Salinity is one of the most important abiotic stress affecting the world rice production. The cultivation of salinity-tolerant cultivars is the most costeffective and environmentally friendly approach for salinity control. In recent years, CRISPR/Cas9 systems have been widely used for target-site genome editing; however, their application for the improvement of elite rice cultivars has rarely been reported. Here, we report the improvement of the rice salinity tolerance by engineering a Cas9-OsRR22-gRNA expressing vector, targeting the OsRR22 gene in rice. Nine mutant plants were identified from 14 T 0 transgenic plants. Sequencing showed that these plants had six mutation types at the target site, all of which were successfully transmitted to the next generations. Mutant plants without transferred DNA (T-DNA) were obtained via segregation in the T1 generations. Two T2 homozygous mutant lines were further examined for their salinity tolerance and agronomic traits. The results showed that, at the seedling stage, the salinity tolerance of T2 homozygous mutant lines was significantly enhanced compared to wild-type plants. Furthermore, no significantly different agronomic traits were found between T2 homozygous mutant lines and wild-type plants. Our results indicate CRISPR/ Cas9 as a useful approach to enhance the salinity tolerance of rice.

The drought tolerance (DT) of plants is a complex quantitative trait. Under natural and artificial selection, drought tolerance represents the crop survival ability and production capacity under drought conditions (Luo, 2010). To understand the regulation mechanism of varied drought tolerance among rice genotypes, 95 diverse rice landraces or varieties were evaluated within a field screen facility based on the ‘line–source soil moisture gradient’, and their resistance varied from extremely resistant to sensitive. The method of Ecotype Targeting Induced Local Lesions in Genomes (Ecotilling) was used to analyze the diversity in the promoters of 24 transcription factor families. The bands separated by electrophoresis using Ecotilling were converted into molecular markers. STRUCTURE analysis revealed a value of K = 2, namely, the population with two subgroups (i.e., indica and japonica), which coincided very well with the UPGMA clusters (NTSYS-pc software) using distance-based analysis and InDel markers. Then the association analysis between the promoter diversity of these transcription factors and the DT index/level of each variety was performed. The results showed that three genes were associated with the DT index and that five genes were associated with the DT level. The sequences of these associated genes are complex and variable, especially at approximately 1000 bp upstream of the transcription initiation sites. The study illuminated that association analysis aimed at Ecotilling diversity of natural groups could facilitate the isolation of rice genes related to complex quantitative traits.

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