Using CRISPR/Cas Technology Editing Maize D8 Gene To Generate Dwarf Germplasm

Maize is the most widely crop in the world.The annual planting area is more than 2 billion mu and the total output has reached 1 billion tons.Currently,the improvement of maize yield is mainly through increasing plant density.The plant will display shadeavoidance syndrome(SAS)under high density conditions,a typical phenotype of SAS is the increased plant height.The excessively high plant height of maize will to cause maize lodging,which results in the loss of yield and increasing the difficulty of machine harvest.Therefore,moderately reducing plant height is an important goal of maize breeding to reduce SAS,and increase the lodging resistance of maize.Gibberellin is one of the phytohormones that promote plant growth,and the DELLA protein is a negative regulator in GA signal transduction pathway.In this study,the dwarf8(D8)gene encoding the DELLA protein in maize was edited using CRISPR/Cas technology in the maize inbred lines Zheng58,Q319 and XCW175 to increase the D8 protein stability and decrease plant height.The main results are as follows:1.Taking 6 maize inbred lines in the laboratory as the object,the +1-+500 bp segment of D8 gene was sequenced.The sequencing results showed that the DELLA domain is relatively conserved in the 6 inbred lines.2.Using target Design website,5 sgRNAs were designed in the +1-+500 bp segment of D8 gene,and sgRNA2-sgRNA4 were respectively combined with sgRNA1 to construct 4 double-sgRNA gene editing vectors.3.The T-DNAs were inserted into the maize genome using Agrobacteriummediated transformation of maize immature embryos under Zheng58,Q319 and XCW175 background.287 positive plants were obtained from Zheng58 and 95 plants among them were edited,the editing efficiency reached 33.10%(95/287).The genome change with 3n bp was generated in 27 plants and the mutation efficiency was 9.41%(27/287).12 positive plants were obtained from the inbred line Q319 and 4 plants among them were edited,the editing efficiency was 33.33%(4/12).The genome change with 3n bp was generated in 1 plant and the mutation efficiency was 8.33%(1/12).89 positive plants were obtained from Q319 and 32 plants among them were edited,the editing efficiency reached 35.96%(32/89).The genome change with 3n bp was generated in 6 plants and the mutation efficiency was 6.74%(6/89).4.We obtained 16 T-DNA free mutation lines with 3n bp changes in T1 generation.The genotypes were as follows: the 3 bp,6 bp,12 bp,24 bp deletions at sgRNA2 target site and a 38 bp deletions;51 bp deletion at the target site of sgRNA3;21 bp and 27 bp deletions at sgRNA4 target site;3 bp,15 bp,27 bp and 60 bp deletions at sgRNA5 target site;15 bp deletions at sgRNA1 target site combined with 3bp deletions at sgRNA2 target site;and 120 bp deletions between sgRNA1 and sgRNA3 and 240 bp deletions between sgRNA1 and sgRNA5 target sites.5.The plant height of mutations with 3 bp and 6 bp deletions at the sgRNA2 target site and 3 bp deletion at the sgRNA5 target site did not change significantly compared with the wild type.The plant height of mutation with 27 bp deletion at target site of sgRNA4 increased about 6% compared with wild type.The plant height of mutations with 15 bp,24 bp and 27 bp deletions at the sgRNA5 target site increased about 8%,12%and 5% compared with wild type.The plant height of mutation with 24 bp deletion at the sgRNA2 target site decreased about 15% compared with wild type.The plant height of mutation with 21 bp deletion at the sgRNA4 target site and 12 bp insertion while 27 bp deletion at the sgRNA5 target site decreased about 48% and 47%.The plant height of mutation with 38 bp deletion at the sgRNA2 target site decreased about 56%compared with wild type.The plant height of mutations with 120 bp and 240 bp deletions decreased about 60% and 55% compared with wild type.The plant height of mutation with 53 bp deletion while 2 bp insertion at the sgRNA2 target site decreased about 80% compared with wild type.6.Dwarf 9(D9)is the homologs of D8.Considering highly similarity between D9 and D8,there is a certain probability that the sgRNA targeting D8 maybe cause mutations in the D9 gene.By sequencing the D9 gene of the edited plants,4 plants were also edited in the D9 gene.In Zheng 58 inbred line,the D8 gene of line D3 happened frame shift mutation,the average plant height of line D3 is 55.67 cm,which is about56% lower than the average plant height of wild-type which is 128.27 cm.We found that the D9 gene has a biallelic deletion of 3 bp and 1 bp.The above results indicate that the mutations of D8 and D9 genes may lead to the appearance of dwarf phenotype.7.According to the comparison of deduced protein sequences of these mutants,the deletions before the DELLA motif have little effect on plant height,while mutations including the conserved DELLA motif deletion make the plant appear significantly dwarfed.In the Zheng 58 inbred line,the average plant height of line N9 is 57.36 cm,which was significantly lower than the average plant height wild-type which is 128.27 cm.In the XCW175 inbred line,the average plant height of the line 1-52 is 59.38 cm,which is significantly lower than the average plant height of wild-type which is 149.83 cm,the average plant height of line 89-1 is 31.67 cm.In the Q319 inbred line,the DELLA conserved domain of line D41-4-16 was not destroyed,but the average plant height of this line is 95.07 cm,which is about 48% lower than the average plant height of wild-type which is 184.82 cm.We found that the D9 gene of line D41-4-16 has a frame shift mutation,and the conserved DELLA domain of the DELLA protein encoded by the D9 gene was destroyed.The above results indicate that if the DELLA conserved domain of the DELLA protein is destroyed,the plant may have a dwarf phenotype.