Confirmation of hybrid origin of Cyrtanthus based on the sequence analysis of internal transcribed spacer

The objectives of this study were to create interspecific hybrids between Cyrtanthus elatus and C. sanguineus and to confirm the hybrid origin of the progeny based on morphological characters and using molecular markers. The tip of the leaves, the shape and size of cells, and stomata distribution in abaxial and adaxial surface of leaf epidermis were analyzed by low temperature scanning electron microscope (LT-SEM). Molecular markers generated from random amplification polymorphic DNA (RAPD) and single nucleotide polymorphisms (SNPs) analysis of internal transcribed spacer 1 and 2 of rRNA gene (ITS 1, 2) were also analyzed to confirm hybrid status. Putative C. elatus × C. sanguineus hybrid A 3 resembled C. elatus and C. sanguineus 'Pringle Bay' (PB) × 'Fred Meyer' (FM) B 23 and was similar to C. sanguineus at flowering. However, the hybrid origin of hybrid A 3 could not be confirmed based on the morphological characteristics of flowers, the tip of the leaves, and cell structure and surface images in both abaxial and adaxial images of LT-SEM. Based on RAPD markers, putative hybrid A 3 and other putative hybrids that did not flower (A 4, A 5, A 13 and D 39, D 40, and D 42) can be considered as a hybrid based on the presence of both species-specific bands for parental taxa. SNP markers of ITS 1, 2 region also revealed that other putative hybrids of A and D that did not flower during the evaluation can be interspecific hybrids of C. elatus and C. sanguineus. Therefore, the use of SNP markers of ITS 1, 2 is considered a more accurate tool to characterize hybrid origin than morphological characteristics and RAPD markers.

► The interspecific hybrids of Cyrtanthus elatus and C. sanguineus were evaluated. ► Origin of interspecific hybrid A 3 could not be confirmed based on morphology. ► Hybrid origin was investigated based SNP analysis of ITS 1 and 2 of rRNA gene. ► The putative hybrid A 3 was confirmed as a true interspecific based on SNP analysis.