Random and targeted CRISPR-Cas mutagenesis to create oilseed rape with reduced seed phytic acid content

Brassica napus L. is an important oil crop in the temperate regions where it is grown for oil production and feed for animals. Breeders aim to reduce the anti-nutritive compounds in rapeseed meal, which is causing serious problems in monogastric livestock. Since the natural variation of phytic acid (PA) contents in oilseed rape is low, the aim of this study was to reduce PA by a mutational approach. Using gene annotations in Arabidopsis, I identified seven genes for key enzymes and one transporter gene. The genes in oilseed rape were named as BnMIPS, BnMIK, BnIMP, Bn2-PGK, BnITPK, BnIPK2ß, BnIPK1 and BnMRP5. Since many PA synthesis intermediates are also substrates for other important metabolic processes, a careful selection of genes was essential in order not to disturb physiological processes. Furthermore, inositol monophosphate is involved in different signaling processes and the basis for purine metabolism and therefore I have not analyzed BnIMP in this study. To identify the total number of genes encoding the seven key enzymes, I performed in silico analysis and found in total 59 genes in oilseed rape that corresponded to eight Arabidopsis genes. To narrow down to the candidate genes for knockout approaches, mRNA expression analysis was performed in developing seed stages. For mutational approach gene editing by CRISPR-Cas9 and TILLING by sequencing (TbyS) in an EMS mutagenized rapeseed population were used. Paralog specific primers for all the genes were developed, which became later also very useful in distinguishing the paralog specific mutations. Finally, twelve highly expressed paralogs were selected for TbyS approach and more than 90% of the coding regions were analyzed to obtain putative knockout mutants. I could identify 1437 putative EMS mutations, out of which 49 were stop codon mutations that resulted in loss of function of the encoded proteins. All identified stop codon mutations along with splice site, missense mutations were confirmed by Sanger sequencing. Since, the BnITPK gene family comprised 14 paralogs, I have chosen these genes for the CRISPR-Cas9 approach and found knockout mutants in three paralogs of BnITPK1 and BnITPK4. I could also show the inheritance of edited alleles from T1 to T4 generation, which was a valuable information for selecting the additional mutant alleles in later generations. Single EMS mutants were combined to produce double mutants of the corresponding gene families. Upon phenotyping, single mutants did not show reduced PA contents supporting the idea of gene redundancy in polyploids. However, in double mutants of bn2-pgk2 I observed a PA reduction of 25.4 %. The reduction of bnitpk was even higher and showed 27.2% - 35.3% less PA in two T4 plants having different mutants alleles. Finally, in double mutants of the PA transporter BnMRP5 15% less PA was observed. EMS knockout mutations in other genes need to be further investigated. In addition to PA reduction, bnitpk mutants also showed an increased Pi contents, which might be an added value for its use as animal feed. Finally, with this study promising candidates for breeding of low PA in oilseed rape could be identified, which will be beneficial for fish and poultry feeding as well as for human diets and could also serve as a substitute for soybean proteins in future.

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