Identification of biochemical and molecular markers of blueability level in Hydrangea macrophylla

In Hydrangea macrophylla cultivars with pink inflorescences, sepals can turn blue in the presence of aluminium (Al). While the formation of the blue complex is well understood, the mechanisms behind variable blueing in pink cultivars remain unclear and this lack of knowledge makes difficult reliably breeding new varieties with good blueability. To investigate this, we studied nine pink cultivars exhibiting different levels of blueability. Sepal color changes after Al supply were used to classify cultivars along a blueability scale. We then assessed Al absorption, transport, and translocation by measuring Al content in vegetative organs and inflorescences, and by analysing the expression of related genes. We also quantified delphinidin (Del) and co-pigment accumulation in inflorescences. Our results show that changes in hue following Al supply are a reliable indicator of blueability. High blueability correlates with both a high Al/Del ratio and a high ratio of stabilizing (5-caffeoylquinic acid + 5-coumaroylquinic acid) to destabilizing (3-caffeoylquinic acid) co-pigments in sepals—defined as the Rcp ratio. Poor blueability is observed when either ratio is below threshold values (Al/Del < 10; Rcp < 0.5). Additionally, the expression of HmCHS (anthocyanin biosynthesis), HmALS3 (Al transport), and HmMYB2 (phenylpropanoid pathway regulator) positively correlates with blueability, suggesting their key roles in regulating blue coloration. Notably, HmALS3 expression in vegetative organs correlates with blueability several months before flowering. Our results provide a valuable program for accelerating blue cultivar screening, saving cultivation space and time: Since the Rcp ratio remains stable regardless of Al supply, it can serve as an early selection marker during the first flowering year, even without Al treatment. Then, measurement of ALS3 expression in the vegetative organs of the selected progenies under Al supply and well before flowering would complete this early screening. Finally, measurement of the [Al]/[Del] ratio in sepals upon flowering and assessment of the blue color at full blooming would confirm good blueability of the few preselected genotypes.