Genome Wide Association Studies Of Alkali Tolerance At Seedling Stage And Transcription Factor OsWRKY70 Negatively Regulates Salt Tolerance In Rice

Rice（Oryza sativa L.）is one of the most important food crops,and is also the staple food for 65%of the Chinese population.In recent years,the area of saline soil has been expanding due to natural factors such as rising temperature,sea level rise and water shortage,as well as human activities such as irrational irrigation,over-fertilization and over-tillage.Saline-alkali stress limits rice development at all growth stages and ultimately affects yield by reducing nutrient solubility,increasing external osmotic pressure,and disrupting ionic balance in many ways.This study focused on two aspects:（1）Phenotypic screening of 203rice Mini-core collections for seedling alkali tolerance and genome-wide association study（GWAS）of QTLs for alkali tolerance;（2）Through salt stress induction analysis of rice WRKY family genes,it was found that OsWRKY70 was induced by salt,genetic transformation,biological function identification and mechanism discussion.The main findings are as follows.1.Genome-wide association study of alkaline tolerance of rice Mini-core collections at seedling stage203 Mini-core collections were phenotypically characterized for alkali tolerance at seedling stage,Two-week-old seedlings were treated with IRRI nutrient solution containing50 mmol/L Na HCO₃（p H=9.0）for 7 days and recovered for 7 days.The survival rate was used as indicator of the strength of tolerance to alkali.The results showed that the alkali tolerance of temperate japonica and tropical japonica rice was generally stronger than that of AUS and indica;meanwhile,a genome-wide association study of alkali tolerance in Mini-core collections was conducted using TASSEI 5.0 software,and two new master QTLs for alkali tolerance in rice were obtained on chromosome 4 and 5,respectively.2.Transcription factor OsWRKY70 negatively regulates salt tolerance in riceSalt stress induction analysis of rice WRKY family genes revealed that OsWRKY70 was salt induced in both roots and shoots of rice,indicating that OsWRKY70 is involved in salt stress response.The identification of subcellular localization and transcriptional activation activity showed that OsWRKY70 is localized in the nucleus,which is a prerequisite for the transcription factor to function;Yeast single-hybrid assay showed that it has transcriptional activation activity.Genetic transformation experiments showed that OsWRKY70overexpressed lines increased salt sensitivity of rice,while knockout lines increased salt tolerance of rice.These results suggested that OsWRKY70 negatively regulated salt tolerance of rice.In order to investigate the mechanism of OsWRKY70,the sensitivity of transgenic lines to Abscisic acid（ABA）was tested.The results showed that:OsWRKY70 overexpressed lines increased the sensitivity of rice to ABA,suggesting that OsWRKY70 may be involved in the negative regulation of salt tolerance in rice through an ABA-dependent pathway..