TI: Identification of random amplified polymorphic DNA markers linked to bacterial wilt

resistance in carnations.
AU: Onozaki,-T; Ikeda,-H; Tanikawa,-N; Shibata,-M; Kudo,-K; Funayama,-T
ED: Forkmann,-G; Hauser,-B; Michaelis,-S
SO: Acta-Horticulturae. 2003; (612): 95-103
PB: Leuven, Belgium: International Society for Horticultural Science (ISHS).
PY: 2003
AB: Bacterial wilt (caused by Burkholderia caryophylli) is one of the most serious diseases
of carnations (Dianthus caryophyllus) in Japan. This disease is very difficult to control with
chemicals once it has occurred. Breeding of resistant cultivars is considered the optimum
strategy to overcome this disease. However, it takes over 3 months to determine the
resistance of breeding materials by inoculation assays. DNA markers, which can be identified
within days, should therefore be a powerful alternative for disease resistance screening.
Therefore, we decided to identify random amplified polymorphic DNA (RAPD) markers
associated with genes controlling wilt resistance in a resistance-segregating population of 134
progenies 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
465 primers to obtain RAPD markers useful for selecting resistant carnation lines, 11 RAPD
markers were linked to genes for resistance to bacterial wilt and 3 linkage groups were
identified by linkage analysis. At least 3 genes are concerned with resistance to bacterial wilt.
In particular, 4 RAPD markers identified by bulked segregant analysis were linked to a major
resistance gene. These markers should be useful for marker-assisted selection in carnation
breeding programmes..

TI: 'Carnation Nou No.1', a carnation breeding line resistant to bacterial wilt
(Burkholderia caryophylli).
AU: Onozaki,-T; Ikeda,-H; Yamaguchi,-T; Himeno,-M; Amano,-M; Shibata,-M
SO: Horticultural-Research-Japan. 2002; 1(1): 13-16
PB: Tokyo, Japan: Yokendo Ltd., Publishers.
PY: 2002
AB: Bacterial wilt (B. caryophylli) is one of the most serious and damaging diseases of
carnations (Dianthus caryophyllus) in Japan. It causes serious crop losses in carnations grown
in warm districts. Therefore, the National Institute of Floricultural Science initiated a
breeding programme for resistance to bacterial wilt in 1988. 'Carnation Nou No.1' is a
carnation breeding line resistant to bacterial wilt and was derived from interspecific cross
between carnation cultivar 'Super Gold' and the highly resistant wild species D. capitatus.
'Carnation Nou No.1' has a perpetual flowering habit. The yield (number of cut flowers/plant)
of this line is very high; the mean yield was 11.5 cut flowers/plant, which was higher than
that of 'Super Gold', 'Scania', and 'Coral'. It has been shown that 'Carnation Nou No.1' has the
ability to produce resistant progeny, indicating that 'Carnation Nou No.1' is available for a
breeding material of carnation and Dianthus cultivars with resistance. The nuclear DNA
amount of 'Carnation Nou No.1' was estimated by flow cytometry to be between that of D.
capitatus and 'Super Gold'..

TI: Male gametophytic generation and a possible approach for selective pollination in
carnation (Dianthus) breeding program.
AU: Tejaswini-
SO: Rostlinna-Vyroba. 2002; 48(8): 368-375
PB: Praha, Czech Republic: Ustav Zemedelskych a Potravinarskych Informaci.
PY: 2002
AB: An experiment was conducted (1) to estimate the morphological and histochemical
variability in pollen populations of carnation, (2) to evaluate the relationship between
morphological/histochemical features and pollen performance, and (3) to evaluate the
relationship between the pollen grain features and resistance to fusarium wilt (F. oxysporum
f.sp. dianthi). Five cultivars of carnation (D. caryophyllus) were used in this study, namely
Golden rush, Internet, Monaco, Regina and Trendy. A plant population of D. chinensis was
grown in the field and three genotypes (G2, G3 and G7) were used for the detailed study of
pollen grains. The result revealed the existence of variability in pollen morphology and
histochemical content among and within the cultivars and species of D. caryophyllus and D.
chinensis. Such variability was found to be sufficient for selection. Pollen grain size and
histochemical content were found to be associated with germination capacity and pollen tube
growth rate. In addition, pollen germination capacity and pollen tube elongation in response
to the presence of culture filtrate of F. oxysporum f.sp. dianthi was found to be governed by
pollen grain size and histochemical content of pollen grains. The results suggest the
possibility of selecting the desired pollen grains from a pollen population and the possibility
of attempting selective pollination in carnation breeding programme..

TI: Use of molecular markers to improve cut flowers longevity in carnation.

AU: Benedetti,-L-de; Burchi,-G; Bruna,-S; Mercuri,-A; Schiva,-T
ED: Blom,-T; Criley,-R
SO: Acta-Horticulturae. 2003; (624): 343-345
PB: Leuven, Belgium: International Society for Horticultural Science (ISHS).
PY: 2003
AB: Postharvest longevity is one of the first and most important traits considered in
carnation breeding. In this work, RAPD analysis was used for the identification of molecular
markers that could be associated with flower vase life. Two cultivars (Roland and Milady)
with different flower longevity and their F1 and backcross offspring were analysed. Our
previous studies on the segregation of the character indicated that flower vase life is probably
a complex quantitative trait in carnation, involving more than a single gene or mechanism,
and that this character is controlled by genes showing predominantly additive effects. Sixty
random primers were initially tested on a few genotypes. DNAs from 70 F1 and 36 backcross
offspring were then analysed with 10 selected primers. For each RAPD band tested, the
progenies were divided into two groups according to the similarity of the band pattern with
Roland or Milady. The statistical significance of the differences in vase life between the two
groups was evaluated. The analysis revealed several bands that allow to discriminate
significantly the individuals with extended longevity. Statistical analysis showed a positive
correlation between the score of each progeny (number of RAPD bands similar to Roland)
and flower longevity..

TI: A RAPD-derived STS marker is linked to a bacterial wilt (Burkholderia caryophylli)

resistance gene in carnation.
AU: Onozaki,-T; Tanikawa,-N; Taneya,-M; Kudo,-K; Funayama,-T; Ikeda,-H; Shibata,-M
SO: Euphytica-. 2004; 138(3): 255-262
PB: Dordrecht, Netherlands: Kluwer Academic Publishers.
PY: 2004
AB: 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..

TI: Construction of a genetic linkage map for the carnation by using RAPD and SSR
markers and mapping quantitative trait loci (QTL) for resistance to bacterial wilt caused by
Burkholderia caryophylli.
AU: Yagi,-M; Onozaki,-T; Taneya,-M; Watanabe,-H; Yoshimura,-T; Yoshinari,-T; Ochiai,-
Y; Shibata,-M
SO: Journal-of-the-Japanese-Society-for-Horticultural-Science. 2006; 75(2): 166-172
PB: Kyoto, Japan: Japanese Society for Horticultural Science.
PY: 2006
AB: We constructed a genetic linkage map for the carnation (Dianthus caryophyllus) on
the basis of random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR)
using a resistance-segregating population of 134 progeny lines that were derived from a cross
between 'Carnation Nou No. 1' (a carnation breeding line resistant to bacterial wilt) and
'Pretty Favvare' (a susceptible cultivar). The linkage map consisted of 137 RAPD and 9 SSR
markers. Linkage analysis revealed that 124 loci could be mapped to 16 linkage groups that
extended for 605.0 cM. The average interval between two loci was 4.9 cM. Quantitative trait
loci (QTL) analysis was applied to evaluations of resistance to bacterial wilt that were
replicated 8 times. The QTL that we reported previously with a large effect on resistance was
detected on group 6 which accounted for 60.5% of the total phenotypic variance with a LOD
score 23.46. Two other QTLs with small effects were detected on groups 2 and 5 with LOD
scores of 2.32 and 2.87, respectively. These results suggest that resistance to bacterial wilt in
carnation is related to one major and at least two minor genes. This study is the first report on
the construction of a linkage map of the carnation.

TI: Amphidiploids produced by natural chromosome-doubling in inter-specific hybrids

between Dianthus x isensis Hirahata et Kitam. and D. japonicus Thunb.
AU: Nimura,-M; Kato,-J; Mii,-M; Katoh,-T
SO: Journal-of-Horticultural-Science-and-Biotechnology. 2006; 81(1): 72-77
PB: Ashford, UK: Headley Brothers Ltd., The Invicta Press.
PY: 2006
AB: Inter-specific crosses were carried out between two lines of Dianthus x isensis
Hirahata et Kitam., and one line of D. japonicus Thunb. Germinating seeds were obtained
from only one line of D. x isensis used as the seed parent, and a total of 46 progeny from this
cross were all confirmed to be hybrids by RAPD analysis and flow cytometry. The hybrids
had intermediate characters of both parents with respect to leaf-width and flower-size, but
flowered at the same time as each other with a uniform flower colour, purplish-pink. All 46
hybrids initially had sterile pollen and seeds, but two later produced some flowers of larger
size with fertile pollen. Flow cytometric analysis revealed that flowers with restored pollen
fertility originated from a chimaeric tetraploid branch induced by natural chromosome-
doubling. Amphidiploid progeny were obtained from seeds produced by self-pollination of
flowers with restored pollen fertility. Since these amphidiploids had some desirable
characters, such as the early flowering of D. x isensis, and the vigorous Summer growth,
robust upright stems and broad leaves of D. japonicus, they can be used in carnation breeding
programmes to incorporate useful traits from these two species such as disease resistance,
heat tolerance and unique flower morphologies..

TI: In vitro culture techniques for carnation breeding.

AU: Radojevic,-L; Djordjevic,-N; Petrovic,-J
SO: Acta-Horticulturae. 1990; (280): 163-168
PY: 1990
AB: Details are given of tests of in vitro response of 7 cultivars to cell suspension, stem
segment and meristem tip cultures on MS media supplemented with various concentrations of
growth regulators. A high percentage of explants of all cultivars developed multiple shoots.
The number of subcultured shoots, transplanted shoots and transferred plants differed
between cultivars, indicating that different cultivars required different culture conditions..

TI: Construction of a reference genetic linkage map for carnation (Dianthus caryophyllus
L.).
AU: Yagi,-M; Yamamoto,-T; Isobe,-S; Hirakawa,-H; Tabata,-S; Tanase,-K; Yamaguchi,-H;
Onozaki,-T
SO: BMC-Genomics. 2013; 14(734): (26 October 2013)
PB: London, UK: BioMed Central Ltd.
PY: 2013
AB: Background: Genetic 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. Results: We 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. Conclusions: The
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.

TI: Characteristics of interspecific hybrids between carnation (Dianthus caryophyllus)

lines with long vase life and D. superbus var. longicalycinus, and their backcrossing
progenies.
AU: Onozaki,-T; Yagi,-M; Fujita,-Y; Tanase,-K
SO: Horticultural-Research-Japan. 2011; 10(2): 161-172
PB: Kyoto, Japan: Japanese Society for Horticultural Science.
PY: 2011
AB: Interspecific hybridization between carnation (Dianthus caryophyllus) lines with a
long vase life and D. superbus var. longicalycinus, a wild Dianthus species native to Japan,
was carried out in order to combine early flowering, high productivity and long vase life.
Although the interspecific hybridization of this combination was very difficult, seed was set
in 4 of 22 crosses. Moreover, all obtained seeds were abnormal; the seeds were imperfectly
solid and had a wrinkled brown surface. However, 15 F1 seedlings were obtained using
normal cross-breeding techniques, without depending on in vitro culture. Selected F1 lines
showed fertility in both seed and pollen parent. SSR analysis proved that all 9 selected F1
lines were interspecific hybrids. In backcrosses between selected F1 or BC1 lines and
carnation lines with long vase life, the percentage of seed setting was markedly increased,
and germination percentage of obtained seeds was also high. The mean vase life of the F1
generation was 7.0 days; in contrast, vase life of the BC2 generation, after two cycles
crossing and selection using carnation lines with long vase life, had improved to 14.7 days, a
net increase of 7.7 days. Carnation breeding using D. superbus var. longicalycinus was
effective for selecting early flowering progenies. The F1 generation showed a very early
flowering habit; average number of days to flowering in 15 F1 seedlings was 135 days. In
BC1 and BC2 generations, segregation of seedlings with early flowering was also observed.
Our findings suggest that flower vase life is not linked early flowering habit. Several selected
BC2 lines with long vase life, early flowering and high productivity could be developed, even
when using D. superbus var. longicalycinus, which has a very short vase life, as breeding
material.

TI: Application of DNA markers for breeding carnations resistant to bacterial wilt.
AU: Yagi,-M
SO: JARQ,-Japan-Agricultural-Research-Quarterly. 2013; 47(1): 29-35
PB: Tsukuba, Japan: Japan International Research Center for Agricultural Sciences.
PY: 2013
AB: Carnation bacterial wilt (CBW), caused by Burkholderia caryophylli, is one of the
most damaging diseases affecting carnations in Japan. In this study, carnation breeding was
conducted using CBW resistance derived from Dianthus capitatus ssp. andrzejowskianus. To
map the genetic loci involved in resistance to CBW and develop the linked markers, the first
molecular linkage map for carnation was constructed by using 134 progeny derived from a
cross between 'Carnation Nou No. 1' (an interspecific hybrid of carnation and D. capitatus)
and 'Pretty Favvare', a susceptible cultivar. The map consisted of 146 DNA markers and
covered 16 linkage groups. QTL analysis identified a QTL with a significant effect and two
QTLs with small effects. Evaluation of disease incidence in relation to the presence of the
STS-WG44 marker, which is linked to a QTL with a large effect, revealed that marker-
assisted selection (MAS) using STS-WG44 enables the tested population to be narrowed
down by half. Repeated crossing and selection via both conventional disease screening and
MAS led to successful development of the first CBW-resistant carnation cultivar, 'Karen
Rouge'.

TI: Construction of a genetic linkage map for the carnation by using RAPD and SSR
markers and mapping quantitative trait loci (QTL) for resistance to bacterial wilt caused by
Burkholderia caryophylli.
AU: Yagi,-M; Onozaki,-T; Taneya,-M; Watanabe,-H; Yoshimura,-T; Yoshinari,-T; Ochiai,-
Y; Shibata,-M
SO: Journal-of-the-Japanese-Society-for-Horticultural-Science. 2006; 75(2): 166-172
PB: Kyoto, Japan: Japanese Society for Horticultural Science.
PY: 2006
AB: We constructed a genetic linkage map for the carnation (Dianthus caryophyllus) on
the basis of random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR)
using a resistance-segregating population of 134 progeny lines that were derived from a cross
between 'Carnation Nou No. 1' (a carnation breeding line resistant to bacterial wilt) and
'Pretty Favvare' (a susceptible cultivar). The linkage map consisted of 137 RAPD and 9 SSR
markers. Linkage analysis revealed that 124 loci could be mapped to 16 linkage groups that
extended for 605.0 cM. The average interval between two loci was 4.9 cM. Quantitative trait
loci (QTL) analysis was applied to evaluations of resistance to bacterial wilt that were
replicated 8 times. The QTL that we reported previously with a large effect on resistance was
detected on group 6 which accounted for 60.5% of the total phenotypic variance with a LOD
score 23.46. Two other QTLs with small effects were detected on groups 2 and 5 with LOD
scores of 2.32 and 2.87, respectively. These results suggest that resistance to bacterial wilt in
carnation is related to one major and at least two minor genes. This study is the first report on
the construction of a linkage map of the carnation.

TI: Application of biotechnology methods in carnation breeding.

AU: Szoke,-A; Kiss,-E; Veres,-A; Kerepesi,-I; Toth,-A; Toth,-E; Toldi,-O; Heszky,-L
SO: Kertgazdasag-Horticulture. 2007; 39(1): 69-77
PB: Budapest, Hungary: Magyar Mezogazdasag Kiado KFT.
PY: 2007
AB: This paper presents the results of two research approaches applied at the SZIE,
Department of Genetics and Plant Breeding in Hungary. First was the investigation of the
effect of the inhibited ethylene biosynthesis on vase life longevity. In this approach,
transgenic lines were produced by the introduction of apple-derived ACC synthase gene in
antisense orientation. In these plants, ethylene production decreased by 12-48% and, in some
cases, vase life longevity was extended by 6 days and the stem steadiness was better. The
second approach was the examination of the effect of carbohydrate composition on
agronomically important traits. In this approach, the concentration of fructose 2,6-
bisphosphate (fru-2,6P2), a regulator molecule of the carbohydrate metabolism, was altered
by the introduction a modified mammalian origin bifunctional enzyme (6-phosphofructo-1-
kinase/fructose 2,6-bisphosphatase) cDNA. This enzyme enabled the increase or decrease of
the fructose 2,6-bisphosphate amount and carbohydrate composition. The decreased fru-
2,6P2 level resulted in higher sucrose concentration and enhanced photosynthesis and
transpiration rates. These effects caused faster growth and the plants flowered 2 weeks earlier,
on average, than the untransformed control. Conversely, the increased fru-2,6P2 content
enhanced the starch accumulation and decreased the photosynthesis and transpiration rates. In
this case, it was expected that these lines grew slower and flowered later. In contrast, the
slower growth could be observed only in the early stage of their development. Later, these
differences were eliminated and the flowering times were identical with those of the
untransformed control.