Anthocyanin raspberry mutants: loss-of-function mutation of anthocyanidin synthase reveals pathway block in yellow Rubus Idaeus L.

Fruit pigmentation in raspberries (Rubus idaeus L.) is a complex phenomenon and one of the most important traits where many colour patterns ranging from deep purple to yellow are available. Anthocyanins and carotenoids are known to be the main pigments, while colour of red fruits is mainly considered due to varying anthocyanin contents (1). However, carotenoids seem to be responsible for the yellow colour and they might be just masked by anthocyanins in the case of red varieties (2). Despite the interest in raspberry anthocyanins, little is known about the genetic control and their regulation process during fruit development. A detailed study was conducted to identify the key structural genes involved in genetic block or control and regulation processes during fruit development in order to investigate the phenomenon involved in pigmentation of yellow raspberry fruits. Raspberry varieties, “Anne” (yellow) and “Tulameen” (red), fruits were analyzed to get insights into the origin of colour differences among raspberry fruits. Here, we describe the molecular cloning and characterization of an ANS gene from raspberry and identified a five base pairs insertion of homozygous nature at position 731 bp of the coding region of “Anne” ANS. The ANS mutant led to a truncated protein of 28.9 kDa (789 bp) in comparison to “Tulameen” ANS (45.4 kDa; 1239 bp) which lacks conserved regions for substrate, co-substrate and iron binding sites in “Anne” (Fig. 1). Most likely the truncated protein is inactive and is apparently the reason for the lack of the red colour at-least in this yellow variety. The expression analysis of key steps of the flavonoid pathway on “Anne” and “Tulameen” also indicates the down-regulation of gene coding for ANS enzyme in later fruit development stages. Furthermore, functional characterization of ANS gene through recombinant protein expression and transformation to ANS mutant line of Arabidopsis thaliana elaborates the role of this gene in the flavonoid biosynthesis pathway in raspberry