CRISPR/Cas9 Targeted Editing Of Hina Gene In Barley Grain Hardness

Barley,cereal plant of the genus Hordeum,in the family Poaceae,is the fourth is the fourth largest food crop in the world.At present,it is mainly used for animal feed and beer brewing,and a small part is used for food and pharmaceutical raw materials.Grain hardness is one of the important traits affecting the processing quality of barley.At present,the research on grain hardness of barley mainly focuses on the molecular and gene level of grain hardness,but there are few studies on grain hardness genes and their different alleles.In this study,we created novel Hina alleles using the CRISPR/Cas9 system in the commercial barley variety ‘Golden Promise’and successfully obtained a barley mutant with increased grain hardness and reduced grain width.Through phenotypic identification,the mutants with increased grain hardness in barley were screened,and the wild-type and mutant with the same genetic background were compared.grain hardness and other traits.The main results are as follows.1.To edit the Hina gene in barley,we designed two target sites for the conserved regions of the coding sequence and assembled these into a single vector to generate the construct p YLH-hina using Golden Gate ligation.p YLH-hina was transformed into immature barley embryos via Agrobacterium-mediated transformation.A total of12 plantlets were obtained from 355 immature embryos.2.Targeted mutagenesis in transgenic plants was examined using Hi-TOM Gene Editing Detection,and the resulting sequences were aligned against the wild-type DNA sequence.Four T0 mutant plants and one T1 mutant plant were identified by Hi-TOM sequencing,indicating that the CRISPR/Cas9 system continued to function in the offspring,with an editing efficiency of 42%.3.According to the mutations of line hina02,specific primers were designed,and homozygous mutant plants were identified by PCR and were verified by Hi-TOM gene editing detection kit.Five homozygous mutant plants were obtained,which were used in the next step analysis.4.Due to a limited quantity of grains,we first analyzed the distribution of single grain HI（Hardness index）in a heterozygous group generated from hina02,hina06,hina07,and hina12 edited plants;these plants produced wild-type grains,heterozygous mutated grains,and homozygous mutated grains.The grain HI of the heterozygous group was from 32.2 to 95.56,with an average of 65,while the HI of wild type was 32.47 to 71.94,with an average of 53.81.The HI of hina10 edited plants was similar to that of the wild type.This result shows that grains of hina mutants,except hina10,possessed a higher HI.5.Scanning electron microscope images of fractured endosperms illustrated differences between the wild type and hina02 mutant.In the wild type,A-type starch granules were smooth and devoid of the associated protein matrix.The starch granule surface in the wild type was relatively free of associated protein material,and the spaces between adjacent starch granules appeared to be largely devoid of matrix protein.In the hina02 mutant,the spaces between adjacent starch granules were filled with protein matrix,which appeared to coat A-type starch granules and to embed B-type starch granules completely.These images indicate that starch granules were loose and separated from the protein matrix in the wild type,while they were deeply trapped and tightly integrated with the protein matrix in the mutant.6.Plants generated from the homozygous hina02 mutant shared the same appearance as the wild type.Phenotypically,there were no differences in plant height,number of tillers,spike length,grains per spike,or grain length.However,the grain width and thousand-grain weight of the hina02 mutant were 14.67% and 22.92%lower than those of the wild type,respectively.The starch and protein content of hina02 were 68.13% and 14.03%,while those of wild type were 63.71% and 11.83%respectively.These results suggested that knocking-out of Hina gene may affect grain development.