Sampling for DUS Test of Flower Colors of Ranunculus asiaticus L. in View of Spatial and Temporal Changes of Flower Colorations, Anthocyanin Contents, and Gene Expression Levels

Sampling for DUS test of flower colors should be fixed at the stages and sites that petals are fully colored, and besides, flower colorations are uniform among individuals and stable for a period of time to allow testers to get consistent results. It remains a problem since spatial and temporal flower colorations are reported a lot but their change traits are little discussed. In this study, expression state, uniformity and stability of color phenotypes, anthocyanin contents, and gene expression levels were taken into account based on measurements at 12 development stages and three layers (inner, middle, and outer petals) of two varieties of Ranunculus asiaticus L. to get their best sampling. Our results showed that, outer petals of L9-L10 (stage 9-stage 10 of variety Jiaoyan zhuanhong') and C5-C6 (stage 5-stage 6 of variety Jiaoyan yanghong') were the best sampling, respectively. For DUS test, it is suggested to track flower colorations continuously to get the best sampling as well as representative colors since different cultivars had different change traits, and moreover, full expression of color phenotypes came later and lasted for a shorter duration than those of anthocyanin contents and gene expressions. Our innovation exists in following two points. Firstly, a model of change dynamic was introduced to illustrate the change traits of flower colorations, anthocyanin contents, and gene expressions. Secondly, genes used for expression analysis were screened on account of tentative anthocyanins, which were identified based on comparison between liquid chromatography-mass spectrometry (LC-MS) results and molecular mass and mass fragment pattern (M-2) of each putative anthocyanin and their fragments deduced in our previous study. Gene screening in this regard may also be interest for other non-model plant genera with little molecular background.