Utilization of intron-flanking EST-specific markers in the phylogenetic analysis and parentage identification of Rhododendron species and hybrids

Abstract

Intron length polymorphisms were used to investigate relationships among eight Rhododendron L. species (R. catawbiense Michaux., R. minus Michaux., R. ponticum L., R. keiskei Miquel., R. arboreum Sm., R. dichroanthum Diels., ssp. scyphocalyx Cowan., R. maximum L. and R. dauricum L.) and two hybrid cultivars [i.e., R. 'PJM' (R. minus van minus × R. dauricum) and R. 'Chionoides' (R. ponticum × unknown)]. A total of 27 of these markers were used to estimate phylogenetic relationships among the species and draw inferences about the parentage of the cultivars, which is partially unknown. In general the expressed sequence tag-polymerase chain reaction (EST-PCR) marker-based phylogenetic map of the eight species is congruent with the currently accepted morphology-based classification of these species at the subgenus as well as the section level. However, the constructed phylogenetic tree revealed that, at the subsection level, two species, R. arboreum (subsection Arborea Sleum.) and R. dichroanthum (subsection Neriiflora Sleum.), are grouped under the same "clade" (80% bootstrap score), suggesting that these species are more closely related than indicated in the current classification system that places them in separate subsections/clades. Moreover, our phylogenetic analysis of the three species belonging to section Ponticum G. Don. demonstrated a closer phylogenetic relationship between R. ponticum and R. maximum (bootstrap score of 74%) than between these species and R. catawbiense; such observation is consistent with a recent phylogenetic analysis of section Ponticum by Milne (2004) using the sequences of a chloroplast gene. Parentage analysis for the two cultivars confirmed the interspecific lineage of R. 'PJM' and provided genetic support for the speculated R. ponticum and R. maximum parentage of K. 'Chionoides'. Our results indicate that, in addition to their use in mapping studies, intron-flanking EST-based PCR markers are valuable tools for conducting phylogenetic and parentage analyses and/or gene flow studies.