OsWRKY49 on qAT5 positively regulates alkalinity tolerance at the germination stage in Oryza sativa L. ssp. japonica

Integrated genome-wide association study and linkage mapping revealed genetic basis of alkalinity tolerance during rice germination. The key gene OsWRKY49 was further verified in transgenic plants. With the widespread use of the rice direct seeding cultivation model, improving the tolerance of rice varieties to salinity-alkalinity at the germination stage has become increasingly important. However, as previous studies have concentrated on neutral salt stress, understanding of alkalinity tolerance is still in its infancy, and the genetic resource data is scarce. Here, we used a natural population composed of 295 japonica rice varieties and a recombinant inbred population including 189 lines derived from Caidao (alkali-sensitive) and WD20342 (alkali-tolerant) to uncover the genetic structure of alkalinity tolerance during rice germination. A total of 15 lead SNPs and six QTLs related to relative germination potential (RGP) and relative germination index (RGI) were detected by genome-wide association study and linkage mapping. Of which, Chr5_28094966, a lead SNP was located in the interval of the mapped major QTL qAT5, that was significantly associated with both RGP and RGI in the two populations. According to the LD block analysis and QTL interval, a 425 kb overlapped region was obtained for screening the candidate genes. After haplotype analysis, qRT-PCR and parental sequence analysis, LOC_Os05g49100 (OsWRKY49) was initially considered as the candidate gene. Having studied the characteristics of rice lines with OsWRKY49 knockout and overexpression, we established that OsWRKY49 could be a positive regulator of alkalinity tolerance in rice at the germination stage. Subcellular localization showed that green fluorescent protein-tagged OsWRKY49 was localized in the nucleus. The application of OsWRKY49 could be useful for increasing alkalinity tolerance of rice direct seeding.