificidade da Espécie

RESUMO

The Thraupidae family is one of the most wanted by bird breeders in Brazil because it is represented by its diverse, colorful and melodious singers. The Great-billed Seed-finch, Sporophila maximiliani, is the only representative of the genus Sporophila considered critically endangered in Brazil. Due to the demands of environmental agencies and of conservation programs, there is a need to increase the number of molecular markers available for the genus and specially for S. maximiliani. Therefore, this work aimed to provide a new set of microsatellite markers for S. maximiliani in order to help bird breeders and environmental agencies on fulfilling its demands as well as contributing with extra genetics tools for conservation programs of the S. maximiliani. Of the 30 markers developed, 25 successfully amplified, and 22 were polymorphic. Annealing temperature varied from 52 to 64 °C, number of alleles from 2 to 13, and the medium allele richness was 7.25 and medium expected, observed heterozygosity and PIC were, respectively, 0.812, 0.661 and 0.752. The probability of identity estimate was 8.54 × 10-27 and all the other probabilities of non-exclusion (sib-identity, parent pair and first-parent) were < 0.001, indicating that this set of microsatellite markers have high genetic variability and high power of individual genetic differentiation for S. maximiliani. Therefore, this work increases the options of molecular markers to be used on inspection for environmental agencies and for conservation programs on analyzing genetic variability and population studies for S. maximiliani.

Assuntos

Tentilhões/genética , Repetições de Microssatélites/genética , Alelos , Animais , Conservação dos Recursos Naturais/métodos , Primers do DNA/genética , Espécies em Perigo de Extinção , Loci Gênicos/genética , Variação Genética/genética , Heterozigoto , Passeriformes/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético/genética , Análise de Sequência de DNA/métodos

RESUMO

Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple loci from many samples. These processes can be laborious and expensive. The choice of microsatellites and PCR calibration for genotyping are particularly daunting. Here we optimized a low-cost genotyping method using multiple microsatellite loci for simultaneous genotyping of up to 384 samples using next-generation sequencing (NGS). We designed primers with adapters to the combinatorial barcoding amplicon library and sequenced samples by MiSeq. Next, we adapted a bioinformatics pipeline for genotyping microsatellites based on read-length and sequence content. Using primer pairs for eight microsatellite loci from the fish Prochilodus costatus, we amplified, sequenced, and analyzed the DNA of 96, 288, or 384 individuals for allele detection. The most cost-effective methodology was a pseudo-multiplex reaction using a low-throughput kit of 1 M reads (Nano) for 384 DNA samples. We observed an average of 325 reads per individual per locus when genotyping eight loci. Assuming a minimum requirement of 10 reads per loci, two to four times more loci could be tested in each run, depending on the quality of the PCR reaction of each locus. In conclusion, we present a novel method for microsatellite genotyping using Illumina combinatorial barcoding that dispenses exhaustive PCR calibrations, since non-specific amplicons can be eliminated by bioinformatics analyses. This methodology rapidly provides genotyping data and is therefore a promising development for large-scale conservation-genetics studies.

RESUMO

Sporophila maximiliani, commonly known as Great-billed Seed-Finch or 'bicudo', is a trafficked bird in Brazil due to the species' beauty and singing, which is appreciated by breeders and collectors. Generally, the Environmental Military Police and IBAMA maintain enforcement actions, rescue work, and seizure of illegally traded of 'bicudo' specimens. The genomic DNA of one specimen was sequenced on MiSeq (Illumina) sequencer. The reads obtained were analyzed, trimmed, and de novo assembled using CLC Workbench® v9.0 (CLC Bio-Qiagen). The mitochondrial genome of S. maximiliani consisted of 16,765 base pairs, 2 ribosomal RNA, 22 transporter RNA, 13 protein-coding genes, and 1 control region. The molecular phylogeny demonstrated that the mitochondrial genome of S. maximiliani diverged from others related Passeriformes.

RESUMO

Among oysters, species of Crassostrea (Sacco, 1897) are the most attractive to aquaculture. In Brazil, the genus is represented by C. rhizophorae (Guilding, 1828) and C. brasiliana (Lamarck, 1819). Because the maturation and breeding technology is not well developed for these species, aquaculturists need a reliable method to decide the correct time to place spat collectors in the field, and to identify both species, which are morphologically similar. In this study a specific Multiplex PCR protocol was developed, using one pair of universal primers from 18S rDNA as a positive control and a pair of specific primers for each target species. The sensitivity and specificity of the protocol was evaluated. It detected C. rhizophorae DNA in low concentrations, and C. brasiliana DNA in even lower concentrations. Further, the Multiplex PCR proved efficient in detecting DNA in concentrations equivalent to that of a single larva of each species, either separated or combined, when mixed with total DNA extract of a plankton sample representing 1000 L of filtered water. Field tests confirmed the applicability of the protocol, which holds the promise to become an important tool for aquaculture or conservation programs, allowing for the continuous monitoring of the life cycle of C. brasiliana and C. rhizophorae, by detecting the right periods of larval release and settlement.

RESUMO

Among oysters, species of Crassostrea (Sacco, 1897) are the most attractive to aquaculture. In Brazil, the genus is represented by C. rhizophorae (Guilding, 1828) and C. brasiliana (Lamarck, 1819). Because the maturation and breeding technology is not well developed for these species, aquaculturists need a reliable method to decide the correct time to place spat collectors in the field, and to identify both species, which are morphologically similar. In this study a specific Multiplex PCR protocol was developed, using one pair of universal primers from 18S rDNA as a positive control and a pair of specific primers for each target species. The sensitivity and specificity of the protocol was evaluated. It detected C. rhizophorae DNA in low concentrations, and C. brasiliana DNA in even lower concentrations. Further, the Multiplex PCR proved efficient in detecting DNA in concentrations equivalent to that of a single larva of each species, either separated or combined, when mixed with total DNA extract of a plankton sample representing 1000 L of filtered water. Field tests confirmed the applicability of the protocol, which holds the promise to become an important tool for aquaculture or conservation programs, allowing for the continuous monitoring of the life cycle of C. brasiliana and C. rhizophorae, by detecting the right periods of larval release and settlement.

RESUMO

Among oysters, species of Crassostrea (Sacco, 1897) are the most attractive to aquaculture. In Brazil, the genus is represented by C. rhizophorae (Guilding, 1828) and C. brasiliana (Lamarck, 1819). Because the maturation and breeding technology is not well developed for these species, aquaculturists need a reliable method to decide the correct time to place spat collectors in the field, and to identify both species, which are morphologically similar. In this study a specific Multiplex PCR protocol was developed, using one pair of universal primers from 18S rDNA as a positive control and a pair of specific primers for each target species. The sensitivity and specificity of the protocol was evaluated. It detected C. rhizophorae DNA in low concentrations, and C. brasiliana DNA in even lower concentrations. Further, the Multiplex PCR proved efficient in detecting DNA in concentrations equivalent to that of a single larva of each species, either separated or combined, when mixed with total DNA extract of a plankton sample representing 1000 L of filtered water. Field tests confirmed the applicability of the protocol, which holds the promise to become an important tool for aquaculture or conservation programs, allowing for the continuous monitoring of the life cycle of C. brasiliana and C. rhizophorae, by detecting the right periods of larval release and settlement.