A strategy for characterization of persistent heteroduplex DNA in higher plants

Heteroduplex DNA (hDNA) generated during homologous recombination (HR) is an important component that shapes genetic diversity in sexually reproducing organisms. However, studies of this process in higher plants are limited. This is because hDNAs are difficult to capture in higher plants as their reproductive developmental model only produces normal gametes and does not preserve the mitotic products of the post-meiotic segregation (PMS) process which is crucial for studying hDNAs. In this study, using the model system for tree and woody perennial plant biology (Populus), we propose a strategy for characterizing hDNAs in higher plants. We captured hDNAs by constructing triploid hybrids originating from a cross between unreduced 2n eggs (containing hDNA information as a result of inhibition chromosome segregation at the PMS stage) with normal male gametes. These triploid hybrids allowed us to detect the frequency and location of persistent hDNAs resulting from HR at the molecular level. We found that the frequency of persistent hDNAs, which ranged from 5.3 to 76.6%, was related to locations of the simple sequence repeat markers at the chromosomes, such as the locus-centromere distance, the surrounding DNA sequence and epigenetic information, and the richness of protein-coding transcripts at these loci. In summary, this study provides a method for characterizing persistent hDNAs in higher plants. When high-throughput sequencing techniques can be incorporated, genome-wide persistent hDNA assays for higher plants can be easily carried out using the strategy presented in this study.