|Wang, Lian yin|
Submitted to: Scientia Horticulturae
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/22/2010
Publication Date: 7/14/2010
Citation: Yuan, T., Wang, L.Y., Roh, M.S. 2010. Confirmation of Clematis hybrids using molecular markers. Scientia Horticultureae. 125:136-145. Interpretive Summary: Clematis is one of the most popular climbing plants used in landscapes as a garden plant or in floriculture as a potted plant. Typically, most commercially available cultivars or species will cease flowering during hot weather. Clematis brevicaudata and Clematis tubulosa have not yet been explored and utilized in breeding programs to obtain heat tolerant Clematis hybrids. Hybrid seedlings require 2 to 3 years to flower so significant time is required to use floral morphology to separate hybrid genotypes. Therefore molecular markers such as randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP), and single nucleotide polymorphisms (SNPs) were used to verify putative hybrid seedlings. The results indicate that RAPD is a simpler, viable, and more convenient tool than AFLP, and with the analysis of SNPs in this study, RAPD effectively verifies the hybrid nature and detects genetic variability of both parental plants. Clematis brevicaudata and C. tubulosa have been successfully hybridized and verified morphologically and molecularly using RAPD, AFLP, and by SNP markers.
Technical Abstract: The hybrid origin of two progeny from reciprocal crosses of Clematis tubulosa and C. brevicaudata was investigated using molecular markers generated by randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and single nucleotide polymorphisms (SNPs). Morphological characters of pollen, seeds, and leaves of flowering parents and hybrids were recorded in 2007. Leaf samples of 10 plants of both parents and 17 and 38 hybrids of C. brevicaudata × C. tubulosa (C. brev × C. tubu) and C. tubulosa × C. brevicaudata (C. tubu × C. brev ), respectively, were collected and DNA was extracted from dried leaves, and used for RAPD, AFLP, and SNP analysis of sequence data for 3 genes. Based on morphological characters, C. brevicaudata was quite uniform in flower color; however, variations were detected in flower color and shape of C. tubulosa. Progeny of hybrids, C. brev × C. tubu and C. tubu × C. brev, showed intermediate growth habit, flower color and shape, leaf shape, and pollen structure. Accessions of C. brevicaudata and C. tubulosa clustered in dendrograms generated by RAPD and AFLP. However, C. tubu × C. brev accessions were clustered into 2 groups in the dendrogram generated by RAPD markers. Clustering of C. tubu × C. brev accessions in the AFLP dendrogram into three sub-groups suggested that this resulted from variations within C. tubulosa. In general, the hybrid origin of all progeny characterized by vegetative and reproductive morphological characters was supported by the RAPD data and also supported by SNPs based on C. brevicaudata and C. lasiandra chloroplast rbcL, accD genes and the C. brevicaudata matK gene. These results indicate that RAPD and SNPs will be useful tools to verify hybrids effectively even when AFLP analysis can not be performed.