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.

Cucurbita L. is a valuable gourd vegetable crop, with high nutritional and economic value. However, the lack of a molecular identification system and population genetic information has impeded the development of proper conservation strategies and marker-assisted genetic breeding for Cucurbita varieties. In this study, we developed a set of simple sequence repeat (SSR) markers for distinguishing the main cultivated Cucurbita cultivars in China and providing technical support for domestic variety preservation, registration, and intellectual property protection. A total of 152 allelic variations and 308 genotypes were identified from 306 Cucurbita cultivars by using 24 SSR markers. Using 24 core markers, we successfully distinguished 300 varieties from 306 Cucurbita varieties, and the identification rate reached 98.36%. The PIC values of the 24 core markers ranged from 0.281 to 0.749, and the average value was 0.643, which was considered high genetic diversity. Based on the results of cluster analysis, principal component analysis, and population genetic structure analysis using 24 pairs of core primers for 306 Cucurbita varieties, the results were basically consistent, all categorized into three genetic clusters, corresponding to the three species: C. moschata, C. pepo, and C. maxima. These results showed a certain correlation with phenotypic traits. DNA fingerprints were constructed for the 306 Cucurbita cultivars based on the core markers. Our research results provide a new tool for population genetic analysis, variety identification, and protection in Cucurbita cultivars with high efficiency, accuracy, and lower costs compared to conventional methods.

Color is of great importance for the testing and breeding of plant varieties, and is known to be varied along with temperature changes. However, at the transcription level, the exact binding sites and corresponding regulation properties in the pathway of anthocyaninn biosynthesis in vivo are rarely reported. In this study, variety ‘Doppio Pandora-purple’ of Ranunculus asiaticus L. whose flower color is sensitive to the decrease in temperature was used. In the results, 27 genes involved in anthocyanin biosynthesis were differentially expressed under cold stress, and 7 MYB genes were up-regulated; the most obviously up-regulated MYB (RaMYB1) gene belongs to R2R3-MYB type. Promoter sequences of the 27 genes were concluded from transcriptome sequences and genome walking, for the purpose of sequence alignment with ChIP-seq/DAP-seq sequences. 7 genes were determined as target genes of RaMYB1 in cold-induced transcription regulation, including 2 PAL (phenylalanine ammonia lyase), 2 CHS (chalcone synthase), 1 F3H (flavanone 3-hydroxylase), 1 DFR (dihydroflavonol 4-reductase), 1 F3’H (flavanoid 3’-hydroxylase). ChIP-PCR indicated that, the binding of RaMYB1 to DFR can be induced by temperature decrease, while the bindings to other 6 genes were constitutive and can be strengthened at 5 °C decrease. Regulation properties of RaMYB1 to the 7 genes were determined as enhancement due to their up-regulation as well as the enhancer in C terminal of RaMYB1. Regulation mode of RaMYB1 was mapped accordingly and validated via HPLC-based anthocyanin content, gene expression level and coloration. The regulation mode can illustrate the binding sites and enhancement property of RaMYB1 in the regulation of anthocyanin biosynthesis under cold stress. It can be applied in color-oriented temperature monitoring and in molecular breeding. The way to establishing the regulation mode may be interest for other non-model plant genera with little molecular background.