Microscopic observation and hormone-targeted metabolomic analysis provide insights into the reproductive barriers of distant hybridization between Rhododendron subgenus Tsutsusi and subgenus Pentanthera.

key messageEmbryo abortion occurred at 20 DAP of HFS × RM, with trans-Zeatin-riboside and brassinolide playing key roles during embryo abortion. Rhododendron is a world-famous flower with high ornamental and economic value. However, hybridization between the Rhododendron subgenus Tsutsusi and subgenus Pentanthera is often hindered by reproductive barriers, which restrict breeding of new cultivars. To determine the reproductive barriers between these two subgenera, artificial crosses were conducted using three cultivars in subgenus Tsutsusi (R. × 'Tiehong' (TH), R. × 'Hongfushi' (HFS) and R. × 'Hongyue' (HY), as the maternal parents), and the wild species R. molle (RM) in subgenus Pentanthera as the paternal parent. The results showed that the pollen tube stopped growing at 1/3 of the style of HY at 72 HAP, indicating a pre-fertilization barrier in HY × RM. The seeds of TH × RM were obtained at 230 DAP, indicating no reproductive barriers. In contrast, HFS × RM embryos developed only globular embryos by 10 DAP and aborted by 20 DAP, indicating a post-fertilization barrier. Hormone-targeted metabolomics determined 21 kinds of plant hormones in the ovaries of TH × RM and HFS × RM. Among them, the contents of trans-Zeatin-riboside (tZr) in TH × RM remained stable from 10 to 20 DAPs; however, in HFS × RM, it decreased significantly by 65% during this stage. Furthermore, Brassinolide (BR) was only detected in the ovaries of HFS × RM at 20 DAP. The results indicated that tZr and BR are likely to be the important hormones responsible for the embryo's abortion, which can aid in breeding of distant hybridization in Rhododendron by adopting suitable strategies to overcome the barriers.