Acta agriculturae slovenica, 83 - 2, november 2004 str. 323 - 329 Agrovoc descriptors: Olea europaea, olives, genotypes, microsatellites, genetic markers, pcr, loci Agris category code: F30 University of Primorska Science and Research Centre of the Republic of Slovenia, Koper COBISS Code 1.01 University of Ljubljana Biotechnical Faculty, Agronomy Department Centre for Plant Biotechnology and Breeding Amplification of fluorescent-labelled microsatellite markers in olives by a novel, economic method Dunja BANDELJ1, Jernej JAKŠE2, Branka JAVORNIK3 Received August 6, 2004; accepted October 15, 2004 Delo je prispelo 6. avgusta 2004; sprejeto 15. oktobra 2004. ABSTRACT A novel method for the economic fluorescent labelling of PCR products and its features are described. To investigate the efficiency of the method for amplification of olive microsatellite markers, PCR conditions of three loci were optimised. The results showed that the method can be successfully applied in genotyping studies of olive varieties. Key words: microsatellite, fluorescent detection, universal M13(-21) primer, olive IZVLECEK NAMNOŽEVANJE FLUORESCENTNO OZNACENIH MIKROSATELITSKIH MARKERJEV OLJKE Z NOVEJŠO, EKONOMICNO METODO Opisana je novejša metoda ekonomicnega fluorescentnega oznacevanja PCR molekul in nekatere njene posebnosti. Za preucitev uspešnosti metode pri namnoževanju mikrosatelitov oljke smo uporabili tri lokuse, katerih PCR pogoje smo predhodno optimizirali. Rezultati so pokazali, da je metoda lahko uspešno uporabljena v študijah genotipizacije sort oljk. Kljucne besede: mikrosateliti, fluorescentna detekcija, univerzalni M13(-21) zacetni oligonukleotid, oljka 1 Research assistant, B. Sc., SI-5000, Koper, Garibaldijeva 18 2 Assistant Prof., Ph. D., SI-1111, Ljubljana, Jamnikarjeva 101, P.O. Box 2995 3 Prof., Ph. D., SI-1111, Ljubljana, Jamnikarjeva 101, P.O. Box 2995 Ac 324 ta agriculturae slovenica, 83 - 2, november 2004 1 INTRODUCTION Microsatellites, as one of the most popular marker systems, are widely used in plant genetic research for diversity studies, genetic map development, linkage analyses, marker-assisted selection and fingerprinting studies. Their wide usage is based on their good properties: co-dominant nature, high abundance in eucaryotic genomes, robustness, hypervariability, high information content, and amenability to automation (Morgante and Olivieri, 1993; Powell et al., 1996; Weising et al., 1998). Microsatellites are particularly attractive for genotyping individuals because the level of polymorphism detected by them is higher than that detected with any other molecular marker assay (Powell et al., 1996; Russell et al., 1997; Jakše et al., 2001). There is great interest in developing a robust, reproducible, cheap and high throughput methodology for the genotyping plants of commercial value. The microsatellite amplification protocol is simple once primers for a defined locus have been designed. After amplification of microsatellites by PCR, the products are separated on high resolution polyacrylamide gels by electrophoresis, and detection of amplified alleles is achieved by three different principles: 1) radioactively, 2) silver staining and 3) a laser induced fluorescence detection system. The employment of fluorescent labelled microsatellite primers and laser detection (e.g. automated sequencer) in genotyping procedures significantly improves the throughput and automatisation (Wenz et al., 1998). The use of microsatellites, however, can be costly due to the high price of the fluorescent label, which must be carried by one of the primers in the primer pair. The cost of microsatellite assay increases substantially if a large number of loci have to be tested. In order to overcome this financial difficulty, Schuelke (2000) introduced a novel procedure in which three primers are used for the amplification of a defined microsatellite locus: a sequence-specific forward primer with M13(-21) tail at its 5' end, a sequence-specific reverse primer and the universal fluorescent-labelled M13(-21) primer. To date, the system has been used successfully with tiger snake (Scott et al., 2001) microsatellite markers and could be a valuable tool for genotyping plants. The objective of this paper is to introduce a simple, less expensive and high-throughput method for amplification and detection of fluorescent-labelled DNA markers and to test its applicability in genotyping olive varieties. 2 MATERIAL AND METHODS 2.1 Plant material Nineteen varieties from the national olive collection orchard in Strunjan, Slovenia, were included in the molecular analysis. Analysed olive varieties are listed in Table 1. BANDELJ, D., JAKŠE, J., JAVORNIK, B.: Amplification of fluorescent-labelled …32 5 Table 1 List of varieties and geographical origin of analysed olives. Variety Origin Arbequina Spain Ascolana tenera Italy Buga Slovenia Cipressino Italy Crnica Slovenia Frantoio Italy-Tuscany Istrska belica Slovenia Itrana South Italy Leccino Italy-Tuscany Leccione Italy-Tuscany Maurino Italy-Tuscany Nocellara del Belice Italy-Sicily Pendolino Italy-Tuscany Picholine France Santa Caterina Italy-Tuscany Štorta Slovenia Zelenjak Uncertain Samo Slovenia Athena Uncertain 2.2 Microsatellite assay Genomic DNA was extracted from fresh olive leaves by a modified CTAB method following the procedure described by Kump & Javornik (1996). Three primer pairs for olive microsatellite loci ssrOeUA-DCA3, ssrOeUA-DCA10 and ssrOeUA-DCA16 (Sefc et al., 2000) were synthesised by MWG-Biotech AG. For each primer pair, the sequence of the original published forward primer was redesigned by adding an universal M13(-21) tail (5'-TGT AAA ACG ACG GCC AGT-3') to their 5' ends (Schuelke, 2000). The third, universal M13(-21) primer was labelled with Cy5, allowing fluorescence detection. The sequences of microsatellite primer pairs used in this study are shown in Table 2. Table 2: Primer sequences of three microsatellite loci and sequence of the fluorescent-labelled universal M13(-21) primer. Primer Primers: 5' to 3' ssrOeUA-DCA3 F: TGTAAAACGACGGCCAGTCCCAAGCGGAGGTGTATATTGTTAC R: TGCTTTTGTCGTGTTTGAGATGTTG ssrOeUA-DCA10 F: TGTAAAACGACGGCCAGTCGTGACCACCTAAATCCGCCCC R: CTGTCCAGAGCTAAAGGTTTCG ssrOeUA-DCA16 F: TGTAAAACGACGGCCAGTTTAGGTGGGATTCTGTAGATGGTTG R: TTTTAGGTGAGTTCATAGAATTAGC Universal M13(-21) Cy5-TGTAAAACGACGGCCAGT Amplification reactions were carried out in a total volume of 10 ”l, containing 20 ng genomic DNA, 1X supplied PCR buffer (Promega), 0.2 mM of each dNTP, (Roche), 0.25 unit of Taq DNA polymerase (Promega), 0.5 ”M of sequence-specific reverse primer, 0.5 ”M of fluorescence labelled universal M13(-21) primer and 0.125 ”M of forward primer with M13(-21) tail. The amplification protocol for loci ssrOeUA-DCA3, ssrOeUA-DCA10, and ssrOeUA-DCA16 according to Bandelj et al. (2002), with some modifications, was performed in a GeneAmp 9700 thermal cycler (Applied Biosystems). The conditions of the two-round PCR amplification were as follows: 94 °C (5 min), then 26 or 30 cycles at 94 °C (30 s) / 50 - 53 °C (45 s) / 72 °C (45 s - 1 min 30 s), followed by 8 cycles at 94 °C (30 s) / 53 °C (45 s) / 72 °C 326 (45 s -1 min 30 s), and a final extension step at 72 °C for 10 min. The PCR products were denaturated by adding an equal volume of formamide loading dye (98 % formamide, dextran blue 5 mg/ml) and by heating at 94 °C for 4 min. The optimal PCR conditions for amplification of microsatellites at each locus are summarised in Table 3. Table 3: Optimal PCR conditions for three microsatellite loci. Locus First round PCR Second round PCR ssrOeUA-DCA3 94 șC/5 min 8 cycles: 26 cycles: 94 șC/30 s 94 șC/30 s 53 șC/45 s 50 șC/45 s 72 șC/45 s 72 șC/45 s 72 șC/10 min ssrOeUA-DCA10 94 șC/5 min 8 cycles: 30 cycles: 94 șC/30 s 94 șC/30 s 53 șC/45 s 53 șC/45 s 72 șC/1 min 30 s 72 șC/1 min 30 s 72 șC/10 min ssrOeUA-DCA16 94 șC/5 min 8 cycles: 26 cycles: 94 șC/30 s 94 șC/30 s 53 șC/45 s 52 șC/45 s 72 șC/1 min 30 s 72 șC/1 min 30 s 72 șC/10 min Amplification products were separated on a 0.5 thick and 7.5% polyacrylamide denaturing gel, containing 7 M urea. Electrophoresis was performed on short glass plates, of 80 mm length, using an automated ALFexpressII sequencer (Amersham Biosciences). Separation was done at 1500 V, 60 mA, and 15 W for 360 min at 55 șC. Fluorescence signals were collected every 1 s and stored in a computer. A fluorescence labelled molecular marker size (Cy5 Sizer 50-500; Amersham Biosciences) comprising 10 fragments in the size range of 50 to 500 bp was used as an external size marker. Allele sizes were determined using the software package ALFwinTM Fragment Analyser 1.01 (Amersham Biosciences). 3 RESULTS AND DISCUSSION To reduce the cost of automated laser detection of fluorescent-labelled PCR fragments, a novel procedure for amplification of microsatellite markers was described (Schuelke, 2000), in which three primers for a single locus are employed. The basis of the approach is two-round PCR. During the first-round PCR (30 cycles), the forward primer with M13(-21) sequence at its 5' end is incorporated into the PCR products. These products are then the target for the fluorescent-labelled universal M13(-21) primer, which is incorporated during the second-round PCR (8 cycles). The thermocycling conditions are first optimised for annealing the forward primer with its M13(-21) sequence, and later, when the forward primer is used up, the annealing temperature is lowered to facilitate annealing of the universal fluorescent-labelled M13(-21) primer. Incorporation of the fluorescent dye into the PCR product is thus obtained, allowing the laser induced fluorescence detection. In order for the procedure to be successfully performed, some important facts should be taken into consideration (Schuelke, 2000): 1) the fluorescent labelled M13(-21) universal primer and sequence specific reverse primer should be used in equimolar amounts, 2) the amount of modified sequence specific forward specific primer should not exceed one-fourth the amount of the reverse primer, so that the universal primer can take over when the forward primer is used up, 3) the second-round PCR (the last eight PCR cycles) should be run with an annealing temperature of 53 șC (the BANDELJ, D., JAKŠE, J., JAVORNIK, B.: Amplification of fluorescent-labelled …32 7 annealing temperature of the universal fluorescent-labelled M13(-21) primer is 53 șC). The main advantage of the method is that the universal fluorescence-labelled M13(-21) primer can be used for amplification of all loci included in the microsatellite assay, so fluorescence-labelling of one primer is no longer necessary, which reduces the cost of analysis. In the case of the ALFexpressII sequencer, where Cy5 dye is used for labelling DNA fragments in the analysis of ten microsatellite loci, the costs can be reduced by half as compared with the classical method. To test the efficiency of the method in amplification of microsatellite markers in olives, three published loci ssrOeUA-DCA3, ssrOeUA-DCA10 and ssrOeUA-DCA16 (Sefc et al., 2000) were chosen. Our previous studies (Bandelj et al., 2002; Bandelj et al., 2004) showed that these microsatellite loci are extremely powerful in genotyping olive varieties, due to their high polymorphic information content (PIC value), low probability of identity (PI value) and high effective number of alleles. For the amplification of microsatellite markers by the novel procedure, the original published sequence of the forward primer was modified according to Schuelke (2000). On the author's recommendation, the PCR mixture contained equimolar amounts of fluorescent-labelled M13(-21) universal primer and sequence specific reverse primer, and one-fourth the amount of sequence specific reverse primer. The optimal two-round amplification thermocycling profiles were determined for each locus independently. The optimisation of the first round PCR conditions mainly comprised adjustment of the primer annealing temperature, an increase of cycle number from 26 to 30 and a prolongation of the annealing and extension time. At locus ssrOeUA-DCA10, weak amplification of long alleles in contrast to short ones was observed. Wattier et al. (1998) explained this problem of short allele dominance as a consequence of competition between short and long alleles, which leads to preferential amplification of short alleles. The problem was only partly overcome by changing the amplification conditions, since the two longest alleles could not be amplified. The protocol for the second round PCR was performed as described by Schuelke (2000) with a modification for loci ssrOeUA-DCA10 and ssrOeUA-DCA16, whereby the extension time was prolonged from 45 s to 1 min 30 s. The PCR products of the three microsatellite loci were separated and visualised on an ALFexpressII sequencer. The separation of microsatellite markers by sequencing apparatus was found to be very suitable, since the detection of alleles was performed automatically using a computer software package and there was no need for manual scoring of the data. Allele sizes were determined by the fragment sizing software Fragment Analyser 1.01, with the help of a fluorescence labelled molecular marker size as an external standard. The smiling effect was eliminated by identification of internal standards. At three loci, all microsatellite alleles were successfully amplified (Figure 1), with the exception of the two above mentioned alleles of the ssrOeUA-DCA10 locus. Allele sizes were 18 bp longer than those determined by silver staining (Bandelj et al., 2002), when genotyping the same set of olive varieties was performed. The differences are due to modification of the forward specific primer by adding 18 bp of the M13(-21) sequence. Comparison of allele sizes determined by silver staining and Ac 328 ta agriculturae slovenica, 83 - 2, november 2004 fluorescent detection showed that in some cases two bases longer alleles were determined by silver staining. Differences in allele sizes are frequently reported in literature and can be explained by different electrophoresis and detection systems (Weber, 1990; Bowers et al., 1996; Kline et al., 1997; Kozjak et al., 2003). In conclusion, the reported method can be successfully used for amplification of microsatellites in plants. In genotyping studies, where many samples are analysed, this procedure reduces the overall costs, which is of great importance with large scale projects. Bases j 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 211 | 400 350 300 200 Internal standard (203 bp) Figure 1: Electrophoregram of 19 olive varieties genotyped at locus ssrOeUA-DCA3: 1-Size marker (50 - 500 bp), 2-Arbequina, 3-Ascolana tenera, 4-Samo, 5-Štorta, 6-Picholine, 7-Santa Caterina, 8-Nocellara del Belice, 9-Itrana, 10-Cipressino, 11-Frantoio, 12-Buga, 13-Zelenjak, 14-Leccione, 15-Athena, 16-Crnica, 17-Leccino, 18-Pendolino, 19-maurino, 20-Istrska belica. 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