The light-dependent coloration of the vital organs of horticultural crops affects multiple parts of production and sales. The simplicity of the metabolic pathways of anthocyanins and the characteristics of light-dependent coloration make chrysanthemum (Chrysanthemum ×morifolium) an ideal subject for studying the mechanism of light-regulated anthocyanin biosynthesis. In this study, real-time quantitative reverse transcription–polymerase chain reaction (PCR) was used in the analysis of the expression levels of anthocyanin biosynthesis genes in C. ×morifolium ‘Reagan’. The reference genes selected were those assumed to remain at constant levels in three flower color lines at five floral developmental stages and at two light conditions. Using digital gene expression technology, we selected nine reference genes with moderate expression in the chrysanthemum ray florets at various floral developmental stages under illuminated and dark conditions as the candidate reference genes for further study. After comprehensively analyzing the stability of gene expression with three distinct statistical algorithms, geNorm, NormFinder, and qBase plus, we found that F-box and PP2A were the most stable genes in all of the samples. In addition, we analyzed the relative expression level of the CmF3H gene in different samples to verify the reference genes that we selected. This study provides a consensus list of validated reference genes that will benefit future studies of the expression of chrysanthemum genes involved in anthocyanin biosynthesis and floral development under various light conditions. Moreover, this information will also promote the molecular breeding of horticultural crops for their color modification.
Yuan Zhang, Chen Wang, HongZheng Ma and SiLan Dai
The morphological characteristics of chrysanthemum (Chrysanthemum ×morifolium) are rich in variation. However, as a result of the aneuploid polyploidy of the chrysanthemum genome and the lack of proper tools, the genomic information of this crop is limited. Development of microsatellite markers has been an increasing trend in crop genetic studies because of the applicability of these markers in breeding programs. In this study, we reported the development of a simple sequence repeat in chrysanthemums using a magnetic beads enrichment method. An enriched genomic library with AC and GT microsatellite motifs was constructed, and 53 positive clones were detected by a colony polymerase chain reaction (PCR) technique. Of these clones, 35 showed high-quality sequences, and 35 primer pairs were designed accordingly. Twenty-six (74.29%) of the 35 primer pairs revealed polymorphisms on a set of 40 chrysanthemum cultivars. There were 172 alleles amplified over 26 loci with an average of 6.615 alleles per locus. The mean values of gene diversity corrected for the sample size and the inbreeding coefficient were 0.609 and 0.119 over 26 loci, respectively, which indicated that the majority of the microsatellite loci is highly informative. Cluster analysis based on 26 polymorphic loci demonstrated that the selected cultivars were clustered according to geographical origin. This study shows the isolation efficiency of the magnetic beads technique; the abundance of microsatellites in chrysanthemum; and the potential application for the cultivar classification, the studies on genetic diversity, and molecular breeding of chrysanthemums, which is beneficial to promoting the conservation and sustainable use of this crop.
Mengmeng Zhang, He Huang, Qing Wang and Silan Dai
Conventional crossbreeding remains an effective technique for chrysanthemum (Chrysanthemum ×morifolium Ramat.) breeding. However, there are always many problems when breeding chrysanthemum because of its complex genetic background, such as difficulty matching parents, selecting superior hybrid progenies, quantitatively describing certain target traits, and evaluating breeding results. A recent mathematical analysis method is an effective method for evaluating plant breeding progress. In this study, we used 505 multiflora chrysanthemum germplasm resources as test materials; we divided the flowering time into five groups using a grading analysis method, including extremely early group (genotypes that flowered when daylength was longer than 13.5 hours), early group (genotypes that flowered when daylength was 13.5–12.0 hours), medium group (genotypes that flowered when daylength was 12.0–11.0 hours), late group (genotypes that flowered when daylength was 11.0–10.0 hours), and extremely late group (genotypes that flowered when daylength was shorter than 10.0 hours). Moreover, the breeding objective was to breed early-flowering genotypes. Using 15 phenotypic characters as evaluation factors, 37 excellent genotypes, including four early-flowering genotypes, were screened out from the aforementioned resources according to an analytic hierarchy process (AHP) and weighting of the gray relational grade. We selected one early-flowering genotype and eight medium-flowering genotypes from these 37 genotypes and matched six hybridized combinations based on the genetic distance between genotypes calculated by the Q cluster analysis method. We used a comprehensive evaluation method combining AHP and the gray relational analysis (GRA) method for the evaluation of 367 progenies. Moreover, we screened out 52 superior hybrids, including 36 early-flowering hybrids. The results of this study demonstrate that the mathematical analysis method is an immensely effective method to breed new cultivars of early-flowering multiflora chrysanthemum. This study also provides an effective method to define and improve the flowering time of other cultivated plants.
Dongru Kang, Silan Dai, Kang Gao, Fan Zhang and Hong Luo
In this study, five cultivars of cut chrysanthemum Chrysanthemum ×morifolium Ramat., ‘Jinba’, ‘Yuuka’, ‘Fenguiren’, ‘Xueshen’, and ‘Huangjin’ were used to explore the functions of 5-azacytidine (5-azaC) on chrysanthemum growth and flower development. The results showed that 5-azaC had different effects on the growth of the five cultivars during in vitro culture. The final statistics showed that low concentrations promoted plant growth, whereas high concentrations inhibited growth; however, each cultivar had different growth curves, demonstrating that 5-azaC had no consistent inhibitory actions on growth. On the basis of the squaring time and flowering time statistics, we found that 5-azaC had a certain effect on the flowering time of all cut chrysanthemums, and all of these cultivars showed extremely early strains. Summer chrysanthemum (‘Yuuka’, ‘Fenguiren’, ‘Xueshen’, and ‘Huangjin’) treatments led to both early and delayed flowering. When the statistics were analyzed for different individuals, we found that the treatments shortened the squaring time in early-flowering plants. In ‘Jinba’, an autumn chrysanthemum, the treatment helped broken juvenile limitations and allowed plants to undergo photoperiod induction in the early stage. Additionally, we also determined the flower diameter differences in these treatments; ray florets from ‘Yuuka’ and ‘Huangjin’ trended to show tubular florets, and the location of tubular and ray florets were changed in ‘Xueshen’ capitulum. In conclusion, on the basis of flowering time in five early varieties of cut chrysanthemum, we propose that 5-azaC may regulate the methylation level of genes that control flower induction and flower development. These results provide phenotypic data and material for exploring the function of DNA methylation in regulating flowering.
Xuebin Song, Kang Gao, Guangxun Fan, Xiaogang Zhao, Zhilan Liu and Silan Dai
The large-flowered Chinese chrysanthemum is one of the most morphologically complex ornamental plants, and its identification and classification requires a well-defined and reproducible system. The diversity of the capitulum is determined mainly by multiple shapes of ray florets. However, the existing classification systems for ray floret types are incomplete and unsystematic. In this study, 299 ray florets from 151 large-flowered chrysanthemum varieties in China, as well as 12 related traits of ray florets, were selected for quantitative classification. First, as one of the most important indices of ray floret shape, the corolla tube merged degree (CTMD) was defined as the corolla tube length/ray floret length (CTL/RFL). Combined with a probability grading method and linear regression analysis, the CTMD was divided into three groups, flat, spoon, and tubular, of which the CTL/RFL ranged from 0 to 0.20, 0.20 to 0.60, and 0.60 to 1.00, respectively. Second, Q-mode cluster analysis indicated that each group could be further categorized into three types (straight, curved, and atypical), based on other important variables in the ray floret. Finally, the ray floret was classified into nine types, including flat-straight, flat-curve, flat-atypical, spoon-straight, spoon-curve, spoon-atypical, tubular-straight, tubular-curve, and tubular-atypical. This ray floret classification system will be valuable in the classification of capitulum shape and has significance for the identification, breeding, and international standardization of chrysanthemum cultivars.
He Huang, Yuting Liu, Ya Pu, Mi Zhang and Silan Dai
Plant growth and development are significantly affected by salt stress. Chrysanthemum lavandulifolium is a halophyte species and one of the ancestors of chrysanthemum (C. ×morifolium). Understanding how this species tolerates salt stress could provide vital insight for clarifying the salt response systems of higher plants, and chrysanthemum-breeding programs could be improved. In this study, salt tolerance was compared among C. lavandulifolium and three chrysanthemum cultivars by physiological experiments, among which C. lavandulifolium and Jinba displayed better tolerance to salt stress than the other two cultivars, whereas Xueshan was a salt-sensitive cultivar. Using the transcriptome database of C. lavandulifolium as a reference, we used digital gene expression technology to analyze the global gene expression changes in C. lavandulifolium seedlings treated with 200 mm NaCl for 12 hours compared with seedlings cultured in normal conditions. In total, 2254 differentially expressed genes (DEGs), including 1418 up-regulated and 836 down-regulated genes, were identified. These DEGs were significantly enriched in 35 gene ontology terms and 29 Kyoto Encyclopedia of Genes and Genomes pathways. Genes related to signal transduction, ion transport, proline biosynthesis, reactive oxygen species scavenging systems, and flavonoid biosynthesis pathways were relevant to the salt tolerance of C. lavandulifolium. Furthermore, comparative gene expression analysis was conducted using reverse transcription polymerase chain reaction to compare the transcriptional levels of significantly up-regulated DEGs in C. lavandulifolium and the salt-sensitive cultivar Xueshan, and species-specific differences were observed. The analysis of one of the DEGs, ClAKT, an important K+ transport gene, was found to enable transgenic Arabidopsis thaliana to absorb K+ and efflux Na+ under salt stress and to absorb K+ under drought stress. The present study investigated potential genes and pathways involved in salt tolerance in C. lavandulifolium and provided a hereditary resource for the confinement of genes and pathways responsible for salt tolerance in this species. This study provided a valuable source of reference genes for chrysanthemum cultivar transgenesis breeding.
Yu Liu, Miao He, Fengli Dong, Yingjie Cai, Wenjie Gao, Yunwei Zhou, He Huang and Silan Dai
The NAC transcription factor is a peculiar kind of transcription factor in plants. Transcription factors are involved in the expression of plant genes under different conditions, and they play a crucial role in plant response to various biotic and abiotic stress. We transferred the ClNAC9 gene into Chrysanthemum grandiflora ‘niu9717’ by Agrobacterium tumefaciens–mediated transformation. The results of kanamycin-resistant screening, polymerase chain reaction (PCR) detection, and Northern blot analysis proved that the target gene had been integrated into the genome of the target plants. Wild-type (WT) plants and transgenic plants were treated with different concentrations of NaCl, NaHCO3, and drought stress, and physiological indexes, such as antioxidant system activity (superoxide dismutase, peroxidase, catalase), malondialdehyde accumulation, and leaf relative water content, were measured. We also observed changes in plant morphology. The physiological indexes’ changing range and extreme values suggested that transgenic plants’ resistance to salinity, alkali, and drought stress was significantly higher than WT plants. Transgenic plant growth was less inhibited compared with WT plants, indicating that the ClNAC9 gene increased the resistance of transgenic plants under the stress of salinization, alkalization, and drought.