FishDNAIDs: DNA barcoding as a tool in the development and validation in silico and in vitro of detection systems to four species of Characiformes of commercial importance in the Brazilian Amazon.

BACKGROUND: The COI mitochondrial gene has been chosen as the "DNA barcode in animals" and the large quantity of genetic information in public databanks gives solid support for the use of DNA barcoding as a promising tool for the development of a specific molecular detection system. METHODS AND RESULTS: The present study aimed to develop a Specific Molecular Detection System (SMDS: FishDNAIDs) (primers and probe sets) for the following four target species: Prochilodus nigricans, Potamorhina altamazonica, Psectrogaster rutiloides and Triportheus angulatus, in qPCR assays. In silico and in vitro tests (using gDNA) were performed to test these sets. The database generated contained the cytochrome c oxidase subunit I (COI) nucleotide sequence for 183 specimens of Characiformes, distributed in 34 species representing eight families. In silico, primers designed for the target species amplified different species from the same genus, except for P. rutiloides, which amplified only the target species. In the in vitro test, using the SYBRGreentm and TaqMan® fluorescence systems, both sets detected the respective target species (P. nigricans, P. altamazonica, P. rutiloides and T. angulatus). In the qPCR assays using SYBRGreentm, species considered to be related were also detected, in addition to the target species, with the exception of P. amazonica and P. essequibensis (correlated to P. rutiloides). All target species were detected in the qPCR assays using the TaqMan® system; however, with the SMDS PALT, the target species P. altamazonica was detected with low CT values (22.21 ± 0.17) as well as the correlates of P. latior and P. pristigaster, though with high CT values (23.51 ± 0.19 and 30.21 ± 0.95). This assay uniquely identifies known adult tissue samples from all four species. CONCLUSIONS: The primers and probe sets developed can act as powerful tools for detecting the target Characiformes species.

Microsatellite DNA markers for the commercially important fish Hypophthalmus donascimientoi (Pisces: Siluriformes: Pimelodidae) in the Amazon basin: isolation, characterization and amplification in congeneric species.

BACKGROUND: The genus Hypophthalmus comprises six species (H. edentatus, H. marginatus, H. fimbriatus, and H. oremaculatus), and the recently described: H. donascimientoi and H. celiae. The popular name for Hypophthalmus spp. in Brazil is mapará, this name refers to the six species. This group of fish has commercial importance for the states of Amazonas and Pará and, for this reason, requires studies to identify fish stocks. One approach is to use molecular markers, which have been very useful in studies with identification and population analysis of fish. Microsatellite molecular markers (SSRs) are one of the most informative markers for this purpose. There is little populations study of Hypophthalmus using SSRs, and there are less than six loci for the species Hypophthalmus marginatus available in the literature. With the construction of a genomic library of H. donascimientoi, we aimed to isolate and characterize SSRS markers and evaluate the extent of interspecific amplification. METHODS AND RESULTS: A genomic library was constructed with regions enriched of microsatellite for Hypophthalmus donascimientoi. A total of 126 contigs with 42 SSRs were used to design flanking primers for 39 microsatellites. Fifteen loci were characterized in three locations of the Solimões/Amazonas Rivers. The number of alleles ranged from one to 17 with a total of 126 alleles. The mean observed heterozygosity (HO) and expected heterozygosity (HE) were 0.721-0.692, respectively (S.d. HO 0.061 and HE 0.060). Two loci showed significant deviation in the HWE. The PIC ranged from 0.375 to 0.908. Such identified, 12 highly informative loci, and two moderately informative loci. Among the fifteen loci characterized, seven were successfully amplified in four other species of the genus. CONCLUSIONS: The microsatellite showed promise for estimating the genetic variability of H. donascimientoi and can be used as an efficient tool in population analyses of this species and in congeneric species analyzed.

The most important fishery resource in the Amazon, the migratory catfish Brachyplatystoma vaillantii (Siluriformes: Pimelodidae), is composed by an unique and genetically diverse population in the Solimões-Amazonas River System

The migratory catfish Brachyplatystoma vaillantii is one of the most important fishery resources in the Amazon. Intense capture occurs associated to its life cycle. In order to know the genetic status, we sequenced the mitochondrial DNA control region from 150 individuals of B. vaillantii, collected in five fishing landing locations, covering the length of the Solimões-Amazonas River in Brazil. Genetic diversity parameters suggest there is no genetic differentiation between the five localities. Population's expansion indicated by R 2 and Fu's Fs tests was also confirmed by the high number of unique haplotypes found. The Analyses of molecular variance indicated that nearly all variability was contained within locations (99.86%), and estimates of gene flow among B. vaillantii were high (F ST = 0.0014). These results suggest that Brachyplatystoma vaillantii forms a panmitic population along the Solimões-Amazonas River and, has greater genetic variability than other species of the Brachyplatystoma genus available so far. Although the influence of different tributaries on B. vaillantii migration patterns remains uncertain, a single population in the main channel should be consider in future policies for management of this resource. However, since the species' life cycle uses habitats in several countries, its management and conservation depend greatly of internationally joined efforts.(AU)

O bagre migrador, Brachyplatystoma vaillantii, é um dos mais importantes recursos pesqueiros da Amazônia. Intensa captura ocorre associada ao seu ciclo de vida. Para conhecer seu status genético, sequenciamos a região de controle do DNA mitocondrial de 150 indivíduos, coletados em cinco locais de desembarque pesqueiro, abrangendo toda a extensão do rio Solimões-Amazonas no Brasil. Os parâmetros de diversidade genética sugerem que não existe diferenciação genética entre as cinco localidades amostradas. A expansão populacional indicada pelos testes R 2 e Fs de Fu, também foi confirmada pelo elevado número de haplótipos únicos encontrados. A análise de variância molecular indicou que quase toda a variabilidade estava contida nas localidades (99,86%), e as estimativas de fluxo gênico desta espécie eram altas (F ST = 0,0014). Esses resultados sugerem que Brachyplatystoma vaillantii forma uma população panmítica ao longo do rio Solimões-Amazonas com maior variabilidade genética que outras espécies do gênero Brachyplatystoma disponíveis no momento. Embora a influência dos diferentes afluentes na migração de B. vaillantii permaneça incerta, em futuras políticas de gestão deste recurso deve-se considerá-lo como uma única população no canal principal. Entretanto, uma vez que seu ciclo de vida abrange habitats em vários países, seu manejo e conservação dependem muito de esforços internacionais em conjunto.(AU)