Abstract
BACKGROUND: Alfalfa (Medicago sativa) is one of the most valuable forages in the world. As an outcrossing species, it needs bright flowers to attract pollinators to deal with self-incompatibility. Although various flower colors have been observed and described in alfalfa a long time ago, the biochemical and molecular mechanism of its color formation is still unclear. METHODS: By analyzing alfalfa lines with five contrasting flower colors including white (cream-colored), yellow, lavender (purple), dark purple and dark blue, various kinds and levels of anthocyanins, carotenoids and other flavonoids were detected in different colored petals, and their roles in color formation were revealed. RESULTS: Notably, the content of delphinidin-3,5-O-diglucoside in lines 3, 4 and 5 was 58.88, 100.80 and 94.07 times that of line 1, respectively. Delphinidin-3,5-O-diglucoside was the key factor for purple and blue color formation. Lutein and β-carotene were the main factors for the yellow color formation. By analyzing differentially expressed genes responsible for specific biochemical pathways and compounds, 27 genes were found to be associated with purple and blue color formation, and 14 genes were found to play an important role in yellow color formation. CONCLUSIONS: The difference in petal color between white, purple and blue petals was mainly caused by the accumulation of delphinidin-3,5-O-diglucoside. The difference in petal color between white and yellow petals was mainly affected by the production of lutein and β-carotene. These findings provide a basis for understanding the biochemical and molecular mechanism of alfalfa flower color formation.