Abstract
Microsatellites have been widely used in studies on population genetics, ecology and evolutionary biology. However, microsatellites are not always available for the species to be studied and their isolation could be time-consuming. In order to save time and effort researchers often rely on cross-species amplification. We revealed a new problem of microsatellite cross-species amplification in addition to size homoplasy by analyzing the sequences of electromorphs from seven catfish species belonging to three different families (Clariidae, Heteropneustidae and Pimelodidae). A total of 50 different electromorphs were amplified from the seven catfish species by using primers for 4 microsatellite loci isolated from the species Clarias batrachus. Two hundred and forty PCR-products representing all 50 electromorphs were sequenced and analyzed. Primers for two loci amplified specific products from orthologous loci in all species tested, whereas primers for the other two loci produced specific and polymorphic bands from some non-orthologous loci, even in closely related non-source species. Size homoplasy within the source species was not obvious, whereas extensive size homoplasy across species were detected at three loci, but not at the fourth one. These data suggest that amplification of products from non-orthologous loci and appearance of size homoplasy by cross-amplification are locus dependent, and do not reflect phylogenetic relationship. Amplification of non-orthologous loci and appearance of size homoplasy will lead to obvious complications in phylogenetic interference, population genetic and evolutionary studies. Therefore, we propose that sequence analysis of cross-amplification products should be conducted prior to application of cross-species amplification of microsatellites.
Highlights
Microsatellites have been widely used in studies on population genetics, ecology and evolutionary biology
1.1 Species and phylogenetic analyses Seven species of catfish were used in this study, namely: Clarias batrachus, C. fuscus (Cfu), C. gariepinus (Cga), C. macrocephalus (Cma), Heterobranchus longfilis (Hlo), Heteropneustes fossilis (Hfo), and Phractocephalus hemioliopterus (Phe)
Size homoplasy could only be detected in C. macrocephalus, but not in the source species, C. fuscus or P. hemioliopterus
Summary
Microsatellites have been widely used in studies on population genetics, ecology and evolutionary biology. In order to save time and effort researchers often rely on cross-species amplification (Chang et al, 2008; Küpper et al, 2008; Kayser et al, 1996; Kijas et al, 1995; Lin et al, 2008) This procedure uses PCR primers complementary to the flanking regions of loci from a extensively studied (source) species to amplify microsatellites from closely (Harr et al, 1998) or sometimes quite distantly related species (Gonzalez-Martinez et al, 2004) for which no such markers are described. Cross-species amplification of microsatellites generated polymorphic products from non-orthologous loci, which were revealed by sequence analysis of 240 clones representing all 50 electromorphs from four loci in seven species (Clarias batrachus, C. fuscus, C. gariepinus, C. macrocephalus, Heterobranchus longfilis, Heteropneustes fossilis and Phractocephalus hemioliopterus)
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