Seed Oil Quality of Brassica napus and Brassica rapa Germplasm from Northwestern Spain

The seed oil content and the fatty acid composition of a germplasm collection of Brassica napus and Brassica rapa currently grown in Galicia (northwestern Spain) were evaluated in order to identify potentially interesting genotypes and to assess their suitability as oilseed crops for either edible or industrial purposes. The seeds of the B. rapa landraces had higher oil content (mean 47.3%) than those of B. napus (mean 42.8%). The landraces of both species showed a similar fatty acid profile (12% oleic acid, 13% linoleic acid, 8–9% linolenic acid, 8–9% eicosenoic acid, and 50–51% erucic acid). They were very high in erucic acid content, which is nutritionally undesirable in a vegetable oil, and very low in oleic and linoleic acid contents. Therefore, they could be used for industrial purposes but not as edible oil. The erucic acid content ranged from 42% to 54% of the total fatty acid composition with an average value of 50% in the B. napus landraces whereas in B. rapa, it ranged from 43% to 57%, with an average value of 51%. Considering the seed oil and the erucic acid content together, three varieties within the B. napus collection and two varieties within the B. rapa one seem to be the most promising genotypes for industrial purposes.


Introduction
Brassica oilseed crops have become the third most important source of edible vegetable oils in the world [1]. Although edible oils currently represent the largest market for Brassica oilseed crops, the prevalence of agricultural surpluses in many developed countries has focused attention toward the possible industrial use of Brassica seed oils. The usefulness and quality characteristics of seed oils are determined by the proportion of its main constituent fatty acids [2][3][4]. Consequently, one of the most important objectives in Brassica breeding is the genetic modification of seed oil by maximizing the proportion of specific fatty acids [5][6][7][8].
Brassica oil is considered beneficial from a health point of view. It contains linoleic acid, which is desirable for nutritional purposes, and oleic acid, whose thermostability makes it desirable for cooking oil [9]. High oleic acid oil tastes better and may also have health benefits. The oxidative stability of this fatty acid also makes it suitable for some industrial applications [10]. Nevertheless, Brassica oil is characterised by significant amounts of erucic acid (about 50% of the total fatty acids), which is absent in any other commercial plant oils [11,12]. Erucic acid (cis-13-docosenoic acid, 22:1) has 22 These accessions represent the genetic variability of the B. rapa and B. napus germplasm currently grown in Galicia. The landraces were collected directly from growers at different sites throughout northwestern Spain from the eighties to present, and some of them have been propagated under isolation at the MBG in different years. Seed samples from different genotypes were taken from accessions kept at the germplasm bank at the MBG under the same low temperature and seed moisture conditions. Varieties were multiplied over several years, but always in the same location, with the same experimental plot, and under the same growing conditions. Due to the high number of genotypes evaluated in this study, it would have been impossible to multiply all the varieties in the same year. The geographical distribution of the B. rapa and B. napus landraces is shown in Figure 1. The number of landraces comprising the B. napus collection was lower than the B. rapa ones since the growing region of B. napus is restricted to the coastal area of southern Galicia and to areas near the Portuguese border ( Figure 1). Here, the crop is well adapted and common in the human diet [21]. Five accessions of B. napus were collected in inland areas, probably due to both human migration and the sale of seeds in local markets. All the B. napus landraces come from Galicia, Spain whereas the 3 B. napus commercial varieties were bought in Vila Real (North of Portugal), since commercial varieties of B. napus are not found in Galicia. All the B. rapa entries come from Galicia, Spain. These accessions represent the genetic variability of the B. rapa and B. napus germplasm currently grown in Galicia. The landraces were collected directly from growers at different sites throughout northwestern Spain from the eighties to present, and some of them have been propagated under isolation at the MBG in different years. Seed samples from different genotypes were taken from accessions kept at the germplasm bank at the MBG under the same low temperature and seed moisture conditions. Varieties were multiplied over several years, but always in the same location, with the same experimental plot, and under the same growing conditions. Due to the high number of genotypes evaluated in this study, it would have been impossible to multiply all the varieties in the same year. The geographical distribution of the B. rapa and B. napus landraces is shown in Figure 1. The number of landraces comprising the B. napus collection was lower than the B. rapa ones since the growing region of B. napus is restricted to the coastal area of southern Galicia and to areas near the Portuguese border ( Figure 1). Here, the crop is well adapted and common in the human diet [21]. Five accessions of B. napus were collected in inland areas, probably due to both human migration and the sale of seeds in local markets. All the B. napus landraces come from Galicia, Spain whereas the 3 B. napus commercial varieties were bought in Vila Real (North of Portugal), since commercial varieties of B. napus are not found in Galicia. All the B. rapa entries come from Galicia, Spain.

Lipid Analysis
Bulk samples of seeds of each accession (landraces and commercial varieties) were screened for oil content. The oil content of the seeds was determined by nuclear magnetic resonance (NMR) with an Oxford 4000 Analyzer (Oxford Analytical Instruments Ltd., Abingdon, OX, UK), following desiccation at 50 °C for 72 h. For fatty acid composition, ten seeds were randomly selected and individually analysed for each local and commercial variety. The content of seven major fatty acids present in the oil extracted from Brassica crops (palmitic, stearic, oleic, linoleic, linolenic, eicosenoic,

Lipid Analysis
Bulk samples of seeds of each accession (landraces and commercial varieties) were screened for oil content. The oil content of the seeds was determined by nuclear magnetic resonance (NMR) with an Oxford 4000 Analyzer (Oxford Analytical Instruments Ltd., Abingdon, OX, UK), following desiccation at 50 • C for 72 h. For fatty acid composition, ten seeds were randomly selected and individually analysed for each local and commercial variety. The content of seven major fatty acids present in the oil extracted from Brassica crops (palmitic, stearic, oleic, linoleic, linolenic, eicosenoic, and erucic), as well as the content of other minor fatty acids (arachidic, arachidonic, and behenic), was determined. In order to evaluate the fatty acid composition of seed samples, the lipids were extracted, transmethylated, and purified using the one-step method of Garcés and Mancha [35] with some modifications. Individual seed samples were heated at 80 • C for 2 h in MeOH: toluene: dimethoxypropane: H 2 SO 4 : heptane (33:14:2:1:50; by vol.), and, after cooling, the fatty acid methyl esters were recovered in the upper phase. The analysis of the fatty acid methyl esters composition was developed in a Perkin Elmer Autosystem gas-liquid chromatograph (Perkin-Elmer Corporation, Norwalk, USA) equipped with a flame ionization detector (FID) and a 2 m long column packed with 3% SP-2310/2% SP-2300 on a Chromosorb WAW (Supelco Incorporated, Bellefonte, USA). The gas chromatograph was programmed for an initial temperature of 190 • C for 10 min followed by an increase of 2 • C per min to 220 • C; this final temperature was maintained for a further 5 min. The injector and flame-ionization detector were held at 275 and 250 • C, respectively. The fatty acids were identified by comparison with known fatty acid methyl esters standards (F.A.M.E. Mix, CRM18920 Supelco and ME14-1KT Supelco, Sigma-Aldrich). The analyses were performed at the 'Institute for Sustainable Agriculture (Instituto de Agricultura Sostenible, IAS), Spain.
Individual and combined analyses of variance were performed for each trait of seed composition, using the general lineal model (GLM) procedure of the SAS statistical package [36]. The accessions and the species were considered as fixed effects. Comparisons of means among populations and species were performed for each trait using Fisher's protected least significant difference (LSD) at p = 0.05 [37].

Oil Content in the Brassica Collection
The oil content in the seeds of the B. napus landraces ranged from 29.1% (for MBG-BRS0423) to 50.1% (for MBG-BRS0044), with an average value of 42.5%. The oil content of the B. rapa landraces ranged from 31.4% (for MBG-BRS0285) to 56.3% (for MBG-BRS0245), with an average value of 47.3% ( Table 1). The Brassica rapa genotypes were significantly higher in oil content than the B. napus ones (Table 1). This result agrees with Mandal et al. [38], who found that seed oil content was higher in a collection of B. rapa (about 42%) than in a collection of B. napus (about 35%). The seed oil content from the B. napus landraces was significantly lower than that from the commercial varieties, whereas no significant differences were found between the landraces and the commercial varieties in B. rapa. The genotypes evaluated in this work showed values for oil content similar to those found on cultivars of the major Brassica oilseed crops (B. napus, B. rapa, B. carinata, and B. juncea), with an average oil content between 45% and 50% [30,39], even though the Brassica germplasm from northwestern Spain is not grown as an oilseed crop. Since only one datum per population was obtained, mean oil content comparisons among populations are not reported. Despite this fact, the highest seed oil content (more than 47%) in the B. napus collection was found for landraces MBG-BRS0044, MBG-BRS0087, MBG-BRS0329, MBG-BRS0434, and MBG-BRS0105, along with the commercial variety, MBG-BRS0373. For the B. rapa collection, landraces MBG-BRS0245, MBG-BRS0236, MBG-BRS0125, MBG-BRS0249, and MBG-BRS0139 had the highest levels.

Fatty Acid Composition in the Brassica Collection
Significant differences among the B. napus landraces were found for all the fatty acids analysed, whereas the commercial varieties of this species were not significantly different for linoleic and erucic acid contents ( Table 2). The fatty acid profile observed between the landraces and the commercial varieties of B. napus was different. The average erucic acid content was considerably higher in the B. napus landraces compared to the commercial seeds ( Table 3). The commercial varieties had zero erucic acid and followed the typical profile of canola varieties even though they are commonly used as vegetable crops but not as oilseed crops. The collection of the B. napus evaluated includes all the germplasm currently grown in Galicia, which means that in this region, only the pabularia type is grown, and B. napus is not used for oil production. The significant differences between the B. napus landraces and the commercial varieties do not mean that the commercial pabularia breeding is based on modern zero erucic varieties of B. napus. Since commercial varieties of B. napus are not common in Galicia, the seeds used in this study came from Portugal, where they were bought as 'couve-nabiça' (Portuguese B. napus landrace), but they are probably zero erucic rapeseed varieties.  Within the B. rapa collection, the landraces and the commercial varieties were significantly different for all the fatty acids. The seeds of the B. rapa commercial varieties, which included crops of turnips, turnip greens, and turnip tops, were significantly different in erucic acid content (Table 2), although their values were significantly lower than those found in most landraces (Table 3). Despite this fact, the genotypes of both types of germplasm (landraces and commercial varieties) showed a similar fatty acid profile (Table 3).
Significant differences were found between the B. napus and B. rapa landraces for all the fatty acids, except for oleic and erucic acids ( Table 2). The seeds of the B. rapa landraces were higher than the B. napus ones for stearic, linoleic, linolenic, and eicosenoic acids, while the B. napus landraces were only higher for palmitic acid content ( Table 3).
Since erucic acid was the major fatty acid found, and because it is a trait of large interest for plant breeding, most discussion will be focused on this fatty acid. The erucic acid content ranged from 42% to 54% of the total fatty acid composition, with an average value of 50% in the B. napus landraces and from 43% to 57% of the total fatty acid composition in the B. rapa landraces, with an average value of 51% (Table 3). Similar values for erucic acid content were found by Sharafi et al. (2015) [12] for three rapeseed varieties and for four entries of B. rapa, although crops were different from those evaluated in this study. The lowest content of erucic acid was found within the B. napus species in seeds of MBG-BRS0333 (about 42% of the total fatty acids) and within the B. rapa species in seeds of MBG-BRS0379 (about 43% of the total fatty acids). Both values are still very high, and, therefore, vegetable oil of the genotypes evaluated should be considered unsuitable for edible oil production. On the other hand, oils with high erucic acid content are desirable for industrial purposes. For both species, the landraces with high erucic acid content are included in Table 4. The highest erucic acid composition, more than 53% of the total fatty acids, was found in B. napus landraces MBG-BRS0329, MBG-BRS0041, MBG-BRS0048, and MBG-BRS0105 (Table 4). B. rapa landraces MBG-BRS0235, MBG-BRS0416, MBG-BRS0202, MBG-BRS0139, MBG-BRS0190, and MBG-BRS0239 showed the highest erucic acid composition-more than 55% of the total fatty acids (Table 4). Considering their high oil content and high erucic acid content together, genotypes MBG-BRS0329, MBG-BRS0434, and MBG-BRS0105 within the B. napus collection, and MBG-BRS0139 and MBG-BRS0101 within the B. rapa collection, offer interesting prospects for future industrial applications. The Brassica landraces grown in Galicia have been traditionally improved by growers for vegetable use but not for their use as oil sources. Nowadays, efforts to develop low erucic acid genotypes of both B. napus var. pabularia and B. rapa ssp. rapa have not been undertaken because the main usage of these crops is for leaf consumption. these five accessions was lower (37.6%) than the values found in the present work. Mandal et al. [38] evaluated the fatty acid content of several cruciferous species and reported erucic acid contents of 41% in genotypes of B. napus, 47.4% in B. rapa ssp. dichotoma, and 51.6% in B. rapa ssp. trilocularis. Rapeseed oil genotypes with higher proportions of erucic acid than the levels found in traditional cultivars (about 50%) are sought by breeders for use in well-known industrial products [4,7,13,20]. In this way, the above-mentioned populations of B. napus and B. rapa could be used in industrial processes but they are not appropriate for edible uses. In general, low intra-population variability was observed for the fatty acid composition of the seed oil. However, some landraces displayed an important variation among the ten seeds individually analysed for erucic acid content. Some of them had seeds with either decreased or increased values of that fatty acid. In the B. napus collection, three individual seeds of MBG-BRS0337 were found to have less than 35% erucic acid content (27.2%, 31.7%, and 33.1%). On the other hand, one individual seed of both MBG-BRS0105 and MBG-BRS0329 was found to have more than 56% of erucic acid content. In the B. rapa collection one individual seed of MBG-BRS0431 and two seeds of MBG-BRS0463 had low values for erucic acid content, (32.3%, 33.9%, and 34.1%, respectively), whereas three landraces, MBG-BRS0195, MBG-BRS0224, and MBG-BRS0231 had individual seeds with values higher than 60% of erucic acid content. The decreased content of erucic acid could be due to an unintended cross with a zero or low erucic commercial variety. However, in order to avoid the problem of pollen contamination, the seeds analyzed were directly obtained from growers or from multiplications made in isolation cages at the MBG. The decreased or increased levels of erucic acid content found in some seeds could presumably correspond to heterozygous seeds for that fatty acid. It would be interesting to analyse more seeds from the above-mentioned populations using the half-seed method described for other oilseed crops [41] and to select genotypes in segregating populations with lower or higher erucic acid content than that observed in their respective landraces.
There are numerous studies on the nutritional and industrial value of the seed oil of Brassica oilseed crops, but there are no previous studies on the potential of seed oil in these vegetable Brassica crops. Therefore, the present work provides relevant information and discussion on the potential of the oil of the seeds of two horticultural crops of Brassica, nabicol and turnip greens, for food or industrial purposes. The reported results offer the first insights into the variability of the current gene pool of the B. rapa and B. napus varieties grown in Galicia. These valuable genetic resources will certainly be studied regarding for important traits. The content of glucosinolates should be taken into account, as glucosinolates are important in modern rapeseed breeding and are probably very high in the landraces of both species. As a conclusion, the germplasm evaluated in this work displayed variability in the fatty acid composition of its seed oil. Some accessions of both species could be further used as sources of oil for industrial purposes because their seeds were high in erucic acid content and low in oleic, linoleic, and linolenic acid content. Further research will be needed for some accessions having seeds with reduced or increased values of erucic acid content, in order to select valuable genotypes that could be used for both nutritional and industrial applications.