Integrated multi-omic data and analyses reveal the pathways underlying key ornamental traits in carnation flowers.

Carnation (Dianthus caryophyllus) is one of the most popular ornamental flowers in the world. Although numerous studies on carnations exist, the underlying mechanisms of flower color, fragrance, and the formation of double flowers remain unknown. Here, we employed an integrated multi-omics approach to elucidate the genetic and biochemical pathways underlying the most important ornamental features of carnation flowers. First, we assembled a high-quality chromosome-scale genome (636 Mb with contig N50 as 14.67 Mb) of D. caryophyllus, the 'Scarlet Queen'. Next, a series of metabolomic datasets was generated with a variety of instrumentation types from different parts of the flower at multiple stages of development to assess spatial and temporal differences in the accumulation of pigment and volatile compounds. Finally, transcriptomic data were generated to link genomic, biochemical, and morphological patterns to propose a set of pathways by which ornamental traits such as petal coloration, double flowers, and fragrance production are formed. Among them, the transcription factors bHLHs, MYBs, and a WRKY44 homolog are proposed to be important in controlling petal color patterning and genes such as coniferyl alcohol acetyltransferase and eugenol synthase are involved in the synthesis of eugenol. The integrated dataset of genomics, transcriptomics, and metabolomics presented herein provides an important foundation for understanding the underlying pathways of flower development and coloration, which in turn can be used for selective breeding and gene editing for the development of novel carnation cultivars.

Genome-Wide Identification and Characterization of Aquaporins and Their Role in the Flower Opening Processes in Carnation (Dianthus caryophyllus).

Aquaporins (AQPs) are associated with the transport of water and other small solutes across biological membranes. Genome-wide identification and characterization will pave the way for further insights into the AQPs' roles in the commercial carnation (Dianthus caryophyllus). This study focuses on the analysis of AQPs in carnation (DcaAQPs) involved in flower opening processes. Thirty DcaAQPs were identified and grouped to five subfamilies: nine PIPs, 11 TIPs, six NIPs, three SIPs, and one XIP. Subsequently, gene structure, protein motifs, and co-expression network of DcaAQPs were analyzed and substrate specificity of DcaAQPs was predicted. qRT-PCR, RNA-seq, and semi-qRTRCR were used for DcaAQP genes expression analysis. The analysis results indicated that DcaAQPs were relatively conserved in gene structure and protein motifs, that DcaAQPs had significant differences in substrate specificity among different subfamilies, and that DcaAQP genes' expressions were significantly different in roots, stems, leaves and flowers. Five DcaAQP genes (DcaPIP1;3, DcaPIP2;2, DcaPIP2;5, DcaTIP1;4, and DcaTIP2;2) might play important roles in flower opening process. However, the roles they play are different in flower organs, namely, sepals, petals, stamens, and pistils. Overall, this study provides a theoretical basis for further functional analysis of DcaAQPs.

Leaf-morphology-assisted selection for resistance to two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) in carnations (Dianthus caryophyllus L).

BACKGROUND: The development of a cultivar resistant to the two-spotted spider mite has provided both ecological and economic benefits to the production of cut flowers. This study aimed to clarify the mechanism of resistance to mites using an inbred population of carnations. RESULTS: In the resistant and susceptible plants selected from an inbred population, a difference was recognised in the thickness of the abaxial palisade tissue by microscopic examination of the damaged leaf. Therefore, it was assumed that mites displayed feeding preferences within the internal leaf structure of the carnation leaf. The suitability of the host plant for mites was investigated using several cultivars selected using an index of the thickness from the abaxial leaf surface to the spongy tissue. The results suggested that the cultivar associated with a thicker abaxial tissue lowered the intrinsic rate of natural increase of the mites. The cultivars with a thicker abaxial tissue of over 120 µm showed slight damage in the field test. CONCLUSION: The ability of mites to feed on the spongy tissue during an early life stage from hatching to adult emergence was critical. It was possible to select a cultivar that is resistant to mites under a real cultivation environment by observing the internal structure of the leaf. © 2016 Society of Chemical Industry.

A quantitative framework for flower phenotyping in cultivated carnation (Dianthus caryophyllus L.).

Most important breeding goals in ornamental crops are plant appearance and flower characteristics where selection is visually performed on direct offspring of crossings. We developed an image analysis toolbox for the acquisition of flower and petal images from cultivated carnation (Dianthus caryophyllus L.) that was validated by a detailed analysis of flower and petal size and shape in 78 commercial cultivars of D. caryophyllus, including 55 standard, 22 spray and 1 pot carnation cultivars. Correlation analyses allowed us to reduce the number of parameters accounting for the observed variation in flower and petal morphology. Convexity was used as a descriptor for the level of serration in flowers and petals. We used a landmark-based approach that allowed us to identify eight main principal components (PCs) accounting for most of the variance observed in petal shape. The effect and the strength of these PCs in standard and spray carnation cultivars are consistent with shared underlying mechanisms involved in the morphological diversification of petals in both subpopulations. Our results also indicate that neighbor-joining trees built with morphological data might infer certain phylogenetic relationships among carnation cultivars. Based on estimated broad-sense heritability values for some flower and petal features, different genetic determinants shall modulate the responses of flower and petal morphology to environmental cues in this species. We believe our image analysis toolbox could allow capturing flower variation in other species of high ornamental value.

Evaluation of ploidy level and endoreduplication in carnation (Dianthus spp.).

Carnation (Dianthus caryophyllus L.) is one of the fifth most important ornamental species worldwide. Many desirable plant characteristics, such as big size, adaptation under stress, and intra or interspecific hybridization capability, are dependent on plant ploidy level. We optimized a quick flow cytometry method for DNA content determination in wild and cultivated carnation samples that allowed a systematic evaluation of ploidy levels in Dianthus species. The DNA content of different carnation cultivars and wild Dianthus species was determined using internal reference standards. The precise characterization of ploidy, endoreduplication and C-value of D. caryophyllus 'Master' makes it a suitable standard cultivar for ploidy level determination in other carnation cultivars. Mixoploidy was rigorously characterized in different regions of several organs from D. caryophyllus 'Master', which combined with a detailed morphological description suggested some distinctive developmental traits of this species. Both the number of endoreduplication cycles and the proportion of endopolyploid cells were highly variable in the petals among the cultivars studied, differently to the values found in leaves. Our results suggest a positive correlation between ploidy, cell size and petal size in cultivated carnation, which should be considered in breeding programs aimed to obtain new varieties with large flowers.

Morphological, physiological, and genetic variation between metallicolous and nonmetallicolous populations of Dianthus carthusianorum.

Waste deposits produced by metal mining and smelting activities provide extremely difficult habitats for plant colonization and growth. Therefore, plants spontaneously colonizing such areas represent a very interesting system for studying evolution of plant adaptation and population differentiation between contaminated and noncontaminated environments. In this study, two populations of Dianthus carthusianorum, one originating from Zn-Pb waste deposit (a metallicolous population, M) and the other from unpolluted soil (a nonmetallicolous population, NM), were analyzed in respect of their morphological and physiological traits as well as genetic markers. It was found that the plants inhabiting the waste heap differed significantly from the NM plants in terms of leaf size and shape, and these differences were persistent between the first generation of the plants of both populations cultivated under uniform, controlled laboratory conditions. In contrast with the evident morphological differences, no significant differentiation between the populations regarding the physiological traits measured (accumulation of proline, anthocyanins, chlorophyll, carotenoids) was found. These traits can be regarded as neither population specific nor stress markers. The genetic variability was analyzed using 17 random amplified polymorphic DNA (RAPD) and four inter simple sequence repeat (ISSR) markers; this proved that the differentiation between the M and NM populations exists also at the genetic level. Analysis of molecular variance (AMOVA) showed that 24% of the total genetic diversity resided among populations, while 76% - within the populations. However, no significant differences in intrapopulation genetic diversity (Hj) between the M and NM populations of D. carthusianorum was found, which contradicts the theory that acquisition of adaptation mechanisms to adverse, isolated growth habitats is related to reduction in genetic diversity. Distinct genetic differences between the two populations in combination with evident morphological variation support the proposal to regard the M population of D. carthusianorum as a separate calamine ecotype.

Phenotypic consequences of polyploidy and genome size at the microevolutionary scale: a multivariate morphological approach.

• Chromosomal duplications and increases in DNA amount have the potential to alter quantitative plant traits like flower number, plant stature or stomata size. This has been documented often across species, but information on whether such effects also occur within species (i.e. at the microevolutionary or population scale) is scarce. • We studied trait covariation associated with polyploidy and genome size (both monoploid and total) in 22 populations of Dianthus broteri s.l., a perennial herb with several cytotypes (2x, 4x, 6x and 12x) that do not coexist spatially. Principal component scores of organ size/number variations were assessed as correlates of polyploidy, and phylogenetic relatedness among populations was controlled using phylogenetic generalized least squares. • Polyploidy covaried with organ dimensions, causing multivariate characters to increase, remain unchanged, or decrease with DNA amount. Variations in monoploid DNA amount had detectable consequences on some phenotypic traits. According to the analyses, some traits would experience phenotypic selection, while others would not. • We show that polyploidy contributes to decouple variation among traits in D. broteri, and hypothesize that polyploids may experience an evolutionary advantage in this plant lineage, for example, if it helps to overcome the constraints imposed by trait integration.

Selection toward shorter flowers by butterflies whose probosces are shorter than floral tubes.

Darwin's meticulous observations on the function of floral shape led to his famous prediction of a long-tongued pollinator, which he believed to be the evolutionary trigger for the long-spurred flowers of the Madagascar star orchid. Although tubular flowers are common, long tubes or spurs are an exception, suggesting that selection maintaining short flowers is widespread. Using the butterfly-pollinated carnation Dianthus carthusianorum and two butterfly species differing in proboscis length (Melanargia galathea and Inachis io) as model organisms, we experimentally demonstrate a reduction in pollinator efficiency with an increasing difference between proboscis length and floral tube length. Such a relationship is a prerequisite for the evolution of floral shape in response to pollinator morphology.

Carnation (Dianthus caryophylus L.).

Carnation is a valuable crop for the cut flower industry and demand for new and improved varieties is growing. However, genetic transformation of carnations is currently limited because of a lack of efficient routine technique. In this chapter, we present an easy and effective protocol for gene transfer to carnation node explants and subsequent adventitious shoot regeneration. For high-adventitious shoot regeneration, node explants from first to third node of 5- to 8-cm long shoots were cultured on Murashige and Skoog (MS) medium, containing 1.0 mg/Lthidiazuron (TDZ), 0.1 mg/L alpha-napthalenoacetic acid (NAA), 20 g/L sucrose, and 2 g/L Gellan gum for 10 d. Then the explants were cut into 8 radial segments and subcultured onto MS medium, containing 1.0 mg/L BA, 0.1 mg/L NAA, 20 g/L sucrose and 2 g/L Gellan Gum. For effective genetic transformation, 3- to 5-d precultured node explants were submerged in an Agrobacerium suspension for 10 min, then cocultivated on filter paper soaked with water and 50 microM acetosyringone (AS). After cocultivation, the explants were cut into eight radial segments and subcultured onto selection medium until transformed shoots regenerated from the explants.

[Effect of freezing of seeds on the morphological characteristics of four pink species].

The effect of non-deep freezing (-10 degrees C) and deep freezing (-196 degrees C) on the morphological characteristics of four pink species was investigated. It was shown that the growth and development of plants after cryopreservation changed insignificantly. Rather stable characteristics (diameter of flower, the number of cauline nodes, and root length) did not change. The length of generative shoots decreased a little, and the character of histograms changed. On the other hand, the changeable characteristics (the number of vegetative shoots and flowers) decreased after seed cryopreservation. However, the number of flowers did not vary in the next year. The parameters essential for seed cryopreservation, seed germination and the quantity of fruits, did not changed. It was also found that seed freezing exerts a stimulating effect on seed germination and the top limit of some parameters, including the quantity of generative organs.

A leader intron and 115-bp promoter region necessary for expression of the carnation S-adenosylmethionine decarboxylase gene in the pollen of transgenic tobacco.

The expression of CSDC9 encoding S-adenosylmethionine decarboxylase (SAMDC) is developmentally and spatially regulated in carnation. To examine the regulation of the SAMDC gene, we analyzed the spatial expression of CSDC9 with a 5'-flanking beta-glucuronidase fusion in transgenic tobacco plants. GUS was strongly expressed in flower, pollen, stem and vein of cotyledons. Expression in both anther and stigma was under developmental control; analysis of a series of mutants with deletions of the 5'-flanking region demonstrated differential activation in petal, anther, stigma and pollen grains. All the major cis-regulatory elements required for pollen-specific transcription were located in the upstream region between -273 and -158. This region contains four putative elements related to gibberellin induction (pyrimidine boxes, TTTTTTCC and CCTTTT) and pollen-specific expression (GTGA and AGAAA). In addition, the first 5'-leader intron was necessary for tissue-specific expression.

[Microscopic identification of Herba Dianthi grown in Shandong].

The medicinal materials of four species and two varieties of Herba Dianthi grown in Shandong were identified. The result shows that they are identified easily and accurately according to the outer properties, and they are apparently divided into the Shizhu group, the Qumai group and the Dianthus shandongensis on the basis of their morphological and structural characteristics of the stem and leaf, but they have not obvious distinction among the species of every group.

[Investigation and identification of original plants of Herba Dianthi produced in Shandong].

The original plant of Herba Dianthi in Shandong are divided into 4 species and 2 varieties by investigation and classified identification. Of them Dianthus shandongensis J. X. Li et F. Q. Zhou is a new species and D. chinensis L. var. versicolor (Fich. ex Link) Y. C. Ma is a new recorder in Shandong. D. chinensis L. is the most important source of Herba Dianthi in Shandong and the D. chinensis L. var. versicolor and D. chinensis L. var. liaotungensis Y. C. Chu, occasionally is D. shandongensis, D. superbus L. and D. longicalyx Miq. Haven't grown into commercial medicinal materials.