CRISPR/Cas9 Technology To Improve Heading Date And Plant Height In Rice And The Aggregation Of Wx-mq And ALS Genes

As a model plant,rice uses traditional breeding methods such as hybridization,backcrossing,and selfing to improve the target traits.However,it is difficult to obtain rice plants with the disadvantages of complicated operation,time-consuming,and other improvements.Gene editing of using the CRISPR/Cas9 system and molecular marker-assisted selection have been successfully used in rice.Heading stage and plant height are the main agronomic traits in the rice growth stage,which determine the growth status,adaptability,and high and stable yield of rice.We selected DTH8,Ghd7 and SD1 as candidate target genes,and used the CRISPR/Cas9 system to improve the heading date and plant height traits of the rice.We constructed their CRISPR/Cas9 gene-editing expression vectors,and used Agrobacterium-mediated genetic transformation mutants of these vectors in different rice materials were constructed.In this paper,we used CRISPR/Cas9 multi-target knockout technology to knock out DTH8,Ghd7 and SD1 to obtain dth8 single mutants,ghd7 single mutants,and sd1 singles with japonica backgrounds in japonica varieties mutant.The main experimental results are as follows: Design target sites according to candidate target genes and construct two target sites of the same candidate gene into CRISPR/Cas9 gene editing expression vectors at the same time.These are CRISPR-DTH8-1,CRISPR-Ghd7-1 and CRISPR-SD1-1;CRISPR-DTH8-1-DTH8-2,CRISPR-Ghd7-1-Ghd7-2and CRISPR-SD1-1-SD1-2.The constructed CRISPR/Cas9 dual-target binary expression vector was transformed into Agrobacterium strain EHA105 using Agrobacterium-mediated genetic transformation of rice.Four agrobacterium-infected rice callus tissues carrying the recombinant plasmids were tested for hygromycin gene hpt by PCR to finally obtain 29 Xiangnuo-dth8 positive plants;29 Xiangnuo-ghd7 positive plants;Koshihikari-sd1 positive plants 4 strain;the positive rate was 14.3%-23.3%.The heading dates of the 7 dth-8 mutant lines were screened,and statistical analysis showed that the heading dates of the 7 mutant plants were different from the wild type Xiangnuo 99-25,which further verified the mutants.Changes in heading date weredue to the editing of heading gene DTH8.In order to cultivate a new rice variety that aggregated low amylose content Wx-mq gene and herbicide-resistant ALS gene,single-nucleotide variation in Wx-mq gene and base variation in ALS gene in coding region were performed and four-primer amplification hindered mutation System PCR(Tetra-primer ARMS-PCR)amplification and allele-specific PCR(AS-PCR)amplification.A total of 6 new rice lines with low amylose content and herbicide resistance were obtained.