Heterozygous indels as useful tools in the reconstruction of DNA sequences and in the assessment of ploidy level and genomic constitution of hybrid organisms

Abstract

In this paper we describe a simple approach using double peaks in chromatograms generated as artefacts in the vicinity of heterozygous indels, to identify the specific sequences present in individual strands of a given DNA fragment. This method is useful to assign bases in individuals that are heterozygous at multiple sites. In addition, the relative sizes of the double peaks help to determine the ploidy level and the relative contribution of the parental genomes in hybrids. Our interpretation was confirmed with the analysis of artificial mixtures of DNA of two different species. Results were robust with varying PCR and sequencing conditions. The applicability of this method was demonstrated in hybrids of the Squalius alburnoides complex and in heterozygotes of Chondrostoma oligolepis. Far from being limited to these fish models and the gene where it was tested (beta-actin), this sequence reconstruction methodology is expected to have a broader application.

• Double peaks
• interspecific hybrids
• haplotype reconstruction
• heterozygotes
• beta-actin gene
• cyprinid fish

Acknowledgements

We thank the help of G. Lemos, Sousa-Santos family, T. Bento and A. Levy. We are also grateful to A. Gomes-Ferreira, M. Gromicho and L. Moreira da Costa for technical assistance with flow cytometry. DGF provided authorizations for field work. Study funded by the FCT Pluriannual Program (UI&D 331/94 and UI&D 329/94) (FEDER participation). C. Sousa-Santos was supported by a PhD grant from FCT (SFRH/BD/8320/2002).