Abstract
Microsatellites are popular genetic markers in molecular ecology, genetic mapping and forensics. Unfortunately, despite recent advances, the isolation of de novo polymorphic microsatellite loci often requires expensive and intensive groundwork. Primers developed for a focal species are commonly tested in a related, non-focal species of interest for the amplification of orthologous polymorphic loci; when successful, this approach significantly reduces cost and time of microsatellite development. However, transferability of polymorphic microsatellite loci decreases rapidly with increasing evolutionary distance, and this approach has shown its limits. Whole genome sequences represent an under-exploited resource to develop cross-species primers for microsatellites. Here we describe a three-step method that combines a novel in silico pipeline that we use to (1) identify conserved microsatellite loci from a multiple genome alignments, (2) design degenerate primer pairs, with (3) a simple PCR protocol used to implement these primers across species. Using this approach we developed a set of primers for the mammalian clade. We found 126,306 human microsatellites conserved in mammalian aligned sequences, and isolated 5,596 loci using criteria based on wide conservation. From a random subset of ∼1000 dinucleotide repeats, we designed degenerate primer pairs for 19 loci, of which five produced polymorphic fragments in up to 18 mammalian species, including the distinctly related marsupials and monotremes, groups that diverged from other mammals 120–160 million years ago. Using our method, many more cross-clade microsatellite loci can be harvested from the currently available genomic data, and this ability is set to improve exponentially as further genomes are sequenced.
Highlights
Microsatellites, called simple sequence repeats, consist of short (1–6 bp), tandemly repeated DNA motifs dispersed throughout genomes
Increasing numbers of polymorphic microsatellites are being associated with genetic disorders and variation in gene expression [2], but the high mutation rate at microsatellite loci offers an abundant and readily available polymorphism that has been the foundation for the wide use of microsatellites as neutral molecular markers, especially in applications requiring fine temporal and/or spatial resolution, e.g. population genetics and forensics
Successful transfer of microsatellite markers requires (i) 1:1 orthology of microsatellite loci, (ii) flanking sequences which are sufficiently conserved between species to provide PCR priming sites for cross-species amplification, and (iii) a microsatellite sequence which exhibits an appropriate level of polymorphism in the non-focal species
Summary
Microsatellites, called simple sequence repeats, consist of short (1–6 bp), tandemly repeated DNA motifs dispersed throughout genomes. We created a reproducible and adaptable framework that has allowed us to develop mammal-wide degenerate primers for nine dinucleotide microsatellites, five of which were successfully genotyped across most of a panel comprising 18 divergent species that represent the major mammalian orders, and three of which displayed high intraspecies polymorphism throughout the mammals tested We conclude from this successful initial trial that this approach has much promise and paves the way for equivalent studies in other genera as the push towards obtaining genome sequences from multiple animal, predominantly vertebrate, species becomes a reality [27]. It provides a significant starting resource for those wishing to focus on specific mammalian species or groups of species where large numbers of microsatellite markers with robust cross-species utility are required
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