Plant Molecular Biology 15: 169-171, 1990. © 1990KluwerAcademic Publishers. Printedin Belgium. 169 Plant Molecular Biology Update Rapid and efficient detection of genetic polymorphism in wheat through amplification by polymerase chain reaction R. D'Ovidio, O.A. Tanzarella and E. Porceddu Dipartimento di Agrobiologia e Agrochimica, Universith della Tuscia, Via S. Camillo de Lellis, O1100 Viterbo, Italy Received 15 February 1990; accepted29 March 1990 Key words: polymerase chain reaction, genetic polymorphism, Triticum, seed storage proteins Abstract The polymerase chain reaction (PCR) was used to amplify genomic DNA of several wheat genotypes. The oligonucleotides used as primers were the terminal sequences of a gamma-gliadin gene. The electrophoretic analysis of the PCR products showed specific bands which revealed both inter- and intra-specific genetic polymorphism among the examined genotypes. The technique is proposed as a very simple and efficient alternative to RFLP markers. The exploitation of restriction fragment length polymorphisms (RFLPs) as genetic markers is one of the most powerful and successful applications of recombinant DNA techniques. Cost, technical skill required, and utilization of radioactive isotopes prevented, so far, the spread of RFLPs in many fields of plant genetics and breeding [ 1]. Polymerase chain reaction (PCR) is being widely used for efficient amplification of specific sequences ofgenomic DNA, mostly in the field of early diagnosis of human genetic disorders [2]. In the present paper, the polymerase chain reaction has been used for the amplification of a wheat genomic sequence. The two oligonucleotides used as primers of the PCR were synthesized on the basis of the published sequence of a gene coding for a wheat storage protein belonging to the gamma-gliadin fraction [3]. The two primers represent the first twenty nucleotides of the 5' transcribed region, and nineteen nucleotides of the complementary strand comprised between position 986 and 1004 (Fig. 1). Genomic DNA was isolated from plants of six durum wheat cultivars, four tetraploid and seven diploid species and subspecies of Triticum and Aegilops, by following the procedure reported by Dvorak et al. [4]. 150 ng of DNA were subjected to PCR for 30 cycles in a total volume of 100/~1 with 2 units of Taq DNA polymerase (Promega), 1 x Taq PCR buffer (Promega), 250 ng of each of the two primers (prepared by Dr. J. Wunderlich, University of Georgia) and 300/~M each dATP, dGTP, dCTP, dTTP (Pharmacia). The PCR mixture was covered with 50/zl of liquid paraffin. DNA was denatured by heating to 95 °C for 2 minutes followed by 2 minutes annealing of primers and DNA at 55 ° C, and then incubated at 72 °C for 2 minutes to extend synthesis of the DNA sequence included between the two 170 ATG AAG ACC TTA CTC ArC CT 5' m 3' n 3' 5' GG TAC ACG TTG CAC ATA CA Fig. 1. Sequence of the oligonucleotides used in the PCR and diagram of the DNA-oligonucleotides pairing during the reaction. primers. The PCR products were analyzed by electrophoresis in 1 x TBE buffer on 1.2~o agarose gel whose result is reported in Fig. 2. The PCR amplified few, specific DNA frag- ments, ranging between 750 and 1000 bp, which show polymorphism in different wheat genotypes. The electrophoretic patterns of amplified DNA were similar for Aegilops longissima and A. squar- Fig. 2. 1.2% agarose gel of the amplified DNA; for each sample 1/5 of the reaction was used. 2 Molecular Weight Marker III (Boehringer); A) A. longissima Schweinf. et Musch.; B) A. searsii Feldman & Kislev; C) A. speltoides Tausch; D) A. squarrosa L.; E) T. boeoticum Boiss; F) T. monococcum L.; G) T. urartu Tum.; H) T. turgidum conv. aethiopicum Jakubz.; I) T. turgidum subsp. carthlicum (Nevski) MK.; L) T. turgidum eonv. polonicum (L.) MK.; M) T. turgidum conv. turanicum (Jakubz.) MK.; and the following T. turgidum conv. durum (Desf.) MK. varieties: N) Langdon; O) Lira biotype 42; P) Lira biotype 45; Q) Quadruro; R) Trinakria; S) Valnova. 171 rosa, and for Triticum boeoticum and T. urartu among the diploid species. The amplified DNA of the durum wheat cultivars can be grouped into two patterns differing only for the band at lower mobility. These cultivars belong to the two groups recognizable on the basis of electrophoretic pattern of the gamma-gliadin protein fraction, and commonly identified as 42 and 45 types [5, 6]. The cultivars showing the differing amplified band with greater mobility correspond to type 42, whereas those with lower mobility belong to type 45. This correspondence, in particular the different pattern shown by the two biotypes 42 and 45 belonging to the same variety Lira (Fig. 2), suggests that the amplified DNA represents sequences for the gamma-gliadin components. The subspecies carthlicum and turanicum of T. turgidum show the same pattern of amplified DNA bands of the type 42 varieties group. Results confirm the high specificity and power of PCR, already shown in many fields of molecular genetics, and suggest its potential application for detection of genetic polymorphisms and as genetic markers in plants. The technique has several advantages over traditional RFLP techniques: highest specificity and reliability, speed, simplicity and low cost, due to avoidance of Southern transfer and hybridization with radioactive probes. The numerous nucleotide sequences already published could represent a rich source of oligonucleotides which could be used as new markers through PCR amplification. Acknowledgement This research was supported by the Italian Ministry of Agriculture and Forestry, Research Project 'Sviluppo di tecnologie avanzate applicate alle piante'. References 1. Landry BS, Michelmore RW: Methods and applications of restriction fragment length polymorphism analysis to plants. In: Bruening G, Harada J, Kosuge T, Hallard A (eds) Tailoring Genes for Crop Improvement: An Agriculture Perspective, pp. 25-44. Plenum Press (1987). 2. Vosberg HP: The polymerase chain reaction and improved method for the analysis of nucleic acids. Hum Genet 83(1): 1-15 (1989). 3. Scheets K, Hedgcoth C: Nucleotide sequence of a gamma-gliadin gene: comparisons with other gamma-gliadin sequences show the structure of gamma gliadin genes and the general primary structure of gamma-gliadins. Plant Sci 57:141-150 (1988). 4. Dvorak J, McGuire PE, Cassidy B: Apparent sources of the A genomes of wheats inferred from polymorphism in abundance and restriction fragment length of repeated nucleotide sequences. Genome 30:680-689 (1988). 5. Damidaux R, Autran JC, Grignac P, Feillet P: Mise en 6vidence de relation applicable en selection entre rrlectrophoregramme des gliadines et des proprirtrs viscorlastiques du gluten de Triticum durum Desf. C R Hebd Srances Acad Sci Ser D 287:701-704 (1978). 6. Payne PI, Jackson EA, Holt LM: The association between gamma-gliadin 45 and gluten strength in durum wheat varieties: A direct casual effect or the result of genetic linkage? J Cereal Sci 2:73-81 (1984).