Identification and characterization of phytic acid synthesis mutations in oilseed rape
Final Report Abstract
This study aimed at establishing advanced reverse genetic tools like TILLING by sequencing and CRISPR-Cas9 for identifcation of loss of function mutations in phytic acid pathway in oilseed rape. We could successfully accomplish both the strategies and found knockout mutants in all the eight key biosynthetic and transporter genes of the pathway. Upon identification of the mutations, double and triple homozygous mutants were produced for phenotypic anaylsis. We found that mutants in Bn.2-PGK2, BnITPK, BnMRP5 led to 27%, 30% and ~13% reduction of PA contents, respectively. We could finally reach our main aim by finding low phytic acid mutants in oilseed rape which could be utilized by the breeders for making human grade canola protein sources. This study also adds to our understanding of Cas9-induced mutation in plants. We found loss of mutated alleles from T1 to T2 generation and de novo mutations in T2 and subsequent generations. We propose to use only homozygous mutant lines in the future to avoid misscoring of heterozygous offspring. This study is unique in a way as it displays random (EMS-induced) and targeted (CRISPR-Cas induced) mutations side by side. It clearly demonstrates that CRISPR-Cas induced mutation are superior becasue several allels can be mutagenized at a time thus avoiding trime consuming crossing experiments. Moreover, EMS mutants are suffering from 10,000s of background mutations which significantly reduce plant vigor whereas genome editied plants are devoid of any negative side effects.
Publications
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(2017). Recent developments in genome editing and applications in plant breeding. Plant Breeding, 00:1-9
Jung C, Capistrano-Gossmann G, Braatz J, Sashidhar N, Melzer S
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(2017): CRISPR/Cas9 targeted mutagenesis in phytic acid synthesis genes of Brassica napus PP1530 International Workshop: ‘Genome Engineering’, Gatersleben, 22. - 23.06.2017
Harloff, H., Sashidhar, N., Jung, C.
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(2017): Systematic knock-out study of phytic acid synthesis genes in Brassica napus, Botanikertagung 2017, Kiel,17. - 21.09.2017
Harloff, H., Sashidhar, N., Blanc, Noelle, Jung, C.
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(2018): Knock-out study of phytic acid synthesis genes in Brassica napus, German Plant Breeding conference-Wernigerode, 28.02.2018
Harloff.H., Sashidhar, N., Jung,C.
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(2019). High throughput reverse genetic tools for knocking out several genes of the phytic acid pathway in Brassica napus, International Plant & Animal Genome XXVII, San Diego, 12.-16.01.2019
Sashidhar, Harloff, H.-J., Jung, C.
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(2019). Identification of phytic acid mutants in oilseed rape (Brassica napus L.) by large scale screening of mutant populations through amplicon sequencing. New Phytologist
Sashidhar N, Harloff H, Jung C
