Screening The Genes Regulating Trichome Development In Actinidia And Their Genome Editing In‘Hayward'Kiwifruit

The kiwifruit has been called‘the king of fruits' because of its remarkably high vitamin C content and balanced nutritional composition of minerals,dietary fibre and other health-beneficial metabolites.The fruit hair is one of the factors affecting the appearance quality and consumption of kiwifruit.Kiwifruit with less or no fruit hair is one of the main goals of kiwifruit breeding.In this paper,the Actinidia chinensis cv.‘Hongyang',A.eriantha and A.rufa were selected as materials,we screened out two candidate genes which regulate development of trichomes of kiwifruit with the help of the second and third generation transcriptome data of ‘Hongyang' kiwifruit,and the kiwifruit genome database,then the candidate genes were edited by the gene editing systerm CRISPR/Cas9 to validate whether they have functions in the development of trichomes of hexaploid cultivar ‘Hayward' kiwifruit or not,and lay the foundation for gene editing breeding.The main results in this paper were as follows:Through the alignment of relevant protein sequences,We attained the homologous genes of five targeted genes AtGL1,AtGL2,AtGL3,AtTTG1 and AtSAD2,which regulate the initation and development of leaf trichomes in Arabidopsis based on the kiwifruit genome database.Then,we obtained eight candidate genes through the alignment of protein sequences and phylogenetic tree analyses,and the candidate gene of AtGL1 was Achn116791,the candidate gene of AtGL2 was Achn198951,the candidate genes of AtGL3 were Achn244201,Achn302211 and Achn071731,the candidate gene of AtTTG1 was Achn045741,the candidate genes of AtSAD2 were Achn379131 and Achn379141.The expression levels of homologous genes of the five targeted genes in young fruits,mature leaves and shoot apexes of the A.chinensis cv.‘Hongyang',A.eriantha and A.rufa,as well as in the different developmental stages of‘Hongyang' fruit were analyzed.Finally,the two genes Achn116791 and Achn302211 were selected as final candidate genes which regulate the development of trichomes of kiwifruit.However,the gene Achn379131 might be not the key gene regulating the development of trichomes of kiwifruit.The sgRNAs of the three genes,Achn116791,Achn302211 and Achn379131 were designed using the software CRISPRdirect according to the design principles of sgRNA,then three gene editing vectors of CRISPR/Cas9 were successfully constructed using the pKSE401 vector.The leaf discs of kiwifruit cultivar ‘Hayward' were used for tissue culture and Agrobacterium-mediated transformation.Through the resistance selectionsand PCR identification of ‘hayward' kiwifruit,we finally obtained sixteen transgenic lines of gene Achn116791,in which nine lines had the base insertions,deletions or substitutions,and compared to WT,the trichome density of leaf was decreased and trichome length of leaf was shorter of Line4,Line5 and Line6 through the observation of ‘Hayward' kiwifruit leaf.We finally obtained four transgenic lines of Achn302211,in which one line had the base insertions,and compared to WT,the trichome density of leaf was decreased and trichome length of leaf was shorter.We finally also obtained four transgenic lines of Achn379131,in which all lines had no base insertions,deletions or substitutions in the targeted region.These results not only indidated that the two genes Achn116791 and Achn302211 have functions in regulating the leaf trichomes development of ‘Hayward' kiwifruit,but also confirmed that the CRISPR/Cas9 gene editing system could also be used in hexaploid cultivar ‘Hayward' kiwifruit,and in this paper,the efficiency of CRISPR/Cas9 gene editing system in transgenic lines was41.7%.