Harloff, Hans-Joachim

Jung, Christian

Sashidhar, Niharika

English

JKI Open Journal Systems (Julius Kühn-Institut)

11th Young Scientists Meeting 2018, Braunschweig, Germany, November 14-16 Sashidhar et al. High throughput reverse genetic tools for knocking out several genes of the phytic acid pathway in Brassica napus  Niharika Sashidhar, Hans-Joachim Harloff and Christian Jung Christian-Albrechts-University Kiel, Institute of Crop Science and Plant Breeding, Kiel E-mail of corresponding author: c.jung@plantbreeding.uni-kiel.deBrassica napus L. (oilseed rape) is an im-portant oil crop in the temperate regions. After oil extraction, the seeds are used as a feed for livestock due to their rich pro-tein and balanced amino acid content. However, the meal is not utilized effi-ciently because of the presence of high quantities of anti-nutritive substances. One of those is phytic acid, accounting for 2-4 % in oilseed rape cultivars. Phytic acidis an important source of inorganic phos-phate for plants and is involved in manybiological functions. It is still not knownto what extent it is required to maintainthe basic physiological functions. Identifi-cation of low phytic acid mutants maynot only provide valuable information forunderstanding the biological function ofthe genes involved in the phytic acidpathway, but also result in improvedseed quality. However, mutational anal-yses in oil seed rape is challenging due topolyploidization. Since gene functions are often encoded by several paralogs, more than one gene has to be knocked out to study the underlying effect. In this project, we adopted two different strate-gies for the mutational analysis. One ap-proach is TILLING by sequencing and the other is genome engineering by using Streptococcus pyogenes Cas9 endonucle-ase. We chose most of the functional pa-ralogs of seven crucial gene families (ITPK, MIPS, MIK, IMPK, PGK2, MRP5, IPK1) of the pathway. We were able to es-tablish a high throughput mutant screen-ing by sequencing, which resulted in an average mutation density of 1/18 kb in all the targeted genes. Furthermore, by tar-geting the conserved regions of two sub-families of the ITPK gene family we ob-tained gene editing in the spring rape-seed cultivar Haydn by using hypocotyl transformation.  Berichte aus dem Julius Kühn-Institut 200 47

High throughput reverse genetic tools for knocking out several genes of the phytic acid pathway in Brassica napus

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High throughput reverse genetic tools for knocking out several genes of the phytic acid pathway in Brassica napus

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