Analiza genotypów mutanta rzepaku ozimego (Brassica napus L.) o obniżonej zawartości kwasu linolenowego z zastosowaniem markerów DNA.

Katarzyna Mikołajczyk, Stanisław Spasibionek, Iwona Bartkowiak-Broda

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 low-linolenic 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 zawar-tością 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 pod-wójnie 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

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

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.

Materials and Methods

Plant Material

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

double-low 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

C16:0 — palmitic acid — kwas palmitynowy C18:0 — stearic acid — kwas stearynowy

C18:1 — oleic acid — kwas oleinowy C18:2 — linoleic acid — kwas linolowy

C18:3 — linolenic acid — kwas linolenowy C20:1 — eicosenoic acid — kwas eikozenowy

C22:1 — erucic acid — kwas erukowy

Fatty acids — Kwasy tłuszczowe Line Linia _C 16:0 C18:0 C18:1 C18:2 C18:3 C20:1 C22:1 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

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

_C,

30 s at 94

_{C, 1 min. at 35}

_{C, 2 min. at 72}

_{C for 45 cycles and 5 min. at 72}

_C.

Reaction mixture, in a final volume of 12,5 µl, contained: PCR reaction buffer

[1×conc.], MgCl

[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.

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

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.

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