ABSTRACT

Traditional rapeseed and mustard oils are characterized by high contents of erucic acid (22:1) and other monounsaturated fatty acids with chain lengths of C20 to C24. Breeding of low erucic acid cultivars of Brassica napus, B. rapa, B. juncea and B. carinata has led to an almost complete abolishment of 22:1 synthesis. In general, erucic acid content in the genus Brassica varies with the allelic constitution of the genotype, differences in the ploidy level, the genetic background and environmental impact. Series of alleles have been identified in B. napus (genome AACC) and B. rapa (AA), which make it possible to breed strains containing almost any level of 22:1 from less than 1% to about 60% of total fatty acids. Regarding B. oleracea (CC), which normally displays a 22:1 content ranging from 28 to 63%, we were able to identify individual plants being nearly free of erucic acid. However, only in a few cases evidental data is available indicating the presence of true alleles which for example in B. napus reside either in the A or C genome. With regard to the molecular basis of erucic acid allelism evidence exists that biosynthesis of 22:1 is controlled through the expression of the elongase condensing enzyme (β-ketoacyl-CoA synthase, KCS), which is encoded by gene(s) homologous to the Arabidopsis thaliana FAE1 gene. In the course of genomic Southern hybridization analyses different oilseed rape and Brassica genotypes - possessing a specific 22:1 content due to a particular genetic constitution - have been evaluated towards the presence of erucic acid genes/alleles. The results of hybridization with a KCS specific probe manifest our hypothesis, that variation in 22:1 content in Brassica is mainly due to individual alleles, which can be distinguished on a molecular level.

INTRODUCTION

Due to substantial progress in breeding and cultivation practice rapeseed and mustards - derived from several locally distributed members of the genus Brassica - have become one of the worldwide most important source of vegetable oil. Especially in several European countries with cool-temperate climates oilseed rape (B. napus) with ‘double-low‘ seed quality (Canola) dominates field crop production. Since erucic acid (22:1) and other very long-chain fatty acids (VLCFA), the major oil components of traditional high-erucic acid rapeseed (HEAR) and other Brassica oilseed species, are considered as detrimental to the food quality of the oil low-erucic acid varieties of B. napus, B. rapa, B. juncea and B. carinata were successfully bred showing an almost complete abolishment of VLCFA synthesis (cf. Lühs and Friedt 1994, Rakow and Getinet 1998). Contradictory, for industrial purposes the 22:1 content of rapeseed oil should be as high as possible. With regard to this breeding objective it has become obvious that natural variation for high 22:1 content is limited in B. napus to a level of about 55-60%, while some accessions of related Brassica species possess 60% 22:1 and even more in their seed oil (Lühs and Friedt 1995a). Regarding the inheritance it is well known, that erucic acid content in B. napus (high-erucic acid genotype: E_AE_AE_CE_C) and other amphidiploid Brassica species is controlled by two genes acting in an additive manner, which are derived from the progenitor species, viz. B. rapa (turnip rape, turnip, sarson, Chinese cabbage etc.; E_AE_A), B. oleracea (C genome; cabbage, kohlrabi, cauliflower etc.; E_CE_C) and B. nigra (B genome; Black mustard; E_BE_B), respectively. However, only in a few cases evidental data is available, which confirms the presence of true alleles residing in the A or C genome of B. napus for example (Jönsson 1977, Chen and Heneen 1989). Following B. napus resynthesis experiments we generated HEAR lines via interspecific hybridization by using B. rapa and B. oleracea genotypes with the most effective alleles for erucic acid synthesis as parents. We were able to show that 22:1 content can be modified by accumulating highly active alleles displaying an average additive contribution of 16-17% of 22:1 per allele, which leads to a total 22:1 content of about 60% in the resynthesized rapeseed (Res) lines (Lühs and Friedt 1995b). The opportunity to improve erucic acid synthesis by accumulation and combination of highly effective alleles through wide Brassica is facilitated by the molecular characterization of relevant genes/alleles.

EXPERIMENTAL

Brassica collection and genetic analyses

A set of 30 Brassica genotypes consisting of 15 B. napus, 5 B. rapa, 5 B. oleracea and 3 B. juncea accessions as well as B. nigra cv. Giebra’ and an Ethiopian B. carinata accession was included in this investigation. The seed samples were part of our own collection or were purchased at local seed stores. A large number of B. oleracea accessions was provided by Drs. J.R. McFerson and L.D. Robertson from USDA-ARS, Plant Genetic Resources Unit, Cornell University, Geneva, NY, USA. Artificial biennal B. napus material was derived from interspecific crosses between Chinese cabbage (B. rapa ssp. pekinensis) and kohlrabi (’Res QLB 1’), cauliflower (’Res QLB 2’) or curled kitchen kale (’Res QLB 11’), respectively. This material was developed and kindly provided by Dr. E. Clauss, BAZ Quedlinburg/Germany. Following a screening for fatty acid composition of Brassica species artificial spring HEAR (’Res 91’, ’Res 125’, ’Res 241’) was created by interspecific hybridization of Indian ’Yellow Sarson’ (B. rapa ssp. trilocularis) and the cauliflower (B. oleracea convar. botrytis var. botrytis) cultivar ’Otecestvennaja’ (’BK 2287’) as described earlier (Lühs and Friedt 1995a, 1995b). Doubled-haploid rapeseed lines (’DH 98/56’, ’DH 90.8/80’) were derived from intraspecific crosses between resynthesized lines (’Res 125’, ’Res 241’; high-erucic acid genotype: E_AE_AE_CE_C) and Canadian spring Canola cultivars (’Profit’, ’Excel’; erucic acid genotype: e_Ae_Ae_Ce_C) (Weber et al. 1995). The genetic constitution of the DH lines of the type E_AE_Ae_Ce_C / e_Ae_AE_CE_C was evaluated by test crossing and analyzing the fatty acid segregation in F₁ and F₂. The fatty acid composition was determined on the basis of half-seed analyses according to a method as outlined in Lühs and Friedt (1995b). Fatty acid content is expressed as a percentage of total fatty acids.

Molecular characterization

By using a PCR amplified fragment of the Arabidopsis thaliana FAE1 gene (James et al. 1995) as probe a promising B. napus cDNA clone (KCSb5) with a fragment size of 1.6kb was isolated from a rapeseed (cv. ’Askari’) silique cDNA library (Han et al. 1998). Genomic DNA from young green leaf tissue was extracted and digested with restriction enzyme BamH1. In the course of genomic Southern analysis the KCSb5 gene was used as a probe for the detection of erucic acid genes/alleles according to Lühs et al. (1998, 1999). PCR amplified fragments generated by deduced primer pairs and covering a range of 501bp (KCSs1/as1), 381bp (KCSs2/as2) and 1251bp (KCSs1/as2) of KCSb5 were separated on a 1.5% agarose gel and visualized by ethidium bromide staining in order to detect polymorphisms among the Brassica genotypes.

RESULTS AND DISCUSSION

Molecular basis of erucic acid biosynthesis

The biosynthesis of very-long chain fatty acids (VLCFA) in higher plants depends on membrane-bound, multienzyme acyl elongase systems which catalyze a series of biochemical reactions similar to those of de novo fatty acid synthesis. These steps consist of the condensation between an acyl primer and a donor malonyl-CoA, following by a reduction, dehydration and a second reduction, leading to an acyl-CoA which is two carbons longer. For example, cis-11-eicosenoic (20:1), erucic and nervonic acid (24:1), which are typical cruciferous long-chain (n-9) fatty acids, are formed by successive 2-carbon chain extension of the precursor oleic acid (Cassagne et al. 1994). The activity of the condensing enzyme (β-ketoacyl-CoA synthase, KCS), representing the first and rate-limiting step of the microsomal fatty acid elongation reaction, determines the acyl chain length of the VLCFA produced by developing seeds (Millar and Kunst 1997). The KCS component of seed-specific fatty acid elongases has been cloned from Arabidopsis thaliana (FAE1, James et al. 1995), jojoba (Simmondsia chinensis, Lassner et al. 1996), honesty (Lunaria annua, Lassner 1997), as well as different Brassica species (Clemens and Kunst 1997, Barret et al. 1998, Han et al. 1998, Fourmann et al. 1998). Due to the high degree of both, homology of nucleotide sequences and identity of the deduced amino acids, there is strong evidence that 22:1 biosynthesis in rapeseed and other Brassica species is controlled through the expression and property of KCS enzymes being encoded by gene(s) homologous to the FAE1 gene from A. thaliana. The introduction of KCS genes cloned from S. chinensis or B. napus confirmed this assumption by showing complementation of the Canola fatty acid elongation mutation (fae) leading to the restoration of erucic acid synthesis in transgenic rapeseed. This means, that the mutation(s) which gave rise to the low-erucic acid phenotype are associated with either the structural gene encoding KCS or with genes regulating the expression of KCS (Lassner et al. 1996, Han et al. 1998, Roscoe et al. 1998). Here, we display polymorphisms in the FAE1 gene that are associated with variation in erucic acid content in the genus Brassica including both high- and low-erucic acid genotypes.

Molecular characterization of erucic acid alleles

In the course of genetic studies a B. napus homolog of FAE1 gene (Han et al. 1998) was used as a probe for the molecular evaluation of different rapeseed and Brassica genotypes, which were known to possess a specific erucic acid content due to a particular genetic constitution (Jönsson 1977, Lühs and Friedt 1995a). Regarding B. napus the material chosen for this experiment displayed a wide variability: HEAR and low-erucic acid rapeseed (LEAR), resynthesized and conventional oilseed rape, spring and winter types were included. Figure 1 illustrates the polymorphisms we have obtained by a genomic Southern analysis with the KCSb5 probe after DNA digestion with BamH1 (top panel) and PCR with a highly specific primer pair for the KCS gene (middle panel). Corresponding to the amphidiploid origin of B. napus and the digenic control of erucic acid content we were able to differentiate two fragments in most of the cases, whereas for the progenitor species B. rapa and B. oleracea we observed one band only (Figure 1, top). This is shown for our resynthesized B. napus lines, such as ’Res 91’ (E_AE_AE_CE_C; 57.1% 22:1), displaying a combination of the parental genomes, i.e. ’Yellow Sarson’ (B. rapa ‘Y.S.‘, E_AE_A; 54.8% 22:1) and cauliflower ’BK 2287’ (B. oleracea, E_CE_C; 58.0% 22:1), respectively. Regarding the B. napus cultivars ’Bronowski’ (10% 22:1) and ’Moana’ (33.1% 22:1), showing a monogenic control of high erucic acid content (Lammerink and Morice 1971, Anand and Downey 1981), both loci - the one with the homozygous ”effective” erucic acid allele and the other with low erucic alleles - display distinct fragments. The same should be true for the doubled-haploid rapeseed lines ’DH 98/56’ (e_Ae_AE_CE_C; 23.3% 22:1) and ’DH 90.8/80’ (E_AE_Ae_Ce_C; 37.8% 22:1), which possess one of the newly generated B. napus alleles, viz. E_A from B. rapa (‘Yellow sarson‘) and E_C from B. oleracea (cauliflower ‘BK 2287‘). However, in the case of ’DH 98/56’ the two fragments were not separated due to much similarity between the cauliflower E_C-allele and the e_A-allele derived from spring Canola (’Profit’, e_Ae_Ae_Ce_C; 0.2% 22:1). The same result was observed for ‘Askari‘ (E_AE_AE_CE_C; 54.5% 22:1), used as a check for KSCb5, the spring rapeseed ‘Golden‘ (E_AE_AE_CE_C; 41.1% 22:1) as well as the low-erucic acid varieties ’Profit’ and ’Excel’ (e_Ae_Ae_Ce_C; 0.5% 22:1), indicating a high grade of homology between the erucic acid genes/alleles residing in the A and C locus due to minor differences in the nucleotide sequence and the deduced amino acids of the KCS polypeptid (cf. Clemens and Kunst 1997, Fourmann et al. 1998).

Figure 1: Molecular characterization of Brassica erucic acid alleles by Southern analysis (top) and KCS-specific PCR (middle) in comparison to the proportion of (n-9) fatty acids (bottom); *non sufficient DNA quality in Southern analysis

Comparing low and high erucic acid types of B. rapa and B. oleracea differences between alleles for high and low erucic acid content become obvious. Regarding the low-erucic turnip rape cultivar ‘Asko‘ (e_Ae_A; 0.2% 22:1) and the B. rapa varieties ‘Arktus‘ (E_AE_A; 44.8% 22:1), ‘Granat‘ (E_AE_A; 51.5% 22:1) and the tetraploid ‘Perko PVH‘ (E_AE_AE_AE_A; 47.1% 22:1) one can detect a polymorphism between the genotypes through different band positions. Following half-seed selection in a seed sample of the Russian cabbage accession ‘Ladozhskaya‘ (26.4% 22:1) we were able to distinguish low-erucic acid plants (e_Ce_C; 0.1% 22:1) from those with high erucic content (E_CE_C; 42.3% 22:1). As far as we know, this is the first time, that low-erucic acid mutants of B. oleracea have been identified so far. Furthermore, as compared to the other low-erucic acid Brassica genotypes mentioned above there was no signal in this ‘selection zero‘ of B. oleracea which indicates a partial or complete deletion of the KCS gene. The latter result was verified through a PCR analysis with a KCS-specific primer pair (Figure 1, lower panel), where the 1.25kb fragment characteristic for all other genotypes was not amplified in the case of ‘Ladozhskaya selection zero‘. In order to preclude misinterpretation DNA was checked for its ability to amplify with the RAPD primer OPS09 (data not shown). Up to now it is not clear if the whole sequence is absent or just the part being not PCR amplified by the primer pair. Recently, Roscoe et al. (1998) found that the mutation(s) which manifest the low-erucic acid rapeseed (LEAR) phenotype act(s) in a post-transcriptional manner. The authors suggest that variation in the sequence of the FAE1 gene can lead to an apparently inactive protein and resulting in deficiency of elongation activity in developing rapeseed (Roscoe et al. 1998). We assume that the low-erucic acid mutation we have detected in the B. oleracea genotype is acting in another genetic fashion leading to the absence of an intact KCS protein. The characterization of this low-erucic mutant will be the matter of further investigations.

CONCLUSION

With regard to the breeding of HEAR this work was aimed in the identification, molecular characterization and accumulation of more effective alleles for erucic acid synthesis in B. napus and related species (Friedt and Lühs 1998, Lühs et al. 1999). The results manifest our hypothesis, that variation in erucic acid content is mainly induced by different alleles, which can be detected on a molecular level as homologs of the Arabidopsis thaliana FAE1 gene (James et al. 1995). A low-erucic acid mutant of B. oleracea was detected being different from low-erucic acid Brassica genotypes known before. Actually, the characterization of the different mutants resulting in the low erucic acid phenotype will be of prime importance to obtain more insight in which way condensing enzymes of the HEAR genotypes are capable in assembling a functional elongase complex.

ACKNOWLEDGEMENT

The authors gratefully acknowledge financial support of their projects dealing with industrial rapeseed breeding by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF), the Bundesministerium für Ernährung, Landwirtschaft und Forsten (BML), and the Gemeinschaft zur Förderung der privaten deutschen Pflanzenzüchtung e.V. (GFP). We thank Drs. F.-P. Wolter and J. Han, Institute of General Botany and Botanical Garden, University of Hamburg, for providing the KCSb5 probe. The excellent assistance of Mrs. Rosa Allerdings, Mrs. Petra Degen and Ms. Sonja Weber is gratefully acknowledged.

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