Analysis of the low-linolenic mutant genotypes of winter oilseed rape (Brassica napus L.) with the use of DNA markers *

Tom XXV ROŚLINY OLEISTE 2004 Katarzyna Mikołajczyk, Stanisław Spasibionek, Iwona Bartkowiak-Broda Instytut Hodowli i Aklimatyzacji Roślin, Oddział w Poznaniu Analysis of the low-linolenic mutant genotypes of winter oilseed rape (Brassica napus L.) with the use of DNA markers * Analiza genotypów mutanta rzepaku ozimego (Brassica napus L.) o obniżonej zawartości kwasu linolenowego z zastosowaniem markerów DNA Key words: winter rapeseed (Brassica napus L.), quality breeding, DNA markers, fatty acids, linolenic acid One of the selection goals in the oilseed rape quality breeding is obtaining of plant genotypes characterized by the specific fatty acids composition. In the Poznań Branch of the Plant Breeding and Acclimatization Institute chemical mutagenesis was applied to double-low winter oilseed rape (10% of linolenic acid) and resulted, among others, in low-linolenic mutant (2% of linolenic acid). The main aim of presented research is to develop DNA markers specific for the low-linolenic mutant genotypes. RAPD method was applied to analyse DNAs obtained from non-mutated and lowlinolenic mutant plants. It revealed the presence of some polymorphic bands which will be the subject of further research. Słowa kluczowe: rzepak ozimy (Brassica napus L.), hodowla jakościowa, markery DNA, kwasy tłuszczowe, kwas linolenowy Olej otrzymywany z nasion rzepaku (Brassica napus L.) znajduje coraz szersze zastosowanie nie tylko jako produkt spożywczy lecz także jako surowiec stosowany w przemyśle oraz technologii do produkcji biopaliwa. W zależności od sposobu wykorzystania wymagany jest zróżnicowany skład kwasów tłuszczowych w oleju rzepakowym. W Oddziale Poznańskim Instytutu Hodowli i Aklimatyzacji Roślin od szeregu lat prowadzone są prace, w wyniku których otrzymano genotypy rzepaku ozimego charakteryzujące się niską zawartością kwasu linolenowego. Jednak proces hodowlany jest utrudniony w związku ze złożonym charakterem dziedziczenia tej cechy, modyfikowanej w znacznym stopniu przez czynniki środowiska. Markery DNA, umożliwiające precyzyjną analizę genotypu niezależnie od zmiennych warunków środowiska, stanowiłyby dogodne narzędzie selekcyjne. Celem prowadzonych badań jest opracowanie markerów DNA dla cechy niskiej zawartości kwasu linolenowego. Wyizolowano DNA genomowy z młodych listków roślin rzepaku: linii podwójnie ulepszonej PN 1775/02 (około 10% kwasu linolenowego), linii wsobnej PN 1712/02 mutanta M-681 uzyskanego z PN 1775/02 w wyniku mutagenezy chemicznej (około 2% kwasu linolenowego) oraz z odmiany jarej Apollo (około 2% kwasu linolenowego). Zastosowano metodę RAPD w celu * This research is partially supported by the 3 P06A 011 25 KBN grant

244 Katarzyna Mikołajczyk... określenia rejonów DNA charakterystycznych dla cechy niskiej zawartości kwasu linolenowego. Przy użyciu pięciu starterów firmy Operon Technologies: OPK-01, OPL-13, OPP-05, OPP-08 oraz D-25 zidentyfikowano prążki polimorficzne umożliwiające rozróżnienie pomiędzy roślinami o niskiej zawartości kwasu linolenowego (typ mutanta) a roślinami linii podwójnie ulepszonych. Wyniki te stanowią podstawę do dalszych badań, mających na celu analizę rejonów DNA charakterystycznych dla cechy niskiej zawartości kwasu linolenowego jak również opracowanie specyficznych markerów DNA. Introduction Rapeseed (Brassica napus L.) is one of the main oil sources cultivated in the moderate climate regions of the world. Its seeds contain more than 40% of oil, the quality of which depends mainly on specific fatty acids composition (Scarth, McVetty 1999; Thelen, Ohlrogge 2002). Rapeseed oil is used not only for human nutrition but also as a raw material in industry and technology for biofuel production (Töpfer et al. 1995; McDonnell et al. 1999; Altin et al. 2001). Differentiated fatty acids composition is required due to the means of rapeseed oil application (Mikołajczyk, Bartkowiak-Broda 2003). One of the main advantages of the oil, while used as nutrition product, is the presence of polyunsaturated fatty acids: linoleic and linolenic, which makes it a valuable source of essential for human health exogenic fatty acids (Fitzpatrick, Scarth 1998; Scarth, McVetty 1999; Simopoulos 2000; Leckband et al. 2002). However, this characteristic could be a disadvantage, provided such oil was applied in industry and technology. Polyunsaturated fatty acids cause the flexibility and oxidative rancidity of the oil. When used for commercial frying, such oil requires partial hydrogenation. Given the health risk associated with hydrogenated oils and also the possibilities of economical losses caused by the instability of the oils with the high level of polyunsaturated fatty acids, it would be beneficial to breed the rapeseed cultivars with lowered linolenic acid level (from about 10% to about 3%). Different approaches have been performed for introducing the low-linolenic acid trait into rapeseed genotypes. However, the breeding process is complicated by the fact that the trait has a complex genetic inheritance being highly influenced by the environment (Bartkowiak-Broda, Krzymański, 1983). DNA markers appear as an accurate and environment independent tool to be used in breeding of the low linolenic oilseed rape (Snowdon, Friedt, 2004). There are several breeding organizations in the world having low linolenic oilseed rape cultivars in development and production (Scarth, McVetty 1999; Rakow, Raney 2003) with less than 7% current level of linolenic acid achieved through selection. In Poland, in the Plant Breeding and Acclimatization Institute in Poznań, attempts to obtain the low-linolenic winter oilseed rape genotypes have been successfully undertaken for several years. Stable inbred lines of about 3% linolenic acid content have been obtained as a result of crosses between double-low

Analysis of the low-linolenic mutant genotypes of... 245 winter oilseed rape lines with the low-linolenic summer oilseed rape cultivars Stellar and Apollo. Moreover, chemical mutagenesis was performed on double-low winter oilseed rape line and resulted in low-linolenic mutant plants which were further used in recombinant breeding programmes (Spasibionek et al. 2000). Significant improvement of the efficiency in breeding of the low-linolenic acid winter oilseed rape cultivars could be achieved with the use of specific DNA markers; FAD-3 desaturase gene seems to be a target for possible mutation (Jourdren et al. 1996; Barret et al. 1999; Hu et al. 2003). The aim of this work is to develop DNA markers for the low linolenic acid content in obtained mutants. Plant Material Materials and Methods Oilseed rape plants were used as follows: double-low winter line PN 1775/02 (obtained in the Plant Breeding and Acclimatization Institute in Poznań) characterized by the typical for doublelow varieties fatty acids composition (Tab. 1); inbred line PN 1712/02 of mutant M-681 which was obtained from PN 1775/02 line throughout chemical mutagenesis (Spasibionek, Krzymański 2000) and characterized by the low linolenic acid content in seed oil (Tab. 1); summer oilseed rape cultivar Apollo obtained from M11 mutant of Oro variety (Rakow 1973; Röbbelen, Nitsch 1975) and which is characterized by the low linolenic acid content (Tab. 1). Table 1 Fatty acids composition [%] in the seed oil obtained from: double-low PN 1775/02 and mutant PN 1712/02 lines and from summer rapeseed cultivar Apollo Skład [%] poszczególnych kwasów tłuszczowych w oleju nasion: linii rzepaku ozimego podwójnie ulepszonego PN 1775/02, mutanta PN 1712/02 oraz rzepaku jarego Apollo C 16:0 palmitic acid kwas palmitynowy C 18:0 stearic acid kwas stearynowy C 18:1 oleic acid kwas oleinowy C 18:2 linoleic acid kwas linolowy C 18:3 linolenic acid kwas linolenowy C 20:1 eicosenoic acid kwas eikozenowy C 22:1 erucic acid kwas erukowy Line Fatty acids Kwasy tłuszczowe Linia C 16:0 C 18:0 C 18:1 C 18:2 C 18:3 C 20:1 C 22:1 PN 1775/02 4,6 1,6 64,0 18,1 9,5 2,3 0 PN 1712/02 3,6 1,8 65,7 25,1 1,7 2,1 2,1 Apollo 3,4 1,3 67,5 24,1 1,7 1,1 0

246 Katarzyna Mikołajczyk... Fatty Acids Composition Analysis Was performed on the rape half-seeds with the use of gas chromatography analysis (Byczyńska, Krzymański 1969); those plants which revealed the same content of particular fatty acids were then subjected to further analysis. Genomic DNA Isolation DNA was isolated from ten day old leaves with the use of the method described by Doyle, Doyle (1990). DNA samples quality was analysed on 0.8% agarose gel electrophoresis. Analysis of Genomic DNA In order to investigate DNA regions specific for the low linolenic acid trait, RAPD method was applied. Operon Technologies primers: OPD-08, OPP-05, OPP-08, OPK-01, OPL-13, and D-25 were used. They were chosen from the literature data concerning the use of RAPD markers for screening oilseed rape populations segregating in respect of linolenic acid content (Jourdren et al. 1996; Thorman et al. 1996; Sommers et al. 1998). Amplification reactions were performed in Perkin Elmer and Biometra thermocyclers, programmed as follows: 30 s at 94 o C, 30 s at 94 o C, 1 min. at 35 o C, 2 min. at 72 o C for 45 cycles and 5 min. at 72 o C. Reaction mixture, in a final volume of 12,5 µl, contained: PCR reaction buffer [1 conc.], MgCl 2 [2 mm], dntps mixture [0,1 mm], primer [0.2 µm] and 0.4 enzymatic units of Taq DNA polymerase from MBI Fermentas. Each reaction was repeated five times on DNA samples obtained from independent preparations. Amplification products were resolved by 2% agarose gel electrophoresis. Results Genomic DNAs isolated from the oilseed rape plants characterized by differentiated linolenic acid content, i.e., from mutants, non-mutated plants as well as from cultivar Apollo (Tab. 1) were analysed with the use of RAPD method. Polymorphic bands, which enabled the distinction between plants of the low (mutant type) and double-low linolenic acid content, were obtained with the use of five, among six used, primers: OPK-01, OPL-13, OPP-05, OPP-08 and D-25 (Fig. 1). Two primers: OPP-08 (Fig. 1B) and OPK-01 (Fig. 1C) revealed bands which were characteristic for the mutant low-linolenic type, whereas the other revealed bands characteristic for non-mutated double-low plants (Fig. 1A, D, F, respectively). In addition, OPK-01 primer seems to display a difference between the low-linolenic plants of PN 1712/2 line and the low-linolenic cultivar Apollo (Fig. 2). In other cases, cultivar Apollo revealed the same pattern as winter oilseed rape mutants (data not shown), which suggests similar genetic background of these two mutations.

Analysis of the low-linolenic mutant genotypes of... 247 A B 1 2 3 4 5 6 7 8 9 10 M 1 2 3 4 5 6 7 8 9 10 M C D 1 2 3 4 5 6 7 8 9 10 M 1 2 3 4 5 6 7 8 9 10 M E F 1 2 3 4 5 6 7 8 9 10 M 1 2 3 4 5 6 7 8 9 10 M Fig. 1. 2% agarose gel electrophoresis of RAPD products obtained with the use of primers: A L-13, B P-08, C K-01, D P-05, E D-08, F D-25; Numbers from 1 to 4 indicate DNA samples obtained from non-mutated plants and numbers from 5 to 10 from mutants, respectively Rozdział elektroforetyczny na 2% żelu agarozowym produktów reakcji PCR-RAPD z zastosowaniem starterów: A L-13, B P-08, C K-01, D P-05, E D-08, F D-25. Kolejnymi numerami oznaczono: od 1 do 4 DNA roślin wyjściowych, od 5 do 10 DNA mutantów M molecular size marker: phage λ DNA hydrolised with endonucleases Eco RI and HindIII; polymorphic bands are indicated by arrows M marker wielkości DNA faga λ hydrolizowany enzymami EcoRI oraz HindIII; prążki polimorficzne oznaczono strzałkami

248 Katarzyna Mikołajczyk... Ap m n M Fig. 2. 2% gel electrophoresis of PCR/RAPD products obtained with the use of K-01 primer; DNA samples from, respectively: Ap Apollo, m low-linolenic mutant, n double-low plant; M molecular size marker phage λ DNA hydrolised with endonucleases Eco RI and Hind III; arrow indicates polymorphic band Elektroforetyczny rozdział na 2% żelu agarozowym produktów reakcji PCR/RAPD z zastosowaniem startera K-01; próbki DNA oznaczono odpowiednio: Ap Apollo, m niskolinolenowy mutant, n roślina nie poddawana mutagenezie; M marker wielkości DNA faga λ lambda hydrolizowany enzymami restrykcyjnymi Eco RI oraz Hind III; prążek polimorficzny oznaczono strzałką Perspective The obtained results make a background for further research in order to analyze DNA regions characteristic for the low-linolenic acid trait as well as to develop the specific DNA markers which would be of great importance for improvement of the low-linolenic acids genotypes breeding efficiency. Polymorphic bands, specific for mutated and non-mutated genotypes, are going to be cloned, sequenced and analyzed; SCAR markers will be designed. Doubled haploid (DH) lines of segregating population derived from the reciprocal crosses between low-linolenic mutated plant with the non-mutated parent are being obtained. Genomic DNA from parental plants and from DH segregating population plants will be analyzed with the use of the SCAR markers as well as microsatellite primers (SSRs) in order to establish DNA characteristics, specific for the low linolenic mutant genotype. Similar approach will be undertaken in order to analyse another winter oilseed rape mutant plants and DH segregating population, characterized by low linolenic and high oleic acid content. References Altin R., Cetinkaya S., Yűcesu H.S. 2001. The potential of using vegetable oil fuels as fuel for diesel engines. Energy conversion and management, 42: 529-538. Bartkowiak-Broda I., Krzymański J. 1983. Inheritance of C-18 fatty acids composition in seed oil of zero erucic winter rape Brassica napus L. 6th International Rapeseed Conference Paris 17-19 Mai 1983 (1): 477-482.

Analysis of the low-linolenic mutant genotypes of... 249 Byczyńska B., Krzymański J. 1969. Szybki sposób otrzymywania estrów metylowych kwasów tłuszczowych do analizy metodą chromatografii gazowej. Tłuszcze Jadalne, XIII: 108-114. Doyle J.J., Doyle J.L. 1990. Isolation of plant DNA from fresh tissue. Focus, 12: 13-15. Fitzpatrick K., Scarth R. 1998. Improving the health and nutritional value of seed oils. P.B.I. Bulletin, NRC-CRC, January: 15-19. Hu X., Sullivan M.L., Gupta M., Thompson S.A. 2003. Cloning of Fad2 and Fad3 Genes and Development of Gene-Specific Markers for High Oleic and Low Linolenic Acids in Canola (Brassica napus L.). 11th International Rapeseed Congress, Copenhagen, Denmark, 6-10 July 2003, BO5.2: 186-189. Jourdren C., Barret P., Horvais R., Delourme R., Renard M. 1996. Identification of RAPD markers linked to linolenic acid genes in rapeseed. Euphytica, 90: 351-357. Leckband G., Frauen M., Friedt W. 2002. NAPUS 2000. Rapeseed (Brassica napus) breeding for improved human nutrition. Food Research International, 35: 273-278. McDonnell K., Ward S., Leahy J.J., McNulty P. 1999. Properties of Rapeseed Oil for Use as a Diesel Fuel Extender. JAOCS, 76 (5): 539-543. Mikołajczyk K., Bartkowiak-Broda I. 2003. Markery DNA w hodowli jakościowej rzepaku ozimego (Brassica napus L.) w aspekcie modyfikacji zawartości kwasów tłuszczowych. DNA markers in rapeseed (Brassica napus L.) breeding with respect to fatty acids content modification. Rośliny Oleiste Oilseed Crops, XXIV: 33-49. Rakow G. 1973. Selektion auf Linol- und Linolensäuergehalt in Rapssamen nach mutagener Behandlung. Z. Pflanzenzüchtung, 69: 62-82. Rakow G., Raney J.P. 2003. Present status and future perspectives of breeding for seed quality in Brassica oilseed crops. 11th International Rapeseed Congress, Copenhagen, Denmark, 6-10 July 2003, BO5.3: 181-185. Röbbelen G., Nitsch A. 1975. Genetical and physiological investigations on mutants for polyenoic fatty acids in rapeseed (B. napus L.). I Selection and description of new mutants. Z. Pflanzenzüchtung 75: 93-105. Scarth R., McVetty P.B.E. 1999. Designer oil Canola a review of a new food-grade Brassica oils with focus on high oleic, low linolenic types. Proc. 10th International Rapeseed Congress, Canberra, Australia, 26-29.09.1999, CD ROM. Simopoulos A.P. 2000. Human requirement for n-3 polyunsaturated fatty acids. Poultry Science, 79: 961-970. Sommers D.J., Friesen K.R.D., Rakow G. 1998. Marker assisted selection of low linolenic acid in oilseed species. Theor Appl. Genet., 98: 897-903. Snowdon R.J., Friedt W. 2004. Molecular markers in Brassica oilseed breeding: current status and future possibilities. Plant Breeding, 123: 1-8. Spasibionek S., Byczyńska B., Krzymański J. 2000. Mutanty rzepaku ozimego podwójnie ulepszonego o zmienionym składzie kwasów tłuszczowych. Rośliny Oleiste Oilseed Crops, XXI: 715-724. Thelen J.J., Ohlrogge J.B. 2002. Metabolic Engineering of Fatty Acid Biosynthesis in Plants. Metabolic Engineering, 4: 12-21. Thormann C.E., Romero J., Mantet J., Osborn T.C. 1996. Mapping loci controlling concentrations of erucic and linolenic acids in seed oil of Brassica napus. Theor. Appl. Genet., 96: 897-903. Töpfer R., Martini N., Schell J. 1995. Modification of plant lipid synthesis. Science, 268: 681-686.