RESUMO
We report here the development of 65 novel microsatellite loci and construction of a composite genetic linkage map for Culex pipiens complex mosquitoes. Microsatellites were identified by in silico screening of the Culex quinquefasciatus genome assembly. Cross-species utility of 73 microsatellites for population studies in C. pipiens sensu stricto and C. quinquefasciatus was evaluated by genotyping a subset of samples collected in Indiana, United States, and Point Fortin, Trinidad. Allele frequencies of 67 microsatellites were within Hardy-Weinberg expectations in both population subsets. A composite linkage map was constructed based on restriction fragment length polymorphism and microsatellite polymorphisms in 12 independent F1 intercross mapping populations. The composite map consists of 61 marker loci totaling 183.9 cM distributed across the 3 linkage groups. These loci cover 29.5, 88.8, and 65.6 cM on chromosomes I-III, respectively, and allow for assignment of 10.4% of the genome assembly and 13.5% of the protein coding genes to chromosome position. Our results suggest that these microsatellites will be useful for mapping and population studies of 2 pervasive species in the C. pipiens complex. Moreover, the composite map presented here will serve as a basis for the construction of high-resolution genetic and physical maps, as well as detection of quantitative trait loci to aid in the investigation of complex genetic traits influencing phenotypes of interest.
Assuntos
Mapeamento Cromossômico , Culex/genética , Marcadores Genéticos/genética , Animais , Frequência do Gene , Ligação Genética , Genoma , Genótipo , Repetições de Microssatélites/genética , Polimorfismo de Fragmento de Restrição/genética , Trinidad e Tobago , Estados UnidosRESUMO
BACKGROUND: Microsatellite markers have proven useful in genetic studies in many organisms, yet microsatellite-based studies of the dengue and yellow fever vector mosquito Aedes aegypti have been limited by the number of assayable and polymorphic loci available, despite multiple independent efforts to identify them. Here we present strategies for efficient identification and development of useful microsatellites with broad coverage across the Aedes aegypti genome, development of multiplex-ready PCR groups of microsatellite loci, and validation of their utility for population analysis with field collections from Haiti. RESULTS: From 79 putative microsatellite loci representing 31 motifs identified in 42 whole genome sequence supercontig assemblies in the Aedes aegypti genome, 33 microsatellites providing genome-wide coverage amplified as single copy sequences in four lab strains, with a range of 2-6 alleles per locus. The tri-nucleotide motifs represented the majority (51%) of the polymorphic single copy loci, and none of these was located within a putative open reading frame. Seven groups of 4-5 microsatellite loci each were developed for multiplex-ready PCR. Four multiplex-ready groups were used to investigate population genetics of Aedes aegypti populations sampled in Haiti. Of the 23 loci represented in these groups, 20 were polymorphic with a range of 3-24 alleles per locus (mean = 8.75). Allelic polymorphic information content varied from 0.171 to 0.867 (mean = 0.545). Most loci met Hardy-Weinberg expectations across populations and pairwise FST comparisons identified significant genetic differentiation between some populations. No evidence for genetic isolation by distance was observed. CONCLUSION: Despite limited success in previous reports, we demonstrate that the Aedes aegypti genome is well-populated with single copy, polymorphic microsatellite loci that can be uncovered using the strategy developed here for rapid and efficient screening of genome supercontig assemblies. These loci are suitable for genetic and population studies using multiplex-PCR.