De Novo Domestication Of A Salt-tolerant Weedy Rice By Genome Editing

‘Sea rice’ is a type of saline-tolerant rice.‘Sea rice 86’ was bred by a rice breeder Risheng Chen from a kind of semi-wild rice growing in the intertidal zone of rivers.Sea rice 86 has some good properties such as salt resistance,alkali resistance,flood resistance,etc.However,Sea rice 86 also has some wild rice characteristics,such as long awn,seed shattering,high photosensitivity,high stalk and easy lodging,poor plant architecture,etc.These characters make it unsuitable for direct agricultural production.In order to improve this line with excellent comprehensive agronomic traits but retaining its saline-tolerant property,CRISPR/Cas9 technology was used to edit the genes controlling the above wild traits,as well as the key genes controlling yield and grain quality.This research has achieved the following results:(1)Construction of multi-target gene editing vector: This study aimed at improving ten traits of Sea rice 86,including plant height,plant architecture,photoperiod sensitivity,awn length,grain type,flavor,yield,seed-shattering,amylose content,and nitrogen utilization.Thirteen genes for these traits were selected for the editing,including SD1,IPA1 or OTUB1,Hd1,An-2,GS3,GW8,GS9,BADH2,Gn1 a,q SH1(two target sites),Wx,ARE1.In this study,we used a plant multi-target CRISPR/Cas9 genome editing system developed in our lab to assemble 12 target sg RNA expression cassettes into two vectors.(2)Obtaining the transgenenic plants of Sea rice 86: Using these two vectors,four positive transformants were obtained through Agrobacterium-mediated genetic transformation of rice.The types of gene mutations were analyzed via PCR and sequencing,which confirmed that most genes were successfully edited.(3)Examining the targeting traits of the transgenic plants: Six traits were successfully improved,including plant height,plant architecture,awn length,grain length,photoperiod sensitivity,and fragrance.While two traits,nitrogen utilization and total spikletes per plant,need further observation.However,the genetic improvement of the two traits,amylose content and seed-shattering,failed.(4)Selection of transgene-free improved editing lines: Through the molecular detection of the transgenes in the T-DNA in the progeny of the transformants,transgene-free “improved sea rice” lines were obtained in this study.In this study,multi-target gene editing technology was used to accurately knock out several genes that controlling multiple wild traits in Sea rice 86,which established the method for de novo domestication from semi-wild rice to modern varieties,laying a key foundation for its application.