A New Problem with Cross-Species Amplification of Microsatellites: Generation of Non-Homologous Products

Abstract

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.

Using a Multi-Locus Microsatellite Typing method improved phylogenetic distribution of Candida albicans isolates but failed to demonstrate association of some genotype with the commensal or clinical origin of the isolates

“…The use of microsatellite marker in population genetic studies is still a subject of controversy since size homoplasy, i.e., the fact that an allelic class could include alleles identical by descent (truly homologous) as well as alleles that achieved the same length via convergent evolutionary events (Orti et al, 1997), is a limit intrinsic to the microsatellite-based approach (Taylor et al, 1999), mainly due to the fact that mechanisms of mutation in microsatellites are incompletely known (Yue et al, 2010). However some authors reported that the extensive variability at microsatellite loci combined with the increase in the number of microsatellite markers studied often compensated for their homoplasious evolution (Estoup et al, 2002;Koskinen et al, 2004).…”

Isolation and characterization of 21 polymorphic microsatellite loci for the rockpool shrimp Palaemon elegans using Illumina MiSeq sequencing

“…elegans . Nevertheless cross-amplification from congeneric species is not generally feasible because inherent problems like allele size homoplasy, polymorphisms biases, null alleles presence, broken repeats motifs or amplification of non-orthologous loci could arise 15 – 18 . Thus, de novo development of species-specific microsatellite markers is strongly recommended.…”

Development and characterization of 24 polymorphic microsatellitelocifor the freshwater fishIchthyoelephas longirostris(Characiformes: Prochilodontidae)

“…No such markers have been developed to date for congeners of I. longirostris limiting our ability to resolve fine-scale differentiation and subdivision patterns among populations. Moreover, cross-amplification of microsatellite loci developed from other species often presents problems such as allele size homoplasy, unsuccessful amplification in phylogenetically distant species, lower levels of polymorphism, null alleles, broken repeat motifs and amplification of non-orthologous loci (Primmer et al, 2005; Barbará et al, 2007; Rutkowski, Sielezniew & Szostak, 2009; Yue, Balazs & Laszlo, 2010). …”