Takashi Onozaki

Genetic improvement of vase life of carnation flowers by crossing and selection

Abstract We used conventional cross-breeding techniques to develop many carnation lines with long vase life and either low ethylene production or low ethylene sensitivity. Two cycles of selection and crossing to improve vase life led to a 3.6-day increase in mean vase life. All 39 selected lines had significantly longer vase life than the control cultivar, ‘White Sim’. In particular, second-generation lines 63-3, 63-12, 66-15, and 63-41 had a mean vase life of more than 15 days without chemical treatment. Measurements of ethylene production indicated that flowers of all second-generation selected lines had a greatly reduced capacity to produce ethylene. We screened three lines (515-10, 64-13, and 64-54) with low ethylene sensitivity. Evaluation by exposure to ethylene at high concentration showed that 64-13 and 64-54 were less sensitive to ethylene than ‘Chinera’, which is known for it low sensitivity. The vase life of these low-sensitivity lines was about twice that of ‘White Sim’. The extended vase life of selected lines was related to low ethylene production at flower senescence rather than to degree of ethylene sensitivity in young flowers. Ethylene sensitivity decreased with the age of the flower in many selected lines. The results clearly show that vase life of carnation flowers can be extended by crossing and selection.

Flavonoid biosynthesis in white-flowered Sim carnations (Dianthus caryophyllus)

Abstract Analysis of flavonoid composition and gene expression of enzymes involved in anthocyanin synthesis in flowers of four acyanic and one cyanic cultivar of Sim carnation showed that the acyanic flower cultivars are divided into three types. The first includes two normal white cultivars, ‘U Conn Sim’ and ‘White Sim’; the second includes a nearly pure white cultivar, ‘Kaly’; and the third includes a nearly pure white cultivar, ‘White Mind’. ‘U Conn Sim’ and ‘White Sim’ accumulated flavonol glycosides and lacked anthocyanins. The transcription of the several genes of enzymes involved in flavonoid biosynthesis were reduced at a later flowering stage than the cyanic cultivar, especially the genes encoding dihydroflavonol 4-reductase and anthocyanidin synthase. ‘Kaly’ accumulated flavanone glycosides and a small amount of flavonol and flavone glycosides by blocking the transcription of the gene encoding flavanone 3-hydroxylase, in addition to the transcriptional reduction of the genes for flavonoid biosynthesis at a later flowering stage. Although ‘White Mind’ contains little flavonoid, the position of the block on flavonoid biosynthesis in ‘White Mind’ is not known.

Sequence Analysis of the Genome of Carnation (Dianthus caryophyllus L.)

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp.

Transcriptome analysis of carnation ( Dianthus caryophyllus L.) based on next-generation sequencing technology

BackgroundCarnation (Dianthus caryophyllus L.), in the family Caryophyllaceae, can be found in a wide range of colors and is a model system for studies of flower senescence. In addition, it is one of the most important flowers in the global floriculture industry. However, few genomics resources, such as sequences and markers are available for carnation or other members of the Caryophyllaceae. To increase our understanding of the genetic control of important characters in carnation, we generated an expressed sequence tag (EST) database for a carnation cultivar important in horticulture by high-throughput sequencing using 454 pyrosequencing technology.ResultsWe constructed a normalized cDNA library and a 3’-UTR library of carnation, obtaining a total of 1,162,126 high-quality reads. These reads were assembled into 300,740 unigenes consisting of 37,844 contigs and 262,896 singlets. The contigs were searched against an Arabidopsis sequence database, and 61.8% (23,380) of them had at least one BLASTX hit. These contigs were also annotated with Gene Ontology (GO) and were found to cover a broad range of GO categories. Furthermore, we identified 17,362 potential simple sequence repeats (SSRs) in 14,291 of the unigenes. We focused on gene discovery in the areas of flower color and ethylene biosynthesis. Transcripts were identified for almost every gene involved in flower chlorophyll and carotenoid metabolism and in anthocyanin biosynthesis. Transcripts were also identified for every step in the ethylene biosynthesis pathway.ConclusionsWe present the first large-scale sequence data set for carnation, generated using next-generation sequencing technology. The large EST database generated from these sequences is an informative resource for identifying genes involved in various biological processes in carnation and provides an EST resource for understanding the genetic diversity of this plant.

A RAPD-derived STS marker is linked to a bacterial wilt (Burkholderia caryophylli) resistance gene in carnation.

Bacterial wilt caused by Burkholderia caryophylli is one of the most important and damaging diseases of carnations (Dianthus caryophyllus) in Japan. We aimed to identify random amplified polymorphic DNA (RAPD) markers associated with the genes controlling bacterial wilt resistance in a resistance-segregating population of 134 progeny plants derived from a cross between ‘Carnation Nou No. 1’ (a carnation breeding line resistant to bacterial wilt) and ‘Pretty Favvare’ (a susceptible cultivar). We screened a total of 505 primers to obtain RAPD markers useful for selecting resistant carnation lines: 8 RAPD markers identified by bulked segregant analysis were linked to a major resistance gene; of these, WG44-1050 had the greatest effect on resistance to bacterial wilt. A locus with large effect on bacterial resistance was mapped around WG44-1050 through QTL analysis. The RAPD marker WG44-1050 was successfully converted to a sequence-tagged site (STS) marker suitable for marker-assisted selection (MAS). Five combinations of primers were designed for specific amplification of WG44-1050. In addition, the STS marker we developed was useful and reliable as a selection marker for breeding for resistance to bacterial wilt, using a highly resistant wild species, D. capitatus ssp. andrzejowskianus and a resistant line, ‘Carnation Nou No. 1’, as breeding materials.

Construction of a reference genetic linkage map for carnation (Dianthus caryophyllus L.).

BackgroundGenetic linkage maps are important tools for many genetic applications including mapping of quantitative trait loci (QTLs), identifying DNA markers for fingerprinting, and map-based gene cloning. Carnation (Dianthus caryophyllus L.) is an important ornamental flower worldwide. We previously reported a random amplified polymorphic DNA (RAPD)-based genetic linkage map derived from Dianthus capitatus ssp. andrezejowskianus and a simple sequence repeat (SSR)-based genetic linkage map constructed using data from intraspecific F2 populations; however, the number of markers was insufficient, and so the number of linkage groups (LGs) did not coincide with the number of chromosomes (x = 15). Therefore, we aimed to produce a high-density genetic map to improve its usefulness for breeding purposes and genetic research.ResultsWe improved the SSR-based genetic linkage map using SSR markers derived from a genomic library, expression sequence tags, and RNA-seq data. Linkage analysis revealed that 412 SSR loci (including 234 newly developed SSR loci) could be mapped to 17 linkage groups (LGs) covering 969.6 cM. Comparison of five minor LGs covering less than 50 cM with LGs in our previous RAPD-based genetic map suggested that four LGs could be integrated into two LGs by anchoring common SSR loci. Consequently, the number of LGs corresponded to the number of chromosomes (x = 15). We added 192 new SSRs, eight RAPD, and two sequence-tagged site loci to refine the RAPD-based genetic linkage map, which comprised 15 LGs consisting of 348 loci covering 978.3 cM. The two maps had 125 SSR loci in common, and most of the positions of markers were conserved between them. We identified 635 loci in carnation using the two linkage maps. We also mapped QTLs for two traits (bacterial wilt resistance and anthocyanin pigmentation in the flower) and a phenotypic locus for flower-type by analyzing previously reported genotype and phenotype data.ConclusionsThe improved genetic linkage maps and SSR markers developed in this study will serve as reference genetic linkage maps for members of the genus Dianthus, including carnation, and will be useful for mapping QTLs associated with various traits, and for improving carnation breeding programs.

Identification of tightly linked SSR markers for flower type in carnation (Dianthus caryophyllus L.)

Single or double flower type is one of the most important breeding targets in carnation (Dianthus caryophyllus L.). We mapped the D85 locus, which controls flower type, to LG 85P_15–2 using a simple sequence repeat (SSR)-based genetic linkage map constructed using 91 F2 progeny derived from a cross between line 85–11 (double flower) and ‘Pretty Favvare’ (single flower). A positional comparison using SSR markers as anchor loci revealed that the map positions of the D85 locus corresponded to the single locus controlling the single flower type derived from wild D. capitatus ssp. andrzejowskianus. We identified four co-segregating SSR markers on the D85 locus. Verification of the SSR markers in commercial cultivars revealed that two of the four SSR markers (CES0212 and CES1982) were tightly linked to the D85 locus, and amplified a 176-bp and 269-bp allele, respectively, which were common and unique to double flower cultivars. The map positions of the D85 locus and the tightly linked SSR markers will be useful for determining the genetic basis of flower type and for marker-assisted breeding of carnations.

Analysis of genomic DNA of DcACS1, a 1-aminocyclopropane-1-carboxylate synthase gene, expressed in senescing petals of carnation (Dianthus caryophyllus) and its orthologous genes in D. superbus var. longicalycinus.

Carnation (Dianthus caryophyllus) flowers exhibit climacteric ethylene production followed by petal wilting, a senescence symptom. DcACS1, which encodes 1-aminocyclopropane-1-carboxylate synthase (ACS), is a gene involved in this phenomenon. We determined the genomic DNA structure of DcACS1 by genomic PCR. In the genome of ‘Light Pink Barbara’, we found two distinct nucleotide sequences: one corresponding to the gene previously shown as DcACS1, designated here as DcACS1a, and the other novel one designated as DcACS1b. It was revealed that both DcACS1a and DcACS1b have five exons and four introns. These two genes had almost identical nucleotide sequences in exons, but not in some introns and 3′-UTR. Analysis of transcript accumulation revealed that DcACS1b is expressed in senescing petals as well as DcACS1a. Genomic PCR analysis of 32 carnation cultivars showed that most cultivars have only DcACS1a and some have both DcACS1a and DcACS1b. Moreover, we found two DcACS1 orthologous genes with different nucleotide sequences from D. superbus var. longicalycinus, and designated them as DsuACS1a and DsuACS1b. Petals of D. superbus var. longicalycinus produced ethylene in response to exogenous ethylene, accompanying accumulation of DsuACS1 transcripts. These data suggest that climacteric ethylene production in flowers was genetically established before the cultivation of carnation.

Significance of CmCCD4a orthologs in apetalous wild chrysanthemum species, responsible for white coloration of ray petals

Most chrysanthemum (Chrysanthemum morifolium Ramat.) flowers have a central capitulum, composed of many disc florets that is surrounded by ray petals. CmCCD4a, a gene that encodes a carotenoid cleavage dioxygenase (CCD), is expressed specifically in the ray petals of chrysanthemum cultivars, and its expression leads to white ray petals as a result of carotenoid degradation. Here, we show that wild chrysanthemums with white ray petals have CmCCD4a orthologs, whereas those with yellow ray petals lack these orthologs, as is the case in chrysanthemum cultivars. CmCCD4a orthologs also exist in some lines of Chrysanthemum pacificum and Chrysanthemum shiwogiku, even though these species lack ray petals. Interspecific hybridization between C. shiwogiku and a yellow-flowered chrysanthemum cultivar showed that the CmCCD4a orthologs from C. shiwogiku lead to the development of white ray petals. This indicates that the translation products of the CmCCD4a orthologs maintain enzymatic activity that can degrade carotenoids in chrysanthemums, irrespective of whether or not the ray petals that CmCCD4a expression actually occurred.

Current researches in ornamental plant breeding

Human beings are instinctively drawn to beauty, as it gives joy, eases our minds, and makes us happy. A representative of beauty in nature is ornamental plants. We can live without ornamental plants, but when we are surrounded by flowers and greenery, it makes for a better, more joyful life. Ornamental plants are also important in the agricultural industry. In Japan, 3.8 billion cut flowers and 226 million potted flowers were produced in 2016. Breeding new and attractive cultivars is necessary for further growth of the flower industry. Consumers are always seeking new varieties of ornamental plants. Breeders are asked to create new flower colors, attractive shapes, improved longevity, better fragrance, etc., and basic research on physiological and genetic mechanisms on such target characteristics is very important for breeding new cultivars. Development and adaptation of new breeding methods such as genetic transformation and mutation using ion beams are also useful for efficiently creating epoch-making unique cultivars. A large number of plant species are used as ornamentals, and these plants harbor a wide range of breeding targets. The current special issue presents fourteen recent works selected from the numerous research subjects related to ornamental plant breeding. It is our pleasure to introduce these research studies to everyone who is interested in ornamental horticulture. Ornamental plants provide comfort and peace in our everyday lives. Progress in ornamental plant breeding research will lead to the creation of more ornamentals, thus contributing to the development of the flower industry while making human lives more pleasant.