Genetic Improvement Of Panicle Neck Length And Other Traits Of Rice Varieties Based On CRISPR/Cas9 System

Rice（Oryza sativa L.）is the world’s leading food crop,and it is the unremitting pursuit of scientists to improve the important agronomic traits of rice and increase production.In recent years,the use of CRISPR（clustered regularly interspaced short palindromic repeats）/Cas9（CRISPR-associated protein 9）technology developed by the acquired immune system of bacteria for gene editing,for the innovation of rice germplasm resources to provide a new safe and efficient way.In this study,the CRISPR/Cas9 system developed in our laboratory was used to improve the important agronomic traits of several rice cultivars,the genes involved include:rice Long spike neck gene Eui1,rice blast resistance gene Os ERF922,rice amylose synthase gene Waxy,rice broad-spectrum resistance to rice blast gene Bsr-d1 and rice ideal plant type gene Os OTUB1,the main results are as follows:1、Twelve gene editing vectors were successfully constructed,which were ten different single-target vectors of Eui1 gene,one double-target vector KO-Os ERF922-Waxy for simultaneously editing of Os ERF922 and Waxy genes,and one double-target vector KO-Bsr-d1-Os OTUB1 for simultaneously editing of Bsr-d1 and Os OTUB1 genes.2、The rice cultivars Tianfeng B,Huaguang and Huazhan were transformed with Twelve gene editing vectors to obtain transformants.Sequencing analysis of the T₀generation showed that all targets were successfully edited and the target mutation rate was high.3、Two eui1 homozygous mutant Tianfeng B strains were screened,and the three indexes of plant height,panicle neck length and seed setting rate basically met the rice breeding requirements,and these mutant alleles were backcrossed to the sterile line Tianfeng A.4、The main agronomic traits of the homozygous mutant strains of oserf922 in Huaguang background are normal,and the resistance to rice blast is being tested,while the amylose content of the waxy homozygous mutant strains is reduced to about 3%（the wild type is about 16%）,becoming a glutinous rice variety.5、The complete loss of Bsr-d1 function in Huaguang and Huazhan background has adverse effects on rice growth and development,and is not suitable for practical application.The osotub1 homozygous mutant strains showed an ideal plant type,but further investigation was needed.In this study,several important genes of rice were edited,and a beneficial attempt was made to realize its practical application.This study provided an important reference basis for the genetic improvement of rice varieties.