Download PCR-based Markers and Cut Flower Longevity in Carnation

PCR-based Markers and Cut Flower Longevity in Carnation
Laura De Benedetti, Luca Braglia, Simona Bruna, Gianluca Burchi*, Antonio Mercuri
and Tito Schiva
Istituto Sperimentale per la Floricoltura, Corso Inglesi 508, 18038 Sanremo (IM), Italy
*Istituto Sperimentale per la Floricoltura, Pescia (PT), Italy
Keywords: Dianthus caryophyllus,
polymorphism, assisted selection
post-harvest,
RAPD
analysis,
ethylene,
Abstract
In carnation, the identification of molecular markers linked to flower vase life
character could be an important tool to improve the efficiency of breeding
programs, considering that this is one of the most important traits selected by
breeders. Longevity is probably a complex quantitative trait, involving several genes
showing predominantly additive effects. A previous study carried out on cv. Roland,
cv. Milady, and their progenies showed that some RAPD bands significantly
discriminated a population with longer vase life. With the aim to verify the general
use of these markers for assisted selection, 12 commercial varieties of carnation were
collected and analyzed with the RAPD technique. The 23 fragments produced with
ten decamers were not able to discriminate the genotypes with greater vase life. In
order to identify more effective markers, preliminary analyses were also conducted
on four genotypes, using 30 primer sets designed to amplify internal sequences from
ethylene biosynthesis and response pathway genesPCR products were obtained with
22 primer pairs, and some polymorphic fragments were observed even in the
agarose gels.
INTRODUCTION
The post-harvest longevity of flowers is of crucial importance in determining the
value of an ornamental crop. Carnation (Dianthus caryophyllus L.) is one of the leading
commodities in the ornamental industry worldwide. In this species, vase life is one of the
most important traits considered by breeders.
Research on post-harvest physiology in carnation has been carried out in our
Institute since 1992: the role of ethylene in flower senescence was investigated and
several genotypes with different post-harvest life and climateric behaviours, were
identified (Burchi et al., 1993). Analysis of the segregation of flower vase life indicated
that this character is probably a complex quantitative trait, involving more than a single
gene or mechanism, and that these genes show predominantly additive effects (Burchi et
al., 1999).
Molecular markers associated with this character were previously identified in the
progenies of a cross between two cultivars with different longevity (‘Roland’ and
‘Milady’), using the RAPD technique (De Benedetti et al., 2003).
The aim of this study was to evaluate if these markers, tested on other collected
genotypes, could be proposed as a general model for the assisted selection of vase life in
carnation. Another approach, in order to identify other useful and effective markers, was
the use of specific primers to obtain amplification of ethylene biosynthesis and response
pathway genes.
MATERIALS AND METHODS
Plant Material and DNA Extraction
Twelve Dianthus caryophyllus genotypes, obtained from the private breeder
“Hybrida”, Sanremo-Italy, and 2 cultivars (‘Roland’ and ‘Milady’), belonging to the
Experimental Institute of Floriculture collections, were analysed. ‘Roland’ had the longest
vase life with late and very low ethylene production in comparison with other cultivars,
Proc. Vth IS on New Flor. Crops
Eds.: A.F.C. Tombolato and G.M. Dias-Tagliacozzo
Acta Hort. 683, ISHS 2005
437
such as ‘Milady’, in which ethylene released by the flower promoted and accelerated
senescence (Burchi et al. 1999). The commercial varieties included standard and spray
Mediterranean ecotypes. Six varieties (‘200013’, ‘200020’, ‘200046’, ‘98076’, ‘200101’
and ‘200147’) were characterized by extended longevity (>16 days; trials conducted in
March 2003, at 23 °C with 75% relativity humidity); the remaining individuals (‘99109’,
‘96118’, ‘200108’, ‘92027’, ‘97116’ and ‘97142’) showed lower values (≤12 days).
DNA was extracted from 100 mg of young leaves using a commercial kit
(DNAeasy Plant Mini Kit, Qiagen), according to the manufacturer’s instructions.
RAPD Analysis
RAPD markers previously identified were analyzed in all genotypes using ten
random decamers (De Benedetti et al., 2003). Conditions for PCR (Polymerase Chain
Reaction) and electrophoresis separation were previously described (De Benedetti et al.,
2001).
Specific Primer Amplifications
Sequences of carnation ethylene biosynthesis and regulation genes were
downloaded from the NCBI database. Nine different accessions were selected and used
for designing primers using the PRIMER 3 software. Primers were designed to be
between 18-24 bases long and to amplify internal regions of 400-800 base pairs. Thirty
primer sets were tested on four individuals (‘Roland’, ‘Milady’, ‘200013’ and ‘96118’).
PCR was carried out in 50 μl reaction mixture containing 100 ng of template
DNA, 1X buffer, 1.5 mM MgCl2, 200 μM each of dCTP, dGTP, dATP and dTTP, 10
picomoles of each primer, and 1.5 units of Taq-polymerase (Gibco B.R.L.) A thermal
cycler PCR Express (Hybaid) was programmed for 30 cycles with the denaturation
temperature at 94°C for 1 min and the extension temperature at 72°C for 1 min. The
annealing temperature was calculated for each primer pairs using PRIMER 3.
Amplification products were separated by electrophoresis on 1.2% agarose gels using
TAE buffer (40 mM Tris-acetate, 1mM EDTA) and visualized by ethidium bromide
staining.
RESULTS AND DISCUSSION
A previous study carried out on ‘Roland’, ‘Milady’ and their progenies showed
that some RAPD bands significantly discriminated a population with greater flower
longevity (De Benedetti et al., 2003).
The amplification patterns of the commercial varieties were compared to Roland
and Milady fragments (Table 1). A score was calculated based on the similarity of each of
23 bands analyzed with ‘Roland’ (1) or ‘Milady’ (0). The individuals with longer vase life
did not show higher scores compared to the genotypes with shorter longevity. These
RAPD bands were not able to discriminate the two groups. This could be explained by the
high genetic similarity of this material, coming from the same breeder and selected since
1950 for longevity.
More effective markers should be identified for the assisted selection of vase life
characters in carnation genotypes. Molecular markers for candidate gene analysis can be
identified, looking for polymorphisms in the genes involved in the expression of the
character of interest and analyzing their cosegregation with the trait in a set of individuals
with different phenotypes (Arus, 2000). With this aim, we started to analyze ethylene
biosynthesis and regulation gene sequences using specific primer sets.
Amplification products were obtained in four individuals using 22 primer pairs
(Table 2). For three primer pairs (n. 8, 11 and 19) the agarose gel separation showed
polymorphic fragments in cv. Roland (data not shown). The amplification of all
individuals with these primers sets, showed polymorphic fragments for one combination
in one of the varieties (‘20020’) with greater vase life (Fig. 1).
Further analyses, such as restriction site and SSCP (Single Stranded Conformation
Polymorphism) analyses, will be performed on all samples to detect more polymorphism
438
and to find a possible correlation between different alleles and the cut flower longevity.
The identification of markers linked to longevity could allow the early screening
of a population with a long flower vase life and could make the selection procedure more
effective.
ACKNOWLEDGEMENTS
Thanks to Dr. Flavio Sapia (Hybrida, Sanremo, Italy) for providing plant material.
Literature Cited
Arus, P. 2000. Molecular Markers for Ornamental Breeding. Acta Hort. 508:91-97.
Burchi, G., Mensuali-Sodi, A., Panizza, M. and Bianchini, C. 1993. Preliminary results of
molecular studies on senescence in carnation flowers ageing on plant or in vase: 1.
Role of ethylene. Proc. XVIIth EUCARPIA Symposium, Sanremo, Italy, 1-5 March,
p.199-206.
Burchi, G., Bianchini, C., Mercuri, A., Foglia, G., Rosellini, D. and Schiva, T. 1999.
Analysis of postharvest flower life in a cross between carnation cultivars with
different ethylene responses. Journal of Genetics and Breeding 53: 301-306.
De Benedetti, L., Mercuri A., Bruna, S., Burchi, G. and Schiva, T. 2001. Genotype
Identification of Ornamental Species by RAPD Analysis. Acta Hort. 546: 391-393.
De Benedetti, L., Burchi, G., Bruna, S., Mercuri A. and Schiva, T. 2003. Molecular
Markers and Cut Flower Longevity in Carnation. Acta Hort. 624: 343-348.
439
Tables
Table 1. RAPD markers obtained in 14 genotypes with different vase life.
More Longeve
92027
97116
97142
200147
200108
200101
96118
98076
99109
200046
Milady
200020
Roland
RAPD
Marker
200013
440
Less Longeve
59-1
59-2
59-2.0
60-2
60-3
60-5
61-5
61-6
66-4
70-3
70-9
72-2
91-3
91-4
91-9
98-2
98-3
98-5
103-2
103-3
104-2
104-3
104-4
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Score
6 4 4 6 6 7 13
0
+/- : presence or absence of an RAPD band.
5
5
5
6
6
6
Table 2. Primer pairs used to amplify carnation ethylene biosynthesis and regulation genes.
Primer
Primer sequences (5’-3’)
Gene
set
3
ttc agg gag caa aag ttc aaa
cgg tgc atc aca ctc ttg ta
cct ggg cct tca ttt tga aa
gtg ctc tcc caa tca atg tca t
aac aat tat tcc gca gtt atg a
ata caa ata ctg cgg gtg gg
4
act ccg aaa tta gaa gcc gc
ttg taa ttt gaa tga ata ctc cg
1
2
6
ttc agg gag caa aag ttc aaa
cgt tat tta tag ttc att tga tta g
atg atg gcg acc ttt gtg tt
ttt gaa ctt ttg ctc cct gaa
7
caa acc cgt caa atc cct ta
taa ggt cgc att gtc cat gt
5
8
9
10
11
12
aat taa cga aca tgg caa aca
aca atg gag tgt ttc atg gga
cag ctc ctc aag gat ggt ca
att gta att tga atg aat act ccg t
gct ttt tga aac ttg att ttt ctt tt
ttt cgg ctt att gca gct aaa
ttc aac tcc aac aaa tcc acc
ttt gtt cac gct tca ttt cg
tct tga tat tgt tgt aga acc gtc tt
cca gtc atg cac att tcc ag
DC-ACO1
(ACC oxidase)
Accession
AB042320
Similar to
DC-ACO1
AB042321
CARACC
(ACC synthase)
M66619
Senescence
related protein
M62380
gcsdc 9 (SAM decarboxylase)
U94786
441
Table 2. (Continued)
Primer
set
Gene
gaa atg gca cgt tta ctc gg
gaa aca atc aat tat tcc aaa cca
14
cgc aaa ctt ctg aag ctg ct
cca acc gat aag ctg cca a
CTR1 (Putative proteine kinase)
AF261147
15
aaa ctg cta agc tgc ttc tgc
ctt tta att tgg cat cac tac gac
CTR2 (Putative proteine kinase)
AF261148
DCERS (putative ethylene receptor)
U83237
EIL1(ethylene-insensitive3-like
protein 1)
AF261654
17
18
19
20
21
22
gat ttg gct tag aac gct gc
tcg agg gtg ctt ctg atc tt
aac aga acg aca tgt aag aat gc
ata act gac aag aat tcg tta cga g
cac aat gtc gct tta gat tta gca
ttg agc tca caa acc tgc ac
ccg tct taa gcg atg gct att
cag ggg aac ctt caa aaa gt
tca tca tca tct atg atc acc gt
gca ctt tct ttg gca gtc atc
gta cgt cag tca aag tgc ttg c
cct ggt tat tgt ttt tcc cg
tcg cag ttt caa atc gac aa
tgc ata ctg ttt act aac gga ttt
gcsdc 9 (SAM decarboxylase)
Accession
13
16
442
Primer sequences (5’-3’)
U94786
Figures
Fig. 1. Amplification of an internal region of the “senescence related protein” gene.
(primer pair number 8). M: PCR marker (Sigma-Aldrich).
443