Confirmation of Clematis hybrids using molecular markers

The hybrid origin of progeny from crosses of Clematis tubulosa and Clematis brevicaudata was investigated using randomly amplified polymorphic DNA (RAPD) and single nucleotide polymorphisms (SNPs) from sequence analysis of chloroplast rbcL, accD genes, and the C. brevicaudatamatK gene. Plants collected from three and four populations of C. brevicaudata (C. brev) and C. tubulosa (C. tubu), respectively, from Mt. Songshan, Beijing, China were used as parents for hybridization. Morphological characters of pollen, seeds, and leaves were recorded in 2007. DNA from leaf samples of both parents and of C. brev × C. tubu and C. tubu × C. brev were extracted, and used for RAPD and SNPs from sequence data. A dendrogram was constructed by the branching neighbor-joining (IB-NJ) method. Proportionate population scores were generated by the admixture model using the STRUCTURE software. Based on morphological characters, C. brevicaudata was quite uniform. However, variations were detected in C. tubulosa. Hybrids of C. brev × C. tubu and C. tubu × C. brev showed intermediate morphological characters of the parents. Accessions of C. tubu × C. brev were clustered into 2 groups, with the majority of hybrids belonging to group IV b in the RAPD dendrogram, suggesting that this resulted from variations within C. tubulosa. In general, the hybrid origin of all progeny characterized by morphological characters was supported by the RAPD and SNPs data. These results indicate that RAPD results supported by SNPs data will be useful tool to verify hybrids.