PCR-based Markers and Cut Flower Longevity in Carnation

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1 PCRbased 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, Sanremo (IM), Italy * Istituto Sperimentale per la Floricoltura, Pescia (PT), Italy Keywords: Dianthus caryophyllus, postharvest, RAPD analysis, ethylene, polymorphism, assisted selection 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 postharvest 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 postharvest 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 postharvest 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, SanremoItaly, 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. V th IS on New Flor. Crops Eds.: A.F.C. Tombolato and G.M. DiasTagliacozzo Acta Hort. 683, ISHS

2 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 ( , , , 98076, and ) were characterized by extended longevity (>16 days; trials conducted in March 2003, at 23 C with 75% relativity humidity); the remaining individuals ( 99109, 96118, , 92027, and ) 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 1824 bases long and to amplify internal regions of base pairs. Thirty primer sets were tested on four individuals ( Roland, Milady, and ). PCR was carried out in 50 μl reaction mixture containing 100 ng of template DNA, 1X buffer, 1.5 mm MgCl 2, 200 μm each of dctp, dgtp, datp and dttp, 10 picomoles of each primer, and 1.5 units of Taqpolymerase (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 Trisacetate, 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 ( ) 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

3 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 Molecular Markers for Ornamental Breeding. Acta Hort. 508:9197. Burchi, G., MensualiSodi, A., Panizza, M. and Bianchini, C 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, 15 March, p Burchi, G., Bianchini, C., Mercuri, A., Foglia, G., Rosellini, D. and Schiva, T Analysis of postharvest flower life in a cross between carnation cultivars with different ethylene responses. Journal of Genetics and Breeding 53: De Benedetti, L., Mercuri A., Bruna, S., Burchi, G. and Schiva, T Genotype Identification of Ornamental Species by RAPD Analysis. Acta Hort. 546: De Benedetti, L., Burchi, G., Bruna, S., Mercuri A. and Schiva, T Molecular Markers and Cut Flower Longevity in Carnation. Acta Hort. 624:

4 Tables Table 1. RAPD markers obtained in 14 genotypes with different vase life. RAPD Marker More Longeve Roland Less Longeve Milady Score / : presence or absence of an RAPD band. 440

5 Table 2. Primer pairs used to amplify carnation ethylene biosynthesis and regulation genes. Primer set Primer sequences (5 3 ) Gene Accession 1 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 act ccg aaa tta gaa gcc gc ttg taa ttt gaa tga ata ctc cg DCACO1 (ACC oxidase) AB 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 Similar to DCACO1 AB caa acc cgt caa atc cct ta taa ggt cgc att gtc cat gt CARACC (ACC synthase) M 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 Senescence related protein M 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 gcsdc 9 (SAM decarboxylase) U

6 Table 2. (Continued) Primer set 13 gaa atg gca cgt tta ctc gg gaa aca atc aat tat tcc aaa cca Primer sequences (5 3 ) Gene Accession gcsdc 9 (SAM decarboxylase) U cgc aaa ctt ctg aag ctg ct cca acc gat aag ctg cca a CTR1 (Putative proteine kinase) AF aaa ctg cta agc tgc ttc tgc ctt tta att tgg cat cac tac gac CTR2 (Putative proteine kinase) AF 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 DCERS (putative ethylene receptor) U 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 EIL1(ethyleneinsensitive3like protein 1) AF

7 Figures Fig. 1. Amplification of an internal region of the senescence related protein gene. (primer pair number 8). M: PCR marker (SigmaAldrich). 443

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Electronic Supplementary Material (ESI) for Molecular BioSystems. This journal is The Royal Society of Chemistry 2017 Electronic Supplementary Information Dissecting binding of a β-barrel outer membrane