Phylogenetic analysis of salt tolerant genes in local Thai rice and salt tolerant gene identification by F2 bulk-segregant analysis

Genetic diversity is important for developing salt-tolerant rice varieties. This research used the existing whole-exome sequences of eight Thai rice varieties, including the standard checks for tolerance and susceptibility, and the previously reported salt-tolerant genes to understand the mechanisms of salt tolerance in Thai rice, as well as the bulk-segregant analysis (BSA) to identify salt-tolerant QTLs in ‘Jao Khao’, the salt tolerant Thai rice. Salt responsive characteristics were used to classify tolerant and susceptible genotypes at the seedling stage. Phylogenetic analysis of SNPs from exome sequences showed that the Thai rice varieties were monophyletic and distantly related to 'Pokkali' and IR29. 'Lai Mahk' and 'Luang Pratahn', which were characterized as Thai salt tolerant varieties, were closely related based on either the exome SNPs or SNPs from 164 salt tolerant genes. Genomic regions conferring salt tolerance traits were identified in an F2 population of the Thai landrace salt tolerant variety, 'Jao Khao', and the salt susceptible line, IR29, via BSA. In total, 600 F2 individuals were phenotyped in the hydroponic system. The 14-day old seedlings were treated with salt stress in a stepwise manner to reach 12 dS.m-1 for 12 days. The F2 members showed a high level of distribution in the standard salt injury evaluation system (SES), cell membrane stability (CMS), relative water content (RWC), whole plant fresh weight (WFW), plant height (PH), and root length (RL). SES showed a strong negative correlation with CMS, RWC, and WFW, but it showed less of a negative correlation with PH and RL. The BSA predicted 4 QTLs harboring the putative salt-tolerant regions on chromosome 3 (CMS trait) and chromosomes 2, 6, and 12 (RWC trait). Three of the identified QTLs overlapped with some reported salt-tolerant QTLs, while the QTLs on chromosome 6 overlapped with drought-tolerant QTLs. The validation of the QTLs was carried out by designing four molecular markers. Their possible involvement in salt tolerance was assessed in an F3 population. The findings revealed that F3 individuals with the ‘Jao Khao’ allele were more tolerant to salt stress than the ones with the allele from IR29 or containing the heterozygous allele, indicating that these QTLs are important in salt tolerance. In the future, the 4 salt stress-tolerant Thai rice varieties can be employed in rice breeding. Moreover, 4 salt tolerant QTLs identified in the study will be used to breed salt tolerant rice, and the developed genetic markers can be used in the marker assisted-breeding program.