ITS sequence variation supports the hybrid origin of Malus toringoides Hughes

Malus toringoides (Rehd.) Hughes was suggested to have originated from hybridization between Malus transitoria Schneid. and Malus kansuensis Rehd., followed by repeated backcrossing to one of the putative parents. In the present study, the sequence information of the internal transcribed spacer (ITS) of nuclear ribosomal DNA (nrDNA) was used to re-examine the origin of this species. A total of 69 accessions from three natural populations (Maerkang, Xiaba and Kehe, Aba Autonomous Region, Sichuan, China) of M. toringoides and 10 accessions of its putative parents were analyzed. Using Malus angustifolia (Ait.) Michx., Malus ioensis (Wood) Britt. and Malus doumeri Chev. as outgroups, our phylogenetic analysis of the ITS sequences of M. toringoides and its putative parents showed that M. toringoides was not monophyletic, and two different types of ITS sequences which were obtained from each of the six accessions of M. toringoides were found to have clustered separately with those of the two putative parent species on the gene tree. A comparison of the sequence variation between M. toringoides and its putative parents revealed an additive variation pattern of ITS sequences in the putative hybrid species. These results are consistent with the previous morphological and amplified fragment length polymorphism (AFLP) data which suggested that M. toringoides was of hybrid origin. Our ITS data provide new molecular evidence for the hybrid origin hypothesis of M. toringoides and these results are of great importance for future study on hybridization, polyploid speciation and evolution of the genus Malus Miller.