RESUMEN
The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.
Asunto(s)
Prueba de Histocompatibilidad/métodos , Complejo Mayor de Histocompatibilidad/genética , Alelos , Animales , Biología Computacional/métodos , Exones , Genes MHC Clase II , Genómica/métodos , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Prueba de Histocompatibilidad/normas , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Recombinación Genética , Rupicapra/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), the main vector of visceral leishmaniasis in the Americas, is a putative species complex. Molecular polymorphism was characterized in a 266 bp fragment of L. longipalpis homologous to period, a 'speciation gene' from Drosophila. Samples from the Brazilian localities of Jacobina (BA), Lapinha (MG) and Natal (RN) were analysed and the data indicate that the three populations are highly differentiated, with a very low level of gene flow between them. These results are in agreement with published pheromone and copulation song studies that suggest the existence of a sibling species complex in Brazil.
Asunto(s)
Proteínas Nucleares/genética , Psychodidae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Brasil , ADN/química , ADN/genética , Proteínas de Drosophila , Femenino , Variación Genética/genética , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Circadianas Period , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Genes involved in the reproductive isolation are particularly useful as molecular markers in speciation studies. Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), a putative species complex, is a vector of visceral leishmaniasis in Latin America. We isolated from this species a fragment homologous to cacophony, a Drosophila gene that encodes features of the lovesong, an acoustic signal that is important in the sexual isolation of closely related species and known to vary considerably among L. longipalpis putative siblings species. Using an intron of the sandfly cacophony as a marker, we analyzed the molecular variation and sequence divergence among five populations of L. longipalpis from Brazil, three allopatric (Jacobina, Lapinha and Natal) and two putative sympatric sibling species from the locality of Sobral. A high level of polymorphism was found and analysis of the data indicates that very little gene flow is occurring among the populations of Jacobina, Lapinha, and Natal. A high level of differentiation was also observed between the two putative sympatric species of Sobral, one of which seems to be the same sibling species found in Natal, while the other is somewhat more related to Jacobina and Lapinha. However, the amount of estimated gene flow among the Sobral siblings is about seven times higher than the previously estimated for period, another lovesong gene, perhaps indicating that introgression might be affecting cacophony more than period. The results suggest that L. longipalpis is not a single species in Brazil, but it is yet not clear whether the different populations studied deserve species status rather than representing an incipient speciation process.