Increase Of Pod Shattering Resistance In Brassica Napus L.by Using CRISPR/Cas9 System

Pod shattering prior to and during harvest is extremely detrimental to oilseed rape.It is associated with the degradation of cells within the dehiscence zone.Several genes such as JAGGED,SHATERPROOF,INDEHISCENT and ALCATRAZ were revealed to be related to the cellular growth and cell identity in the dehiscence zone of Brassica napus pods.To develop the understanding and potential variations in plant material for pod shattering resistance;Bn JAG,Bn SHP,Bn IND and Bn ALC genes were targeted by CRISPR/Cas9 system.Knockout mutagenesis in all homoeologs of Bn JAG gene drastically affect the development of the lateral organs in organizing pod shape and size.A cylindrical body of the pod was generated,comprised of an undifferentiated bunch of cells “like a callus” without distinctive valves,replum,septum,and valve margins.Pseudo-seeds were produced in pods;divided into two halves with an incomplete layer of cells,probably septum that separates the undifferentiated cells.However,one mutant line was identified in which only Bn JAG.A08-NUB-Like paralogous of Bn JAG gene was mutated.The replum region of the mutant was increased to a great extent with enlarged cell size,bumpy fruit with reduced pod length than WT.To exploit the function of Bn SHP genes in pod shattering resistance,Bn SHP1A09 mutated line(Bn SHP1-157)was employed to cross another mutant with four mutated homoeologs(Bn SHP4-484)to create a five homoeologs(Bn SHP5-184)mutated lines.Phenotypic investigation results suggested that five homoeologs mutated line(Bn SHP5-184)exhibit reduced lignified layer and separation layer adjacent to valves and replum,respectively.The SRI was increased to 0.31 in these mutated lines compared with WT(SRI = 0.036).In addition,knocking out of Bn SHP1A09 homoeolog,significantly increase the pod SRI ranged from 0.13 to 0.17 than WT(0.033).Bn SHP1A09 is probably a promising homoeolog for controlling the lignin content at dehiscence zone than all other Bn SHP homoeologs.Loss of Bn IND.A03 and Bn IND.C03 activity has a more significant impact on the development of valve margins,which were absent in Bn IND double mutant line and exhibited higher shattering resistance(SRI = 0.28)than WT(SRI = 0.036).Among the segregating population,we identified that Bn IND.A03 proved to be as promising homoeolog to induce cell differentiation than the Bn IND.C03 homoeolog.Loss of Bn ALC gene appears to have more limited role in valve margin development than other related genes in pod shattering mechanism(Bn JAG,Bn SHP and Bn IND).As mutations in Bn ALC affect the differentiation of separation layer partially but not the lignified layer.Mutagenesis in Bn ALC loci exhibited partial but positive impact on pod shattering(SRI=0.12)than WT pods.In conclusion,pod shattering resistance can be increased to minimize the yield losses by mutating few genes and their homoeologs.Nevertheless,total encapsulation of the seeds in fruit is not required by mutating all homoeologs since significant losses would be incurred while endeavoring to retrieve the seeds.These results establish a framework for the regulation of pod shattering resistance that can be applied to diverse Brassica species.