Genetic variation and QTL analysis of saturated fatty acids in two doubled haploid populations of oilseed rape (Brassica napus L.)

Consumption of foodstuff with low contents of saturated fatty acids is considered beneficial for human health. Reducing saturated fatty acid content in oilseed rape (canola) and other oil and protein crops is a relevant breeding aim. The objective of this work was to study the genetic variation and inheritance of saturated fatty acids in two DH populations of oilseed rape, to map QTL and to identify candidate genes. In addition, the correlation to other seed quality traits was studied. To this end, two half-sib DH populations were tested in up to five field environments in north-western Europe and seeds harvested from open-pollinated seeds were analyzed. Genotyping was performed using Illumina Brassica 15 K SNP chip. In both populations, significant effects for the genotypes and for the environments were detected, and heritability ranged from 68 to 89% for the predominant palmitic acid and stearic acid content. Up to 48 QTL for different fatty acids, oil and acid detergent lignin (ADL) content were mapped in the two populations. Co-locating QTL for palmitic acid, stearic acid, the C16/18 fatty acid ratio, the FATB/A ratio, oil and ADL content were identified on different chromosomes. A large number of candidate genes were identified within the vicinity of QTL flanking markers. Identification of several co-locating QTL positions, of associated candidate genes and SNP markers should facilitate oilseed rape breeding for low saturated fatty acid content.


Introduction
Oilseed rape (Brassica napus L., AACC, 2n = 38) is an allotetraploid species of winter and spring genotypes. It is the third largest source of vegetable oil after oil palm and soybean (FAOSTAT 2020). The production of oilseed rape is predominantly in most part of Europe, Canada, Australia, China and India, with over 36.5 million ha of cultivation worldwide with annual global production of about 72 million tons (FAOSTAT 2020). Even though the by-product after oil extraction from the seed is the protein rich meal, the major interest in oilseed rape used to be the seed oil. Recently, other seed quality traits like increasing the protein and reducing the fibre content are gaining importance (Balalić et al. 2017). The oil quality and its nutritive value is determined by the relative composition of the fatty acid content present in the oil (Vick et al. 2002). Modern cultivars of rapeseed contain about 60-65% monounsaturated fatty Abstract Consumption of foodstuff with low contents of saturated fatty acids is considered beneficial for human health. Reducing saturated fatty acid content in oilseed rape (canola) and other oil and protein crops is a relevant breeding aim. The objective of this work was to study the genetic variation and inheritance of saturated fatty acids in two DH populations of oilseed rape, to map QTL and to identify candidate genes. In addition, the correlation to other seed quality traits was studied. To this end, two half-sib DH populations were tested in up to five field environments in north-western Europe and seeds harvested from open-pollinated seeds were analyzed. Genotyping was performed using Illumina Brassica 15 K SNP chip. In both populations, significant effects for the genotypes and for the environments were detected, and heritability ranged from 68 to 89% for the predominant palmitic acid and stearic acid content. Up to 48 QTL for different fatty acids, oil and acid detergent lignin (ADL) content were mapped in the two populations. Co-locating QTL for palmitic acid, stearic acid, the C16/18 fatty acid ratio, the FATB/A ratio, oil and ADL content were identified acids of mainly oleic acid (18:1), 30-35% polyunsaturated fatty acids of mainly linoleic acid (18:2) and linolenic acid (18:3), and 5-8% saturated fatty acids (SFA) (Chen et al. 2018). Monounsaturated 18:1 and polyunsaturated 18:2 are beneficial to human health and are less susceptible to oxidation during frying (Merrill et al. 2008;Zhao et al. 2019), so are desirable traits for improvement. However, presence of high SFA content in the oil, possess health risk, as it is reported to elevate the Low Density Lipoprotein (LDL) in blood serum causing coronary heart disease (Rebetzke et al. 2001). From the human nutritional point of view, the content of SFA should be as low as possible. Palmitic (16:0) and stearic acid (18:0) are the major components of SFA in Brassica napus (L.) and its composition in the oil content is receiving interest because dietary recommendations limit its intake to less than 7% (Beaith et al. 2005). Like other fatty acids, the variation of SFA content is quantitatively inherited in plants, which depends on the genotype, the environment and their interactions (Rebetzke et al. 2001). As a result, genetic reduction of SFA will increase the integrity of the oil content and the competitive ability with other vegetable oils utilized for nutritional purpose (Gororo et al. 2014). A low SFA content of less than 7% is critical for labelling rapeseed oil as low SFA product in the USA (Rakow and Raney 2005). In plant biosynthesis, fatty acid synthesis (FAS) takes place in the plastid while oil synthesis in form of triacylglycerol (TAG) takes place in the cytosol at the endoplasmic reticulum. Several enzymes are involved in the biosynthetic pathways producing fatty acids, which regulate the composition of different fatty acids in the oil (Gororo et al. 2014). As a result, genetic modifications of fatty acids can be achieved by studying enzyme activities in the respective biosynthetic pathways (Stoll et al. 2005). Mapping of QTL linked to genes encoding enzymes in the fatty acids and TAG synthesis will be of great interest to elucidate the complexity involved in the accumulation of fatty acids in the oil. Genetic basis of fatty acid biosynthesis and pathway have been characterized in Arabidopsis thaliana (Li-Beisson et al. 2013). For the SFAs, they are generally synthesized in the plastid and exported to cytoplasm to be incorporated into TAG. In the plastids, palmitoyl-ACP (16:0-ACP) is the primary product of series of fatty acid synthesis (FAS) catalyzed by two condensing enzymes 3-ketoacyl-ACP synthase I and III (KASI and KASIII).
There have been several studies on the expression of genes encoding some fatty acid synthase enzymes and the phenotypic expression of SFA in the seed oil content of crops like maize (Zheng et al. 2014 In this study, two doubled haploid populations derived from crosses between canola cultivar Adriana with erucic acid DH line SGEDH13 and with the Chinese canola cultivar Zheyou 50, were investigated. The aim of this work was to (1) Study the genetic variation and inheritance of saturated fatty acids and their correlations with other fatty acids and oil content in two DH populations, to map QTL and to identify candidate genes involved in the expression of these traits.

Plant material
The study material consisted of two DH populations. The first population consisted of 170 F 1 derived doubled haploid (DH) lines from a cross between the canola cultivar Adriana and the DH line SGEDH13, using microspore culture. Adriana is a German winter rapeseed line cultivar and is among the canola cultivars analyzed by Dimov and Möllers (2010), characterized by high oil, reduced palmitic acid (16:0) and high oleic acid content (18:1). SGEDH13 is a DH line derived from the cross between DH line SGDH14 (Zhao et al. 2005) and inbred line 617 of the German winter rapeseed canola cultivar Express analyzed by Behnke et al. (2018). SGEDH13 is characterized by high oil content, normal palmitic acid (16:0) and medium erucic acid (22:1) contents caused by the presence of only one erucic acid gene. The DH population was termed ASG, an acronym derived from the letters from both parents Adriana and SGEDH13. The second DH population consisted of 95 F1 derived doubled haploid lines derived by microspore culture from a cross between Adriana and Zheyou 50; henceforth referred to as AZH DH population. Zheyou 50 is a semi-winter cultivar from China characterized by high oil and normal oleic acid (18:1) and palmitic acid (16:0) contents. Both DH populations were developed at the Division of Crop Plant Genetics, Georg-August University, Göttingen, Germany.

Field experiments
The ASG DH lines and the parents were tested in three growing seasons (2015/16, 2016/17, and 2017/18) in five field environments located in northwestern Germany and Poland. The AZH DH population was evaluated in four consecutive seasons in one environment in north-western Germany. The field experiments were conducted in small plots as a Randomized Complete Block design without replication. Each genotype was sown with 100 seeds in a row of 5 m length; distance between the rows was 75-90 cm. At maturity, open pollinated seeds were bulk harvested from each genotype from the terminal raceme and three upper most primary branches of ten healthy plants. The harvested seeds were de-husked and cleaned and stored at room temperature for seed quality trait analysis using gas chromatography (GC) and near-infrared reflectance spectroscopy (NIRS).

Measurement of fatty acid composition using gas chromatography
The fatty acid composition was analyzed using gas chromatography as described by Rücker and Röbbelen (1996) with a few modifications. About 200 mg of seeds per genotype were weighted into a clean propylene tube and a small stainless-steel rod was added to the seeds in the tube. 1000 µl of Na-methylate in 88 Page 4 of 20 Vol:. (1234567890) methanol (0.5 mol/l; 9 ml 5.56 mol sodium methylate in 1 l methanol -71 FLUKA 71748-+ 10 ml isooctane ad 100 ml with methanol p.a., + 100 µl 1% bromocresol green in methanol) was dispensed into each tube with the seeds and tightly closed with screw cap. The sample was then ground for 1 min using a custom-built Wartburg-Engine vertical shaker at the Institute of Applied Plant Nutrition, Georg-August-University Göttingen, in order to extract the oil. The ground samples were then incubated for about 10 min at room temperature. Afterwards, 300 µl each of 5% NaHSO 4 and iso-octane solutions were dispensed into each tube. The tubes were then vortexed to allow a thorough mixture and extraction of fatty acid methyl esters. The tubes were then centrifuged at 1000 rpm for 10 min. Then, about 200 µl of the clear upper phase was removed using a Hamilton syringe and dispensed into septum vials and tightly closed with crimp caps. The extracted fatty acids were then stored in the refrigerator until the gas chromatograph analyzer was ready. Using an auto-sampler 2 µl of the fatty acid extract was injected into the gas chromatograph (Trace GC ultra, Thermo Electron corporation) equipped with a 25 m × 0.25 mm I.D. FFAP column (Macherey & Nagel, 0.25 µm film thickness, 210 °C, split injection ratio (1:70), Hydrogen carrier gas: 150 kPa, injection/detector: temperature 230 °C). Total analytical time for each sample was 6 min. The fatty acids were expressed as the percentage of the total fatty acid content in the seed oil. The fatty acids that were considered to calculate SFA, total fatty acid content, C16/18 (KASII) and FATB/A ratio are listed in Table 1.
Measurement of oil and ADL content using near infrared reflectance spectroscopy (NIRS) Seed oil content was determined by scanning 2-3 g of seed samples with a NIRS monochromator as described in Behnke et al. (2018). Seed oil content was expressed at 9% moisture content. The acid detergent lignin (ADL) content was determined using the calibration equation developed by Dimov et al. (2012).
Correction for the effect of erucic acid on oil and palmitic acid ASG DH population segregated for erucic acid with 102 and 68 genotypes with high and low erucic acid content (< 2%), respectively. The expression of the erucic acid alleles has pleiotropic effect on contents of oil and palmitic acid. The corrected values of these traits were determined using the regression method described in Behnke et al. (2018). The corrected oil and palmitic acid content was estimated by fitting a linear regression as follows: Oil content corrected for Erucic acid (Oilc) = NIRS oil content − (slope of the linear regression between NIRS oil content × GC erucic acid content) and for the palmitic acid: Palmitic acid corrected for erucic acid (16:0c) = Palmitic acid content − (slope of linear regression between palmitic and erucic acid × GC erucic acid content (Fig.  S1).

Statistical analysis
Analysis of Variance (ANOVA) was performed for the data using PLABSTAT version 3A software (Utz 2011), considering location -year as an environment using the following simple linear model: where Y ij is the trait value of ith genotype in jth environment and µ is the overall mean, g i is the effect of the ith genotype (i = 1,2…), while e j is the effect of j environment and ge ij is the interaction between ith genotype and jth environment and the random error. Both genotype and environment were considered as random factors. Broad sense heritability (h 2 ) was calculated for each trait using Where σ 2 g is the variance component for the genotype and σ 2 ge is the variance component for the genotype × environment interaction and random error, and E is the number of environments. The mean values across the environments were used to calculate the Spearman rank correlation coefficient using R 4.0.3 Package (R Core Team 2018). The normality of the distribution was tested using Shapiro test using the R default script.

DNA extraction and SNP markers
The DNA from all the DH and the parental lines was extracted at TraitGenetics GmbH (https:// trait genet ics. com/) using a proprietary protocol. Genotyping of the DH populations and parental lines were

Linkage map construction
The linkage map was constructed using ASMap package in R (Taylor and Butler 2017) based on the minimum spanning tree (MST) algorithm (Wu et al. 2008). In total 43.66% and 44.61% of the markers were polymorphic in the ASG and AZH population, respectively. Heterozygous genotype calls were manually set to missing values and markers with more than 5% missing calls were deleted. Genotypes were checked for too many double cross overs and markers which were not polymorphic between the two parents were all removed before the map construction. Co-segregating markers and those with strongly distorted segregation were also initially excluded from map construction. After the map was constructed, cosegregated and distorted markers that deviated from 1:1 ratio were pushed back to have a full map. Kosambi distance (1944) was used for the final map construction. where q relates to QTL followed by the trait and then the rapeseed chromosome number (A1-A10 and C1-C9). If more than one QTL was identified on the same chromosome for the same trait, a serial number was suffixed.

Phenotypic analysis
The genotypic and environmental variance components were statistically significant for all traits studied in both DH populations (Tables S5, S6, S7). The heritabilities for the saturated fatty acids in both populations ranged from 33% for behenic (22:0) to 89% for palmitic acid (16:0). Since the ASG population Page 7 of 20 88 Vol.: (0123456789) segregated for one erucic acid gene, the heritability was very high for oleic and erucic acid in ASG population. Despite significant differences for all traits, the means of the two populations were quite similar (Table 1). Palmitic acid was the most prominent saturated fatty acid followed by stearic acid. The distribution of all the saturated fatty acids showed transgressive segregation in both populations and were normally or near normally distributed; both populations showed bimodal distributions for ADL content (Figs. S2, S3). ASG population showed a bimodal distribution for 18:1 (Fig. S2) and for 22:1 content that were consistent with 1:1 segregation hypothesis in chi square tests (data not shown). Close negative correlations between saturated fatty acids, the FATB/A ratio and the 20:1 and 22:1 content were found in the ASG population. In contrast a close positive correlation to oil content were found (Table 2). Since the presence of erucic alleles confounded traits like seed oil and 16:0 content, both traits were corrected for 22:1 value using regression method. Following correction, the value for palmitic acid content was higher and for oil content it was lower (Table 1). Through the correction, the negative correlation between 16:0 and oil content remained negative, although insignificantly. The correlation between 16:0 and 18:1 was negative (− 0.43**) among the 68 erucic acid free genotypes (Table S8). Stearic acid and SFA content were negatively correlated with oil content in the ASG population; this remained significant even after correction. Also, stearic acid and SFA contents were negatively correlated with oil content in AZH population (Table 3). The C16/18 ratio was negatively correlated with oleic acid content in both populations (Tables 2, 3), and which remained negative among the erucic acid free genotypes of the ASG population (Table S8). The C16/18 ratio was not correlated to oil content in both populations. Noteworthy, ADL content was negatively correlated to oil content in ASG and AZH population (Tables 2, 3, S8).

Linkage map
The final map covered 19 chromosomes with a distance of 1683 and 1703.2 cM and with 3763 and 5743 SNP markers for ASG and AZH population, respectively (Tables S1, S2). For QTL mapping, a framework map of 870 and 766 markers was used for ASG and AZH population, respectively (Tables S3, S4). In the ASG genetic map, 51.5% SNP markers showed significant deviation from the 1:1 segregation ratio, of which 42.4% favored Adriana alleles (Table S3). For the AZH population, 35.2% SNP markers deviated from the 1:1 segregation ratio of which 51.5% favored Adriana alleles (Table S4).

QTL analysis of the ASG population
There were total of 48 QTL mapped for different fatty acids, oil and ADL content in this population (Table 4). 7 QTL were identified for 16:0 on 7 chromosomes which cumulatively explained 74.6% of the phenotypic variance (Table 4, Fig. S4). Except for QTL q16:0-C3 and q16:0-C6, all QTL showed negative additive effects with Adriana alleles reducing 16:0 content. The position of major QTL q16:0-C3 co-located with positions of QTL q18:0-C3, q18:1-C3, and q16/18-C3 ratio with positive additive effects. QTL q16:0-C3 also co-located with QTL q22:1-C3, q20:1-C03, q18:2-C3, q18:3-C3-1, and qOil-C3 with opposite additive effects, indicating that q20:1-C03 and q22:1-C3 alleles led to a decrease in palmitic, stearic, oleic acid and derived polyunsaturated fatty acid contents, to a decrease in C16/18 ratio and to an increase in oil content. The erucic acid QTL allele q22:1-C3 of SGEDH13 also led to a decrease the FATB/A ratio. Following correction of the 22:1-effect, QTL q16:0-C3, q18:0-C3 and qOil-C3 became insignificant and the additive effect and the percentage of explained phenotypic variance of the remaining four QTL for 16:0c increased. All four remaining QTL following correction had negative additive effects with Adriana alleles leading to a reduction of 16:0 content (Fig. S4). QTL q16:0-A1 and q16:0c-A1 co-located with QTL q18:2-A1 with the same direction of the additive effect, which agrees with the positive correlation between 18:2 and 16:0. QTL q16:0c-A9 and q16:0c-C9 co-located with QTL q16/18-A9 and q16/18-C9 with the same direction of the additive effect. Following correction of the erucic acid alleles, only two QTL for oil content remained significant. Interestingly, QTL qOil-C5 and qOilc-C5 mapped at the same position as QTL qADL-C5 with opposite additive effects, indicating that a reduction in ADL content by SGEDH13 alleles led to an increase in oil content. QTL qADL-C5 mapped also at the same position as QTL q18:0-C5 with  the same sign of the additive effect, which is in line with the positive correlation between these two traits (Table 2). Noteworthy, QTL qADL-C5 also mapped at the same position as QTL q18:2-C5 and q18:3-C5, but with opposite additive effects. The increase in oil, 18:2 and 18:3 content caused by QTL qADL-C5 is congruent with the negative correlation between these traits. For arachidic acid (20:0) and behenic acid (22:0) content no significant QTL were detected. The QTL qFATB/A-A9 with the Adriana allele causing an increase in the ratio co-located with QTL q16:0-A9 and q16/18-A9 with the same direction of the additive effect.

QTL analysis of the AZH population
There were total of 42 QTL mapped for different fatty acids, oil and ADL content in this population (Table 5). Two QTL for 16:0 mapped on chromosome C08 and C09 which cumulatively explained 26.5% of the phenotypic variance (  Fig. S5). This is in line with the transgressive segregation found in the population (c.f. Table 1). QTL q20:0-C9 co-located with QTL q22:0-C9 on C09. QTL q20:0-A2 and q20:0-C6 mapped at same positions as qFATB/A-A2 and qFATB/A-C6, respectively, with opposite direction of the additive effect. There was epistatic interaction between QTL q20:0-A2 and q20:0-A3 reducing 20:0 content by 0.04%. Three QTL for oleic acid content with positive additive effect collectively accounted for 49.6% of the phenotypic variance. The large QTL q18:1-A1 mapped very close to QTL qOil-A1 for oil content with same direction of the additive effect and with QTL q18:2-A1 and q18:3-A1 with opposite direction of additive effects. This is in line with the positive correlation between 18:1 and oil content and their negative correlation to 18:2 and 18:3 (Table 3). Altogether five QTL with additive and two QTL with epistatic effects for oil content were detected. Among these five QTL, confidence intervals of three QTL on A04, A05 and C05 overlapped with QTL for acid detergent lignin content (ADL), partly with opposite signs of the additive effect. This is in support with the transgressive segregation of ADL content in the DH population and with the negative correlation between ADL and oil content. Remarkably is the co-location of QTL qADL-C5 with QTL qOil-C5, q18:1-C5, and with q18:3-C5 with Zheyou 50 alleles leading to an increase in oil and polyunsaturated fatty acid content and a decrease in 18:1 content.

Discussion
To avoid pollen genotype effects (xenia) on seed quality traits in oilseed rape, one usually avoids cross-pollination by bagging inflorescences at the beginning of flowering. Bagging of inflorescences, however, is changing light quality and intensity as well as the microclimate inside the bags. This directly influences different seed quality traits. In addition, bagged plants are usually more prone to Sclerotinia stem rot disease that leads to premature senescence and seed quality changes. To avoid these kinds of negative effects in this study, open pollinated seeds were harvested from main inflorescences of healthy plants at full maturity. With the exception of the xenia effect of the erucic acid alleles on oil content and fatty acid composition, xenia effects appear to be only of minor relevance.
Since the ASG population segregated for erucic acid content, a regression method was applied to correct for strong effects of the erucic acid allele on oil and palmitic acid content. As the conditional mapping approach developed by Zhu (1995)  Genetically reducing saturated fatty acid content and increasing oil content are continuous aims in oilseed rape breeding. In this study, significant and transgressive genetic variation has been found for most of the traits. Heritability values for saturated fatty acids ranged from 33% for 22:0 to 89% for 16:0 in both populations (Table S5), while for seed oil content it ranged from 74% in AZH to 93% in the ASG population. Low heritability values for 20:0 and 22:0 were caused by values of 1% and less at the detection level (Kaur et al. 2020). High heritability values for main fatty acids, oil and ADL content show that contents are generally more influenced by the genotype than by the GxE interaction (Gazave et al. 2020;Behnke et al. 2018;Teh and Möllers 2016).
The only major QTL for the prevailing saturated palmitic acid content (16:0) in the ASG population was caused by the pleiotropic effect of the erucic acid BnaC05G0480500(FAD2) 52625320 BnaC05G0488300(GPAT5) 53204240

BnaC05G0493500ZS(CESA3) 53573712
Bn-scaff_17441_1-p958418 53643948 BnaC05G0498400ZS(PAL4) 53832968 Bn-A05-p22111286 53843948 BnaC05G0501000ZS(CCR1) 53990712 BnaC05G0503400ZS(KASII) 54090360 Bn-scaff_17441_3-p37334 54136696 Bn-scaff_17441_3-p147254 54263828 Bn-scaff_17441_3-p156691 54273244 Bn-scaff_20219_1-p143569 54721592 Bn-scaff_23186_1-p303208 55119872 Bn-scaff_23186_1-p8774 55425312 Bn-scaff_20270_1-p1440999 55921872 Bn-scaff_20376_1-p168831 56145856 Bn-scaff_20376_1-p361323 56363588 Reducing SFA in oilseed rape would involve lowering levels of palmitic acid (Pandian et al. 2003). The negative correlation between the palmitic acid and oleic acid content for the erucic acid free genotypes of the ASG population (− 0.43**, Table S8) and the AZH population ( − 0.38**, Table 3) shows that the palmitic acid content can be reduced by breeding for higher oleic acid content in the seed oil. QTL underlying this negative correlation in ASG population are located on A09 with candidate gene FATB. The negative sign of the additive effects shows that the Adriana allele led to reduced 16:0 contents and C16/18 ratio (Table 4, Fig. S4). Earlier reports (Zhao et al. 2008; Teh and Möllers 2016) also identified QTL for 16:0 on A09 and suggested FATB gene as the underlying candidate gene. Zhao et al. (2019) mapped a QTL for increased 18:1 content on A09 at a different physical region in ZS11, that also did not affect 16:0 or 18:0 content. In the AZH population there were no such QTL on A09. However, in AZH population on C08 there were QTL for 16:0 and C16/18 ratio with the Adriana allele causing reduced contents and ratio. Again, FATB was identified as possible candidate gene on C08 (Fig. 2). Obviously, there are allelic differences in FATB thioesterases preferentially accepting 16:0-ACP or 18:0-ACP as substrates (Bonaventure et al. 2003). Overexpression of Cupea paucipetala FATB (cpFATB) in oilseed rape was found to increase 16:0 and other saturated fatty acids by four times (Nam et al. 2019), while knocking out FATB alleles by genome editing led to a significant reduction in 16:0 and 18:0 contents in soybean seeds (Ma et al. 2021). In a GWAS study Gacek et al. (2017) identified the FATB gene BnaA05g23790D as one promising candidate gene for oleic acid and linoleic acid content. However, a search for FATB genes in the Darmor-bzh reference genome revealed some confusion regarding the annotation of FATB and FATA genes (Gacek et al. 2017). Studies on expression of Brassica napus FATB and FATA in different tissues and stages of growth will help to understand the relationship between their mutation and phenotypic expression of palmitic and stearic acid contents under natural condition. Negative correlation between palmitic acid and oleic acid content was also reported in other studies in oilseed rape (Chen et al. 2018;Zhao et al. 2019). As prevailing saturated fatty acid, palmitic acid is closely positive correlated with SFA content in the complete ASG population and in the erucic acid free sub-population. There is no such correlation in the AZH population which may be explained by closer correlations between SFA and 18:0, 20:0 and 22:0. Other candidate genes that could be responsible for changes in 16:0, 18:0 and the C16/18 ratio are FATB, KASI, KASII, and KASIII. KASI is responsible for condensation cycles of 4:0-ACP to 16:0-ACP, KASII is involved in the elongation of 16:0-ACP to 18:0-ACP, and KASIII is catalyzing in the initial condensation of acetyl-CoA with malonyl-ACP (Hölzl and Dörmann 2019). No FATA candidate gene was identified in any of the QTL confidence intervals in both populations. FATA copies of Arabidopsis thaliana in rapeseed genome ZS11 are located on A07, A09, C03, C04, C06 and C07 (Song et al. 2020), but none was located within the QTL CI identified on A07 and C06 for FATB/A (Tables 4, 5). In contrast, KASII candidate locus was identified in the QTL region on A02 and KASI and KASIII candidate genes were identified in the QTL region on C09 in ASG population. Since KASIII is involved in the initial condensation reaction (Hölzl and Dörmann 2019), it appears unlikely as candidate gene for 16:0 and C16/18 ratio. In the AZH population only KASI was identified as candidate gene for QTL Arachidic-1 on A02 (Fig. 2).
Furthermore, SFA content can be reduced by selecting for higher oil content, since oil content is positively correlated with oleic acid content in the erucic acid free ASG sub-population (Table S8) and in the AZH population (Table 3). In both populations on A01 a QTL region for oleic acid conspicuously overlapped with a QTL for oil content. In this study, FAD2, LPAAT, DGAT2 and GPAAT5 were identified as candidate genes for the QTL on A01. FAD2 gene encodes for oleoyl-CoA desaturase, GPAAT5, and DGAT2 are involved in TAG synthesis taking place in the endoplasmic reticulum. Teh and Möllers (2016) mapped LPAAT and FAD2 on A01 when they analyzed Sansibar × Oase DH population. If TAG synthesis is efficient, oleic acid will be assembled more in TAG and will be less desaturated to 18:2 and 18:3, which finally leads to increased oleic acid content in the seed oil content (Möllers and Schierholt 2002;Zhao et al. 2012). No correlation between 16:0 and oil content have also been reported by Teh and Möllers (2016), whereas Möllers and Schierholt (2002) found a negative correlation in a DH population segregating for a major QTL affecting 18:1 content. Negative correlations between 16:0 and oil content were also found in two different sets of European winter oilseed rape cultivars (Dimov and Möllers 2010). Loose negative correlations between oil content and 16:0c in ASG, 16:0 in AZH population and 18:0 was not reflected by co-locating QTL in both populations. The close correlation between C16/18 ratio and 16:0c in ASG population and 16:0 in AZH population suggests little variation in the KASII activity, and hence flux from palmitic acid to stearic acid, which is evident by the lack of correlation between C16/18 ratio and oil content.
Noteworthy, 16:0, the C16/18 ratio and oil content in both populations were negatively correlated with ADL content. ADL content was positively correlated with 18:0 content; and 18:0 content was negatively correlated with oil content, because it was mainly desaturated to 18:1, which was positively correlated with oil content. Furthermore, reduced ADL content was accompanied by reduced oleic acid content and increased contents of polyunsaturated fatty acids in the AZH (Table 3) and in the erucic acid free ASG subpopulation (Table S8). This finding is opposite to the observed positive correlation between 18:1 and oil content (Möllers and Schierholt 2002). There are a number of candidate genes located within the various overlapping QTL confidence intervals on C05 in both populations. The QTL qADL-C5 for lignin content in SGEDH13 is derived from the ancestor population SGDH14 × Express 617, in which the QTL was mapped exactly at the same position on C05 (Behnke et al. 2018). Surprisingly, Zheyou 50 carries the same QTL at C05. This may be explained by the origin of both SGDH14 and Zheyou 50 from the Zhejiang Academy of Agricultural Sciences (Hangzhou, China). Since Zheyou 50 is of canola quality, it is possible that Zheyou 50 may be derived from one of the DH lines of SGDH14 × Express 617 (Behnke et al. 2018). The low ADL content of DH lines with the SGEDH13 or Zheyou 50 QTL allele at C05 can be caused by allelic differences in the PAL4, CESA3 and CRR1 candidate genes. Synthesis of cellulose is mediated by the cellulose synthase (CESA) gene family, of which CESA3 is one. Both, the Phenylalanine-Ammonium Lyase (PAL) and Cinnamoyl-CoA-Reductase (CRR) are involved in the Phenylpropanoid biosynthesis pathway (Liu et al. 2012). Hence, all three genes are candidates for reduced lignin content. Furthermore, a QTL for reduced seed hull proportion was mapped at C05 at the same position in the DH population SGDH14 × Express617 (Behnke et al. 2018). It may be that a reduced lignin content or reduced seed hull proportion has led to an increased oil content. On the other hand, it is possible that the KASII gene expression caused increased 18:0, decreased 16:0 and C16/18 ratio and higher oil content. Own, unpublished results (manuscript in preparation), has shown that in SGDH14 and SGEDH13 a homoeologous non-reciprocal translocation has occurred by which a 200 kb large fragment carrying the PAL4 gene from A05 has been transferred to C05, replacing the original C05 fragment. Gene conversion or homoeologous nonreciprocal translocations (HNRT) in rapeseed have been described by Stein et al. (2017). Again, the GPAT5 homolog (BnaC05G0482300) may be underlying the QTL for oil content. GPAT is the first transferase involved in the acylation of glycerol-3-phosphate to lysophosphatidic acid in the oil synthesis at the endoplasmic reticulum. In the AZH population there are in addition co-locating QTL for reduced ADL and increased oil content on A04 with a PAL2 candidate gene (Figs. 2,  S5).
The genetic combination of QTL alleles for reduced SFA content could result in further reduced saturated fatty acid contents in canola oil. Additional genetic variation for reduced SFA content may be identified in canola genetic resources which includes oilseed rape resynthesized from its diploid progenitors Brassica rapa and Brassica oleracea (Jesske et al. 2013). In this context, the correction of the Page 17 of 20 88 Vol.: (0123456789) erucic acid allele xenia effects on oil and saturated fatty acid content by regression is valuable in determining the corrected saturated fatty acid content. Homoeologous nonreciprocal translocations in resynthesized rapeseed (Stein et al. 2017) may lead to a replacement of a FATB by a FATA and as a consequence to a decreased 16:0 and increased 18:1 content and probably oil content (Rodríguez-Rodríguez et al. 2014). Recently, Cargill has released specialty low saturate canola oil with 3.5-5% total saturated fatty acid content (Patent US20190174788). In conclusion, the present work has elaborated the interactions between different fatty acids and oil content in rapeseed and has identified QTL alleles at different loci that can be applied to develop new canola breeding material with reduced saturated fatty acid content.