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

Abstract

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 (M²) 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.

Keywords: Ranunculus asiaticus L.; anthocyanin content; change trait; color phenotype; gene expression level.

Figure 1

Flowers and petals at 12 development stages and three layers of two varieties of Ranunculus asiaticus L. (A) flowers; (B) petals; L: ‘Jiaoyan zhuanhong’; C: ‘Jiaoyan yanghong’; 1–12: stage 1–stage 12, time interval between stage 1 and stage 12 is two weeks, among which, intervals between two adjacent stages are two days from stage 1 to stage 4 and one day from stage 4 to stage 12; LOP: outer petals of ‘Jiaoyan zhuanhong’; LMP: middle petals of ‘Jiaoyan zhuanhong’; LIP: inner petals of ‘Jiaoyan zhuanhong’; COP: outer petals of ‘Jiaoyan yanghong’; CMP: middle petals of ‘Jiaoyan yanghong’; CIP: inner petals of ‘Jiaoyan yanghong’.

Figure 2

Colorations and corresponding model of change dynamics of petals of three layers at 12 development stages. L-O, L-M, and L-I: outer, middle, and inner layers of petals of ‘Jiaoyan zhuanhong’, respectively; C-O, C-M, and C-I: outer, middle, and inner layer of petals of ‘Jiaoyan yanghong’, respectively; (A) CIE systems of colorations, in each individual figures, balls named 1–12 reflected the colors of the 12 development stages (Figure 1), and corresponding color chars were shown in the right; (B) measurements of L*, a* and b* at 12 stages; (C) model of change dynamics of petal colorations: $Y_{jv}={{\displaystyle \int }}_j^{j+1}\left[v_{pj}+\left(t-t_j\right)·\left(v_{p,j+1}-v_{pj}\right)/(t_{j+1}-t_j)\right]dt·\frac{\epsilon }{1+e^{-a_{pj}}}$, where measurements of L*, a*, b* as well as E* of two varieties were recorded as l (Jiaoyan zhuanhong) or c (Jiaoyan yanghong), i.e., $l_{p,j+1}=l_p\left(t_{j+1}\right),\left(p=\mathrm{outer}\mathrm{petal},\mathrm{middle}\mathrm{petal}\mathrm{or}\mathrm{inner}\mathrm{petal};j+1=\mathrm{stage}1,\dots ,12\right)$; $v_{pj}=\frac{l_{p,j+1}-l_{pj}}{t_{j+1}-t_j}; a_{pj}=\left|\frac{v_{p,j+1}-v_{pj}}{t_{j+1}-t_j}\right|$; Y1–Y10: corresponded to stage 3–stage 12; negative or positive values of $Y_{jv}$ indicated corresponding status of coloration changes were decreasing or increasing, respectively; big $Y_{jv}$ indicated the tendency was toward great changes while small $Y_{jv}$ meant changes became gentle.

Figure 3

Anthocyanin content and corresponding model of change dynamics of petals of three layers at 12 development stages of both varieties. L: variety ‘Jiaoyan zhuanhong’; C: variety ‘Jiaoyan yanghong’; OP, MP and IP: outer, middle and inner layer of petals, respectively; (A) anthocyanin content measured using pH differential method and displayed with unit (mg 100 g⁻¹ FW), L1–L12, C1–C12: measurements of petals at 12 development stages (Figure 1); (B) Model of Change Dynamics of anthocyanin content: $Y_{jv}={{\displaystyle \int }}_j^{j+1}\left[v_{pj}+\left(t-t_j\right)·\left(v_{p,j+1}-v_{pj}\right)/(t_{j+1}-t_j)\right]dt·\frac{\epsilon }{1+e^{-a_{pj}}}$, where measurements of anthocyanin content of two varieties were recorded as l (Jiaoyan zhuanhong) or c (Jiaoyan yanghong), i.e., $l_{p,j+1}=l_p\left(t_{j+1}\right),\left(p=\mathrm{outer}\mathrm{petal},\mathrm{middle}\mathrm{petal}\mathrm{or}\mathrm{inner}\mathrm{petal};j+1=\mathrm{stage}1,\dots ,12\right)$; $v_{pj}=\frac{l_{p,j+1}-l_{pj}}{t_{j+1}-t_j}; a_{pj}=\left|\frac{v_{p,j+1}-v_{pj}}{t_{j+1}-t_j}\right|$; Y1–Y10: corresponded to stage 3–stage 12; negative or positive values of $Y_{jv}$ indicated change status of anthocyanin content were decreasing or increasing, respectively; big $Y_{jv}$ indicated the tendency was toward great changes while small $Y_{jv}$ meant changes became gentle.

Figure 4

HPLC–MS chromatograms and corresponding structures of major anthocyanins of two varieties of Ranunculus asiaticus L. L1 and L2 were the major anthocyanins of variety ‘Jiaoyan zhuanhong’; C1 and C2 were the major anthocyanins of variety ‘Jiaoyan yanghong’.

Figure 4

HPLC–MS chromatograms and corresponding structures of major anthocyanins of two varieties of Ranunculus asiaticus L. L1 and L2 were the major anthocyanins of variety ‘Jiaoyan zhuanhong’; C1 and C2 were the major anthocyanins of variety ‘Jiaoyan yanghong’.

Figure 5

Gene expression patterns of two varieties of Ranunculus asiaticus L. L1–L2: stage 1–stage 12 of ‘Jiaoyan zhuanhong’ (Figure 1); C1–C12: stage 1–stage 12 of ‘Jiaoyan yanghong’; O: outer petal; M: middle petal; I: inner petal; c72570, c130622, and c83020 were unigenes annotated as encoding 3GT, 3GT, and 3MAT based on transcriptome sequencing.

Figure 6

Heat map of gene expression patterns. 1–12: stage 1–12 (Figure 1); c72570, c130622, and c83020 were unigenes annotated as encoding 3GT, 3GT, and 3MAT based on transcriptome sequencing; LOP, LMP, and LIP stood for outer petals, middle petals, and inner petals of variety ‘Jiaoyan zhuanhong’; COP, CMP, and CIP stood for outer petals, middle petals, and inner petals of variety ‘Jiaoyan yanghong’. The darker the color of the square within the heat map, the higher the expression level.

Figure 7

Correlation between anthocyanin contents and gene expression levels. A: measurements of ‘Jiaoyan zhuanhong’; B: measurements of ‘Jiaoyan yanghong’; IP: inner petals; MP: middle petals; OP: outer petals; L1–L12: stage 1–12 of ‘Jiaoyan zhuanhong’; C1–C12: stage 1–12 of ‘Jiaoyan yanghong’; c72570, c130622, and c83020 were genes annotated as encoding 3GT, 3GT, and 3MAT.