RESUMO
A bulk analysis of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) provides a quick, reliable, and highly informative system for DNA banding patterns that permit species identification. The present study evaluates the applicability of this system to Trichinella species identification. After a single amplification carried out on a single larva with the primer 816([CA]nRY) under high stringency conditions, which provide high reproducibility, we were able to identify by consistent banding patterns 5 sibling species: Trichinella spiralis (ISS48), 2 Trichinella britovi isolates (ISS11 and ISS86), Trichinella murrelli (ISS35), Trichinella nativa (ISS71), Trichinella nelsoni (ISS29); 3 additional Trichinella genotypes: T8 (ISS149), T9 (ISS408 and ISS409), and T6 (ISS34); and the nonencapsulated species Trichinella pseudospiralis (ISS13). Moreover, 33 new Trichinella isolates from 2 zoogeographical regions were unequivocally identified. All Trichinella isolates have shown an identical pattern with those produced by the reference strain. According to these data, we have demonstrated that ISSR-PCR is a robust technique that emerges as a useful new application for the molecular identification of Trichinella isolates in epidemiological studies.
Assuntos
DNA de Helmintos/química , Reação em Cadeia da Polimerase/métodos , Trichinella/genética , Animais , Canidae , Primers do DNA/química , DNA de Helmintos/isolamento & purificação , Eletroforese em Gel de Ágar , Feminino , Genótipo , Masculino , Repetições de Microssatélites/genética , Sus scrofa , Trichinella/classificaçãoRESUMO
The phylogenetic relationships of 10 rye landraces and cultivars from the north of Portugal and from Brazil were analysed using 20 isozyme loci, and a total of 511 PCR markers (342 ISSRs and 169 RAPDs). The isozymes were analysed in at least 100 plants of each population/cultivar and, therefore, we have data about intra and inter population/cultivar genetic variability. However, the analyses with ISSRs and RAPDs were obtained using a mix of 25 plants of each population. Therefore, each population/cultivar was reduced to one tube and we have no data about intra genetic variability. As expected in a cross pollinated crop we found genetic diversity and a larger variation within than among the populations using isozymes. Somewhat unexpectedly, however, we found that the breeding cultivars have the same level of heterozygosity as the landraces. The phylogenetic relationships obtained using isozymes among the landraces, synthetic cultivar and the cultivars from breeding programs do not reflect their origin. Moreover, the cultivar from Brazil is not separated from the remaining populations/cultivars studied. However, the data observed using RAPDs and ISSRs are in agreement with their known origin. The populations maintained by the farmers in the north of Portugal are grouped in a cluster in the phenogram and the C902591 (from Brazil), the Alvão (synthetic variety) and Larouco (a hybrid between Montalegre and Brazil) are in a different cluster. The ISSRs and RAPDs provide a rapid method for the production of polymorphic markers, which appear to correspond to known pedigree information.