Genetic and physical mapping

In this study, QTL analysis identified four main QTL for seed oil content on linkage groups A08, C03, C05 and C07. This number of QTL is comparable to the three distinct QTL detected by Ecke et al. (1995), but generally lower compared to other reports in which QTL for oil content were mapped (see section 2.6). With 31.2 and 50.2% of the phenotypic variance, QTL E_Oil-1 and E_Oil-2 on linkage groups A08 and C03, respectively, were identified as the major QTL for seed oil content in the SGEDH population. The major QTL on A08 overlapped with a major QTL for erucic acid (E_GC22:1-2). While the major QTL on C03 was co-located with one of two QTL for eicosenoic acid (E_GC20:1-2) which explained the highest phenotypic variation of this trait. In agreement with this finding the segregation of erucic acid and its intermediate eicosenoic acid in the SGEDH population shows high eicosenoic acid contents for genotypes with medium erucic acid contents carrying one positive erucic acid gene (Figure 3.9). Ranging from 16.1 to 34.8% the variation of the group of genotypes with medium erucic acid contents is relatively large. In this group genotypes are expected to either carry the positive allele for erucic acid content on A08 or C03, which suggests that this group can be divided into two subgroups. In accordance the marker genotype segregation of the DH lines within this group identified two main groups. One group carried the positive allele for erucic acid content on C03 and a negative allele on A08 (Figure 3.9; red dots), while the other group carried the positive allele for erucic acid content on A08 and a negative allele on C03 (Figure 3.9; grey dots). Furthermore, the presence of the major QTL for eicosenoic acid on C03 assumed that the positive erucic acid allele of this locus has a higher affinity to oleoyl-CoA as substrate, producing predominantly eicosenoic acid. Thus, genotypes carrying only the positive allele of C03 showed relatively low erucic acid contents.

While the allele of A08 seemed to be less selective for its substrates oleoyl-CoA and eicosenoyl-CoA, which results in high eicosenoic acid and higher erucic acid contents for genotypes carrying only the positive allele on A08.

Figure 3.9: xy plot of erucic acid and eicosenoic acid content determined by gas chromatography;

dots represent groups of marker genotypes eAeAeCeC (circles), eAeAECEC (red dots), EAEAeCeC (grey dots) and EAEAECEC (black dots); e represents a negative allele for erucic acid, E represents a positive allele for erucic acid content; A indicates the erucic acid locus of the A genome of B. napus on A08, C indicates the erucic acid locus of the C genome on C03

In accordance with these results, two of the oil-QTL identified by Ecke et al. (1995) showed a strong association to estimated positions of the two erucic acid genes and other subsequent studies, not only identified two major QTL for erucic acid, but also located these QTL on linkage groups N8 (A08) and N13 (C03) (Howell et al. 1996, Thormann et al. 1996, Qiu et al. 2006). For example, Qiu et al. (2006) detected two major QTL for erucic acid co-located with QTL for oil content on N8 and N13, investigating a DH population from a cross between a double low and

‘++’-quality parent independently at four locations. While Radoev (2007), investigating a DH population segregating for only one erucic acid gene, detected major QTL for oil content on N8 and N13, and found one major QTL for erucic acid on linkage group N8 co-located with the oil-QTL on N8. Furthermore, Teh and Möllers (2015) detected a minor oil-QTL for oil content on C03, analysing a DH population of two double low quality cultivars. The SNP marker closest to the peak of this QTL (Bn-scaff_18936_1-p277517) was also found in the SGEDH population, but at the position 12.8cM, while the oil-QTL in the SGEDH population was located at 190.4cM, indicating that the oil-QTL identified in both populations represent different loci. The regulation of erucic acid content in B. napus by the additive action of two genes was already reported previously (Harvey and Downey 1964, Stefansson and Hougen 1964, Siebel and Pauls 1989), and matches the established biosynthesis path, in which oleic acid is first elongated to eicosenoic acid, which in a second step is elongated to erucic acid, by subsequent addition of C2 units. Both elongation cycles are catalysed by the cytoplasmic/ER-bound oleoyl-CoA elongation complex,

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comprising a four enzyme system analogous to the fatty acid synthase complex of the plastid (section 2.4). The four enzymes include the ketoacyl-CoA synthase (KCS) FAE1 which catalyses the rate-limiting step of oleic acid elongation to erucic acid (Millar and Kunst 1997, Fourmann et al. 1998). A comparison of the physical position of the FAE1 candidate genes and confidence intervals of QTL for oil content and fatty acids showed that FAE1 was co-located not only with major QTL for erucic acid on linkage groups A08 and C03 (E_GC22:1-2 and E_GC22:1-3), but also with the major oil-QTL on A08, E_Oil-1. On linkage group C03 the confidence interval of the major QTL E_Oil-2 did not overlap with the confidence interval of E_ GC22:1-3 (peak to peak distance of 10.8cM), but with that of E_GC20:1-2, the major QTL for eicosenoic acid, the elongation intermediate, which might be explained by a higher affinity to oleoyl-CoA of the FAE1 on C03 as discussed before (cf. Figure 3.9). However, these findings suggested, that the addition of carbon units to oleic acid to produce erucic acid, and the consequent increase of molecular mass of the fatty acid chain, might explain the association found for seed oil content and erucic acid content. Besides, identifying FAE1 as the most likely candidate gene, since the number of stored fatty acid chains remains unaffected but the increase in molecular mass is causing an increase of oil content (Ecke et al. 1995). Although this effect is most probably caused by a pleiotropic effect of FAE1, a close linkage between the erucic acid genes and other independent QTL for seed oil content cannot be excluded (Ecke et al. 1995).

Referring to Price (2006) there is evidence that the position of a QTL obtained from a primary mapping population can identify a candidate gene with less than 1cM around the QTL peak for a major QTL, which explains more than 25% of the trait’s variation. Since none of the selected lipid related candidate genes (Appendix 4) were identified for the major oil-QTL E_Oil-2 on linkage groups C03 (cf. Table 3.9), a 1cM region around the E_Oil-2 peak (190.4cM) was scanned for A.

thaliana protein matches on the B. napus Darmor-bzh reference genome (Chalhoub et al. 2014, http://www.genoscope.cns.fr/brassicanapus/). Using the genetic and physical marker information of Figure 3.7, 1cM in proximity of E_Oil-2 equals about 130kbp. As closest marker to the QTL-peak, br-Pb839425 (192.3cM/54303404bp) was used to estimate the +/-1cM-region of E_Oil-2. The scanned region reached from 53910 to 54170kbp on the B. napus genome. In this region 27 A. thaliana protein matches were found (Appendix 5), but none of the proteins was related to lipid metabolism according to the A. thaliana lipid metabolism database (Beisson et al.

2003, http://aralip.plantbiology.msu.edu/locations).

QTL E_Oil-3 on C05 indicating SGDH14 as source of the increasing allele, and E_Oil-4 on C07 indicating Express617 as source of the increasing allele, showed only minor effects and the comparison to physical positions of putative candidate genes to their confidence intervals

remained without results. However, Zhao et al. (2005) identified QTL for oil content with additive x environment interaction effects at a German location on equal linkage groups in the Sollux x Gaoyou DH population, both indicating the Chinese parent Gaoyou as the source of the increasing allele. Delourme et al. (2006) also detected oil-QTL on both linkage groups from field trials conducted in France. While Qiu et al. (2006) in contrast detected a QTL for oil content on N17 (C07), but no QTL on N15 (C05). Referring to Price (2006) also for minor QTL an accuracy of less than 3cM around the QTL peak was detected to identify putative candidate genes. If possible, accuracy to identify a candidate gene for minor QTL was even improved by averaging peak positions from different screens or by combining multiple data sets to increase the heritability of the trait of interest (Price 2006). Hence, a scan with a wider scan region might also be used to identify putative candidate genes for the minor QTL E_Oil-3 and E_Oil-4.

The three independent correction approaches applied to eliminate the effect of erucic acid from oil content revealed a consistent major QTL for oil content, represented by E_Oil-reg_corr-3, E_Oil-mol_corr-5 and E_Oil-cond-3, which was located on C05 at 39.7cM (cf. Table 3.9;

confidence interval 35.7 – 43.7), about 30cM apart from E_Oil-3. Comparison of the physical positions of lipid related candidate genes with the confidence interval of this QTL for oil content identified no co-localisation. Therefore, as described for E_Oil-2, a scan for A. thaliana protein matches on the B. napus Darmor-bzh reference genome (Chalhoub et al. 2014, http://www.genoscope.cns.fr/brassicanapus/) was conducted around the peak of the major QTL for corrected oil contents on C05. The closest marker to the QTL peak (39.7cM) was Bn-A05-p22111286 at 39.9cM with physical position at 39985396bp on C05. 1cM in the confidence interval of the QTL for corrected oil contents equalled around 82kbp, therefore a region between 39.9Mbp to 40.1Mbp on C05 was scanned for suitable A. thaliana protein matches, resulting in more than 40 matches. Closest to the position of Bn-A05-p22111286 two protein matches were identified, AT3G10320.1 a Glycosyltransferase family 61 protein (MUCILAGE-RELATED21) ranging from 39982337 to 39983989bp, and AT3G10310.1 a P-loop nucleoside triphosphate hydrolases superfamily protein with CH (Calponin Homology) domain ranging from 39984486 to 39988842bp. For both proteins an overlapping B. napus gene prediction was identified, BnaC05g42790D alias GSBRNA2T00110924001 and BnaC05g42800D alias GSBRNA2T00110923001, respectively. While AT3G10320.1 is involved in the mucilage biosynthetic process in seed coat development of A. thaliana, AT3G10310 is involved in microtubule-based movement. None of these genes seemed to be directly related to seed oil biosynthesis. Both genes were also not listed in the Arabidopsis acyl-lipid metabolism database (Beisson et al. 2003). More distant to the QTL peak of the major QTL for corrected oil contents, two protein matches were found with entry in the Arabidopsis acyl-lipid metabolism database

(Beisson et al. 2003), AT3G10370.1 the FAD-dependent oxidoreductase family protein (SDP6) ranging from 39950760 to 39953567bp, and AT3G14270.1 a phosphatidylinositol-4-phosphate 5-kinase family protein (FAB1B) ranging from 39908061 to 39910664bp. For both proteins an overlapping B. napus gene prediction was identified, BnaC05g42730D alias GSBRNA2T00110930001 and BnaC05g42630D alias GSBRNA2T00110943001, respectively.

AT3G10370.1 is involved in the mitochondrial phospholipid synthesis and AT3G14270.1 in phospholipid signalling. Thus, a direct relationship to seed oil biosynthesis could not be observed. Regarding position and function no other suitable protein matches were identified.

In total this study identified eight QTL for oil content in the SGEDH population using original and corrected oil contents. This number in general is compared to the number of oil-QTL of previous studies (Zhao et al. 2005, Qiu et al. 2006, Zhao et al. 2012, Teh and Möllers 2015), identifying six to nine QTL. Comparison of QTL for oil content in the SGEDH population to QTL results from other populations indeed identified QTL on same linkage groups, but due to lack of common markers between populations or the use of none sequence-informative markers in previous studies, in most cases it is still unclear if QTL on same linkage groups are identical or not.

Physical mapping in general showed a good correspondence of genetic and physical positions.

Nevertheless, a number of markers showed an incorrect genetic order according to their physical position. This lack of consensus might be one reason for relatively large confidence intervals of some QTL or might even cause errors in QTL detection. Thus, it is recommended to reorder or delete markers with extremely deviating order in the genetic and physical map.

Likewise, sequence informative markers without or with no suitable physical position were detected. To improve the information content of the SGEDH map of individual marker positions and to improve QTL mapping and the search for candidate genes, these markers should also be replaced if a co-segregating marker is available with matching sequence information regarding the physical position. On the other hand using the B. napus reference genome to physically map marker positions as well as candidate genes, it needs to be considered that the reference genome order is not yet fixed, especially the position and orientation of scaffolds only represents a most likely order/anchoring due to the high complexity of the allotetraploid B. napus genome, including multiple homoeologous gene copies, chromosomal rearrangements and amplification of repetitive DNA (Edwards et al. 2012).

To test if the putative candidate genes identified are responsible for changes in oil content in a next step expression analyses could be used to investigate transcriptional differences of the parental lines and other genotypes, or the allelic diversity could be tested by sequencing.

Furthermore, to demonstrate a functional relationship between a candidate gene and its

corresponding QTL a knock-down or knock-out mutants could be produced. Alternatively, a transgenic approach to overexpress a candidate gene or an antisense RNA approach to inhibit or silence a candidate gene post-transcriptionally could shed light on the role of this candidate gene to influence oil content.