Preliminary De Novo Domestication Of Lycium Ruthenicum With Larger Fruit Or Thornless Trait

Lycium ruthenicum is the shrub in the Solanaceae family,which is an important garden plant for soil and water conservation in the northwest region of our country,and an important economic crop for local industrial development.The cultivation of varieties with larger fruit and fewer thorns not only provides excellent flower germplasm for the construction of beautiful western China,but also exerts its good ecological function and economic value in rural revitalization.In this study,the CLAVATA3(CLV3)and its promoter were edited to obtain the larger fruit.Referring to the study on thorn development and regulation mechanism of citrus,the three THORN IDENTITY genes(TI,homologous genes of thorn identity in citrus)were edited to transform the thorns into lateral branches.Based on the mechanism of axillary bud formation,the DWARF(DWF)that involved in brassinolide(BR)synthesis was edited to inhibit the formation of thorns by inhibiting lateral bud germination.The main findings are as follows:1.The leaves of Lycium ruthenicum were genetically transformed,and 51 clv3 mutants were obtained,including 4 single-target editing mutants and 47 dual-target editing mutants,and the editing efficiency was 100%.15 mutants had homozygous mutations at least one target,and 36 mutants had biallelic mutations at least one target.fw2.2 mutants were infected,and a total of 11fw2.2-clv3 double gene mutants were obtained,of which fw2.2 had homozygous mutation and clv3 had biallelic mutation.A total of 66 mutants were obtained,including 29 homozygous mutants and35 biallelic mutants,and the vector editing efficiency was 94.29%.2.The dual-target editing vectors of Lr TI1-1,Lr TI1-2,Lr TI2 and Lr DWF genes were constructed by seamless cloning.A total of 69 ti2 mutants were obtained in the T0 generation,including 55 single-target editing mutants and 14 dual-target editing mutants,and the editing efficiency was 86.25%.A total of 58 ti1-1 mutants were obtained,including 44 single-target editing mutants and 14 dual-target editing mutants,and the editing efficiency was 92.06%.A total of 45 ti1-2 mutants were obtained,including 34 single-target editing mutants and 11 dual-target editing mutants,and the editing efficiency was 90%.A total of 28 dwf mutants were obtained in the T0 generation,of which 12 were homozygous mutant lines and 8 were biallelic mutant lines,and the editing efficiency was 77.78%.The phenotype observation of the tissue culture seedlings showed that some heterozygous dwf mutants were dwarfed and compact,some mutants roots swelled with nodule-like tissues,some were coiled.The phenotype of thorn development in the mutants remains to be further observed.3.In the p DIRECT＿21C-g RNA3-g RNA6-g RNA7 and p DIRECT＿21C-g RNA8-g RNA4-g RNA5 edited plants,a total of 11 ti1-1,16 ti1-2,10 ti2,9 ti1-1 ti2,15 ti1-2 ti2 and 2 ti1-1 ti1-2 ti2 biallelic and homozygous lines were obtained.The phenotype observation of the mutants showed that the ti mutants had more side branches,some mutants further grew new short side branches at the leaf axils of the side branches,and the formation of thorns was rare.4.Transcriptome sequencing of ti mutants showed that the plant hormone signal transduction pathway,sugar signal pathway and transcription factors responded to the mutation of TIs.The upregulated expression of BR,cytokinin(CTK)synthesis genes and the down-regulated expression of strigolactone(SL),abscisic acid(ABA),and gibberellin(GA)synthesis genes corresponded to the branching phenotype of the mutants.Sucrose synthase(SUS),hexokinase(HXK)and trehalose-6-phosphate synthase(T6P)were mainly up-regulated,indicating that the sugar signaling pathway was activated in the ti mutants.Transcription factors such as PIN-FORMED(PIN),AUX/IAA,DELLA,BES1/BZR1,etc.were also involved in the phenotype regulation of ti mutations.5.The expression levels of genes in mutants were analyzed by real-time PCR,and the results showed that the expressions of CLV3 and CLV1 in clv3 mutants were significantly decreased,the expression of RPK2 was significantly decreased in some mutants,and the expression of WUSCHEL(WUS)was increased in some mutants.Among the 7 clv3 promoter mutants,CLV3 expression was significantly decreased in 5 of them.The expressions of TIs and WUS in ti mutants were analyzed.The expression of WUS in some mutants was increased with different degrees,the expressions of TIs decreased in some mutants,but were significantly up-regulated in other mutants.The expression levels of DWF,DET2,CYP90A1,CYP90B1 and CYP724B1 were detected in the dwf mutants.The results showed that CYP724B1 was significantly up-regulated in two mutants,on the contrary,the expressions of DET2,CYP90A1 and CYP90B1 were all decreased.The expression of DWF was significantly down-regulated in one of the mutants,while increased significantly in the other mutant,indicating that different types of mutations at the target site have different effects on the transcription level of the gene.In summary,a large number of mutant germplasms were created by editing the genes regulating fruit size and lateral organ development,and 409 mutant lines were obtained,including 205 homozygous or biallelic lines.Some mutants showed the phenotypes reported previously at the seedling stage.Transcriptome sequencing of the mutants revealed differential expression of pathway genes and transcription factors.Real-time PCR analysis showed that the mutation of the target gene leads to changes in the expression level of related regulatory genes.This study preliminarily realized the de novo domestication of Lycium ruthenicum with larger fruit and fewer thorns,and also provided a reference for targeted improvement of Lycium ruthenicum traits by gene editing technology.