Despite the increasing affordability of next-generation sequencing technologies, there is still no appreciable quantity of sequence data available for many non-model species. The deficiency of genomic resources in wild species has provided the impetus to create a versatile and cost-effective amplicon-based sequencing tool. By exploiting highly conserved genomic elements we have designed a suite of universal nuclear markers that are transportable across amniote orders. This presents a simple method for next-generation sequencing and comparative genomics research in the absence of a reference genome.
Genomic elements with a high degree of similarity between species, or evolutionarily conserved elements, occur throughout the genomes of vertebrates. We utilised these conserved elements to develop an automated BLAST and PRIMER3-based pipeline for the identification of shared priming regions. After filtering we flagged >49,000 regions suitable for primer placement in eutherians, >13,000 in therians (eutherian + metatherian), and >3,000 in amniotes. Primers were designed to amplify unique in placental, marsupial, monotreme, and bird species. The utility of 30 of these candidate primers was established using a long-range PCR approach, resulting in 57% to 89% PCR success in 7 representative amniote species (Canis familiaris, Equus caballus, Bos taurus, Mus musculus, Monodelphis domestica, Ornithorhynchus anatinus, and Gallus gallus). We aim to establish the variability of these markers within and between lineages, therefore we will be conducting deep sequencing of a selection of pooled markers across 12 wild and domestic species using the MiSeq platform (Illumina, Inc.).
The capture of orthologous genome-wide sequence data in evolutionarily diverged species presents a valuable resource, eliminating the requirement for laborious species-specific marker development in non-model organisms, and facilitating phylogenetic inference. This is particularly beneficial for species conservation strategies, where economical approaches are crucial. This tool has applications in areas as diverse as systematics, SNP discovery, diversity screening, kinship studies, and individualisation.