Abstract:

【Objective】 Populus davidiana × P. bolleana (Shanxin poplar) is an ideal material for basic and applied research in tree genetic engineering. This study aimed to determine the ploidy level of Shanxin poplar, reveal the phenomenon of PCR-mediated recombination via a case study of PdbSUS genes, and define two haplotypes of each gene to provide precise sequence information for subsequent genetic engineering studies and serve as a reference for cloning and determining the haplotypes of genes in woody plants. 【Method】 Fresh young leaves of tomato (Lycopersicon esculentum) and Shanxin poplar were separately collected. The genome size and ploidy level of Shanxin poplar were analyzed by influx flow cytometry, using the tomato genome as an internal reference. Primers designed based on the sequences of sucrose synthase (SUS) genes in P. trichocarpa, were used to amplify the full-length genomic DNA and the cDNA sequences of SUS genes in Shanxin poplar. The PCR amplicons were visualized on 1% agarose gels by GelRed staining, then appropriate bands were excised from the gels and purified using the AxyPrep ^TM DNA Gel Extraction Kits. All purified products were separately ligated to the pEASY-Blunt vector and transformed into Escherichia coli strain DH5α competent cells. Colonies of E. coli were randomly selected from individual LB agar plates containing kanamycin and screened for target fragments by using colony PCR. Independent bacterial clones harboring the recombinant plasmids were sequenced using the Sanger method, then the sequences for each gene were aligned and analyzed using DNAMAN (version 8) and SnapGene (version 1.1.3) software with default parameters. 【Result】 Shanxin poplar is a diploid plant. Seven PdbSUS genes were obtained, and the haplotypes of each gene were identified at the genomic and transcriptional levels. Recombination mediated by PCR was verified by analyzing restriction fragment length polymorphisms (RFLP). Among the 209 sequenced clones, 18 harbored chimeric products, accounting for 8.6%, and the frequency of chimera generation in individual genes ranged from 0 to 33.3%. All chimeric products were recombinants derived from heterozygous alleles located at the same locus of homologous chromosomes. Chimeras of different PdbSUS genes were undetectable. 【Conclusion】 The present findings indicated that PCR-mediated recombination is not rare, but is rather a high-frequency event leading to PCR-derived recombinants, which might arise via incomplete primer extension or polymerase template switching. Thus, chimeric products should be differentiated from parental haplotypes when cloning genes from woody plants or other species with highly heterozygous genomes by using PCR.

Key words: Populus davidiana × P. bolleana (Shanxin poplar), sucrose synthase gene, PCR-mediated recombination, template-switching, chimeric product, recombinant molecule