Identification and validation of the first EST-SSR markers based on transcriptome of Anopheles darlingi, the primary transmitter of malaria in Brazil.

BACKGROUND: Anopheles darlingi is a monotypic species in terms of its morphological, genetic, and behavioral aspects and is the primary transmitter of human malaria (99%) in Brazil, especially in the Brazilian Amazon. In this pioneering study, 15 expressed sequence tag (EST)-simple sequence repeat (SSR) markers were obtained and characterized in samples from the municipality of São Gabriel da Cachoeira, Amazonas state, Brazil, with polymorphisms that can be used for further genetic research. METHODS AND RESULTS: The specimens (from egg to larval stage) collected were bred in the insectary at INPA (National Institute for Amazonian Research). The SSR repeats within the contigs of the A. darlingi EST banks were confirmed on the Vector Base site. DNA was extracted and amplified using polymerase chain reaction and then genotyped. Fifteen polymorphic SSR loci were identified and characterized. The number of alleles totaled 76 and ranged from 2 to 9. The observed heterozygosity varied between 0.026 and 0.769, the expected heterozygosity between 0.025 and 0.776, and the mean polymorphism information content was 0.468. Eight loci showed Hardy-Weinberg equilibrium (HWE) after Bonferroni correction (P: (5%) ≤ 0.0033). No linkage disequilibrium was found among the loci. CONCLUSIONS: The polymorphic SSRs of the loci have been shown to be efficient for investigation of the variability and genetic population structure of A. darlingi.

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.

Physical Mapping of the Anopheles (Nyssorhynchus) darlingi Genomic Scaffolds.

The genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.

Phylogenetic analysis of the GST family in Anopheles (Nyssorhynchus) darlingi.

Anopheles darlingi Root, 1926 and Anopheles gambiae (Diptera: Culicidae) are the most important human malaria vectors in South America and Africa, respectively. The two species are estimated to have diverged 100 million years ago. Studies on the phylogenetics and evolution of gene sequences, such as glutathione S-transferase (GST) in disease-transmitting mosquitoes are scarce. The sigma class GST (KC890767) from the transcriptome of An. darlingi captured in the Brazilian Amazon was studied by in silico hybridization, and mapped to chromosome 3 of An. gambiae. The sigma class GST of An. darlingi was used for phylogenetic analyses to understand the GST base composition of the most recent common ancestor between An. darlingi, Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. The GST (KC890767) of An. darlingi was studied to generate the main divergence branches using a Neighbor-Joining and bootstrapping approaches to confirm confidence levels on the tree nodes that separate the An. darlingi and other mosquito species. The results showed divergence between An. gambiae, Ae. Aegypti, Cx. quinquefasciatus, and Phlebotomus papatasi as outgroup, and the homology relationship between sigma class GST of An. darlingi and GSTS1_1 gene of An. gambiae was valuable for phylogenetic and evolutionary studies.