Functional analysis and mutagenesis of GDSL lipase genes for breeding oilseed rape (Brassica napus) with higher oil content

Rapeseed is the third-largest oil crop grown worldwide. Seed oil accumulation is a complex process, which is controlled by many genes. Final oil content in seeds is determined by the balance between both anabolic and catabolic pathways. Therefore, studying the catabolic pathway is important as many researchers until today mainly focused on the synthesis pathway to increase seed oil content (SOC). In this study, SEED FATTY ACID REDUCER (SFAR) genes known to be involved in seed oil degradation in Arabidopsis were knocked out by two different approaches, CRISPR-Cas9 targeted mutagenesis and EMS random mutagenesis. I identified 12 homologous genes for the five known Arabidopsis SFAR genes, AtSFAR1-AtSFAR5. Each gene family had two paralogs in rapeseed except BnSFAR4, which had four paralogs. The BnSFAR4 gene family was subdivided into subfamily a and b, based on their sequence similarity. Their expression was studied during seed development in Express-617 at 15, 25, 35, 45 and 55 days after pollination. Based on relative expression data, I selected BnSFAR1, BnSFAR4, and BnSFAR5 copies for functional analysis. I screened an EMS mutagenized winter rapeseed population using a TILLING platform to identify mutations in BnSFAR1 and BnSFAR4 paralogs. In total, 163 mutations were found in six paralogs comprising 7 nonsense, 103 missense, 46 silent, 6 splice site, and 1 UTR mutations. I found nonsense mutations in all genes except for Bna.SFAR1.Ann. I considered a splice site and one missense mutation in a functional amino acid for the Bna.SFAR1.Ann paralog, and nonsense mutations for all other five paralogs for further analysis. Crosses and backcrosses were performed to reduce background mutation load and to produce double mutants. By CRISPR-Cas9, I knocked out all paralogs in BnSFAR4 and BnSFAR5 simultaneously using specific target regions. After Agrobacterium-mediated hypocotyl transformation of winter-type RS306, I obtained two, five and two transgenic T1 plants for BnSFAR1, BnSFAR4 and BnSFAR5, respectively, representing 0.2 - 1.1% transformation efficiency. I selected two T1 plants from BnSFAR4 and the single BnSFAR5 for further studies. I found complete gene editing on BnSFAR4 T1 plants resulting in quadruple mutants. BnSFAR5 T1 was chimeric, but the mutations were fixed in T2. T2 and T3 generations were used for phenotypic analysis. Six segregating F2 populations derived from crosses either between M3 single mutant plants or M3 single mutant plants backcrossed with Express-617 were analyzed for SOC. In both crossing populations, SOC was increased in BnSFAR4.a double mutants by 12.1% and 8.9% and in BnSFAR4.b double mutants by 10.3 % and 8.7% when compared to wild-type plants segregating within the progenies. However, SOC was not significantly different between BnSFAR1 double mutants and wild-type plants in both populations. Moreover, single mutants did not show a significant increase in SOC either for BnSFAR1, BnSFAR4.a, and BnSFAR4.b. In T2 and T3 BnSFAR4 and BnSFAR5 CRISPR-Cas9 mutants, SOC was increased significantly by 14.5% and 12.9% for BnSFAR4 and 10.4% and 11.2% for BnSFAR5, as compared to the wild-type. Analysis of fatty acid composition in BnSFAR4 T2 showed a significant reduction of erucic acid (C22:1) content and a non-significant increase of C18:1 and C18:3. Oil body size in CRISPR-Cas9 T2 mutants was significantly larger than in wild-type RS306. Seed oil accumulation in CRISPR-Cas9 T3 mutants reached a maximum at 45 DAP in both mutant and wild-type of 40.3% and 37.8%, respectively, but declined more in wild-type (33.7%) as compared to mutants (38.2%) during the seed maturity. In germinating seeds, oil was mobilized with a higher rate in RS306 than in BnSFAR4 and BnSFAR5 T4 mutants. Seed germination rate and seed vigor were not significantly affected T2 BnSFAR4 CRISPR-Cas9 mutants and revealed no pleiotropic effects. Background mutation load was reduced by backcrossing EMS single mutants with fast flowering spring rapeseed variety ‘Peace’, and the reduction was monitored using AFLP markers and a 15K Illumina InfiniumTM SNP array. I could select backcrossed plants (BC1) with 84% share of Peace genomic background. Single mutants selected from SNPs array should be combined to produce double mutants for further studies. In summary, I can conclude that loss of function in BnSFAR4 and BnSFAR5 increases SOC in rapeseed. BnSFAR4 loss of function seems to have no adverse effect on seed germination and vigor. It is vital to combine mutations within BnSFAR gene families. Otherwise, a single gene knock-out can be restored from its functional homoeolog. Backcrossing of EMS mutated winter rapeseed with a spring variety can be applied to reduce undesirable mutations rapidly using marker-assisted genomic background selection.


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