Project: Design and evaluation of molecular breeding tools for cultivated carnation (Dianthus caryophyllus L.)

We aim to develop a collection of molecular tools for cultivated carnation (Dianthus caryophyllus L.) that will enable marker-assisted selection (MAS) as a new breeding strategy in this species. In many ornamental crops, application of MAS is a challenge due to the limited availability of molecular and genetic information. Recent developments in DNA sequencing technology make the identification of molecular polymorphisms among different varieties of a given species feasible, which can then be used for the discovery of trait-linked markers through genome-wide association studies (GWAS) [1, 2]. In this project, GWAS will be performed on a large collection of proprietary carnation varieties, available at Barberet & Blanc (B&B), using polymorphisms that will be identified through genome and transcriptome sequencing. In a first phase of the GWAS, a thorough screening of the available germplasm with a genome-wide set of polymorphic markers will be performed. Later, the genetic information obtained in this way will be used to correlate any desired phenotype with the molecular markers identified. This makes the GWAS approach a valuable strategy for quick identification of trait-linked markers required for MAS in carnation breeding programs. To demonstrate the potential economic benefit of GWAS application, we will address the problem of poor adventitious root formation from propagated stem cuttings in some carnation accessions, to enable early selection of elite plant material optimally suited for mass-production of cut flowers. An efficient propagation of uniform starting material is a strong requirement in mass-production, which is not equally fulfilled among the wide range of commercial carnation varieties. The production of young plantlets is frequently hampered by minimal adventitious root formation from stem cuttings, which has a strong genetic dependency and which leads to production losses in certain carnation cultivars. The problem has been partly overcome by optimizing the storage of fresh cuttings at low temperature [3, 4, 5, 6]. Hence production economics would tremendously benefit from improved growth characteristics of stem cuttings as well as from reduced high-cost and energy-demanding treatments. Current knowledge about adventitious rooting in carnation COly comes from physiological studies showing that root induction is affected by complex interactions between sucrose and hormone levels. The large variation in adventitious root formation is also found to be genotype-dependent [7, 8, 9, 10]. In this project, the genetic determinants responsible for the induction of adventitious root formation and root development in carnation stem cuttings will be identified, and linked molecular markers allocated. The strategy and techniques developed serve as a straightforward approach for genomic selection of desired phenotypes, which will lead to a reduction in breeding time and costs for creating new cut-flower cultivars._x000D_The Ps in this project will collaborate on whole genome and transcriptome analysis for the development of genome-wide single nucleotide polymorphism (SNP) collections for the application of GWAS in carnation. GWAS is already widely applied in field crops, e.g. maize and barley [11], but hardly explored in ornamental crops except for a very recent study in orchids [12]. Recent improvements in sequencing technologies have resulted in reduction of costs and time efforts, rendering the technology now amenable for genetically less studied crops like carnation. Contributions of the project Ps to the project are complementary. Since next-generation sequencing (NGS) technologies are the state of the art [13], these will be used to identify polymorphic markers for the application of GWAS. The genome sequence of a commercial, representative carnation variety will be established and re-sequencing of a few distant varieties will be performed to identify genetic variation. Functional genes will be identified and genetic variation will be assessed between broader ranges of carnation varieties used in breeding programs. Universidad de Murcia (UMU) subcontracted by B&B has already contributed to the understanding of adventitious root formation in carnation cuttings [3,7,8,10,14]. UMU and Universidad Miguel Hernández de Elche (UMH) will contribute to the biological interpretation of the genetic analyses, and effects of polymorphisms in candidate genes which have an impact on root formation and which can be used in association mapping of this complex trait. Genetwister Technologies (GT) currently develops genetic markers for trait selection in a series of horticultural crops and will perform GWAS analysis to identify informative markers to enable early stage selection of carnation varieties with optimal adventitious root formation and root development. This in turn will lead to improved varieties at much lower production costs._x000D__x000D_[Numbered references provided in Appendix I]

Acronym CARNOMICS (Reference Number: 6834)
Duration 01/04/2012 - 31/08/2015
Project Topic Our goal is to develop advanced molecular tools for marker-assisted breeding in carnation. Benefits of this new technology will be validated by the genetic dissection of growth characteristics that limit successful mass production of commercial carnation varieties from stem cuttings.
Project Results
(after finalisation)
The CO results of the CARNOMICS project are categorized as scientific knowledge, new methodologies and molecular breeding tools. The CO goal of the project was the development of a collection of molecular tools for carnation to enable 'marker-assisted selection' (MAS) as a new breeding strategy in this ornamental crop. The emphasis was on the development of SNP markers for the efficiency of rooting of carnation cuttings, a trait which is of major importance in the production practice of carnation. At the onset of the project knowledge was available about the influence of plant hormones on this process. However, little was known about genetic factors influencing adventitious rooting in carnation._x000D_By applying next-generation sequencing (NGS) techniques in this project genome- and transcriptome sequence data of a collection of commercial carnation varieties were generated. The quality of the genome draft of the reference cultivar from this project was not as high as anticipated and in the meantime a genome sequence from another carnation variety became publicly available. However, the transcriptome data have provided a large contribution to the improvement of the gene models of the carnation genome. By comparing 15 different varieties an extensive set of markers was obtained, which is useful for several applications in carnation. The markers were implemented in a custom-made genome-wide array for carnation, which enables efficient genotyping of large numbers of carnation varieties._x000D_The original plan was to find SNP markers associating with rooting efficiency from the collection of carnation varieties ('germplasm') available at Barberet & Blanc (B&B) utilizing GWAS (genome-wide association study). For this purpose large-scale phenotyping of the rooting of carnation cuttings had to be developed. An extensive pilot experiment using various cultivars provided a method to quantify these phenotypes. It became apparent that the phenotyping was very labour-intensive and time-consuming and therefore it was not feasible to apply GWAS to the entire carnation collection of B&B. As an alternative we chose to carry out GWAS on the crossing population between a good and a bad rooting carnation variety, which was made for genetic mapping. The genotyping data from this population on the SNP array were incorporated into a genetic linkage map. However, the GWAS analyses did not yield any significant SNPs associating with the rooting phenotype. Through the knowledge and experience that was gained in this project it is possible to make recommendations for further research into rooting markers in carnation._x000D_An extensive expression profiling study was carried out on the two parents of the cross mentioned above, in order to identify potential candidate genes involved in the adventitious rooting process. A multidisciplinary approach was followed, studying hormone profiles and morphological changes as well. The outcome of this study is a model for genetic and metabolic factors playing a role in rooting. A number of candidate genes was identified, but no markers were found in these genes which showed a clear correlation with rooting._x000D_In conclusion, scientific knowledge, expertise and molecular tools are the CO results from this project. The toolkit can be applied to finding markers for other genetic traits in carnation and subsequent marker-assisted breeding._x000D_
Network Eurostars
Call Eurostars Cut-Off 7

Project partner

Number Name Role Country
2 Genetwister Technologies BV Coordinator Netherlands
2 Barberet & Blanc, S.A. Partner Spain