Genome-Wide Association Study (GWAS) to Identify Salt-Tolerance QTLs Carrying Novel Candidate Genes in Rice During Early Vegetative Stage

Abstract

Background: Rice is considered as a salt-sensitive plant, particularly at early vegetative stage, and its production is suffered from salinity due to expansion of salt affected land in areas under cultivation. Hence, significant increase of rice productivity on salinized lands is really necessary. Today genome-wide association study (GWAS) is a method of choice for fine mapping of QTLs involved in plant responses to abiotic stresses including salinity stress at early vegetative stage. In this study using > 33,000 SNP markers we identified rice genomic regions associated to early stage salinity tolerance. Eight salinity-related traits including shoot length (SL), root length (RL), root dry weight (RDW), root fresh weight (RFW), shoot fresh weight (SFW), shoot dry weight (SDW), relative water content (RWC) and TW, and 4 derived traits including SL-R, RL-R, RDW-R and RFW-R in a diverse panel of rice were evaluated under salinity (100 mM NaCl) and normal conditions in growth chamber. Genome-wide association study (GWAS) was applied based on MLM(+Q + K) model.

Results: Under stress conditions 151 trait-marker associations were identified that were scattered on 10 chromosomes of rice that arranged in 29 genomic regions. A genomic region on chromosome 1 (11.26 Mbp) was identified which co-located with a known QTL region SalTol1 for salinity tolerance at vegetative stage. A candidate gene (Os01g0304100) was identified in this region which encodes a cation chloride cotransporter. Furthermore, on this chromosome two other candidate genes, Os01g0624700 (24.95 Mbp) and Os01g0812000 (34.51 Mbp), were identified that encode a WRKY transcription factor (WRKY 12) and a transcriptional activator of gibberellin-dependent alpha-amylase expression (GAMyb), respectively. Also, a narrow interval on the same chromosome (40.79-42.98 Mbp) carries 12 candidate genes, some of them were not so far reported for salinity tolerance at seedling stage. Two of more interesting genes are Os01g0966000 and Os01g0963000, encoding a plasma membrane (PM) H⁺-ATPase and a peroxidase BP1 protein. A candidate gene was identified on chromosome 2 (Os02g0730300 at 30.4 Mbp) encoding a high affinity K⁺ transporter (HAK). On chromosome 6 a DnaJ-encoding gene and pseudouridine synthase gene were identified. Two novel genes on chromosome 8 including the ABI/VP1 transcription factor and retinoblastoma-related protein (RBR), and 3 novel genes on chromosome 11 including a Lox, F-box and Na⁺/H⁺ antiporter, were also identified.

Conclusion: Known or novel candidate genes in this research were identified that can be used for improvement of salinity tolerance in molecular breeding programmes of rice. Further study and identification of effective genes on salinity tolerance by the use of candidate gene-association analysis can help to precisely uncover the mechanisms of salinity tolerance at molecular level. A time dependent relationship between salt tolerance and expression level of candidate genes could be recognized.

Keywords: Genome-wide association mapping; Molecular breeding; Rice; SNPs; Salinity.

Fig. 1

Manhattan plots of p-values analyzed using mixed linear model (MLM) controlled for population structure and kinship of rice genotypes for different traits under normal condition

Fig. 2

Manhattan plots of p-values analyzed using mixed linear model (MLM) controlled for population structure and kinship of rice genotypes for different traits under salinity condition

Fig. 3

Distribution of GWAS-based detected QTLs on 10 chromosomes of rice. Distances on the map are in Mbp. The position of SalTol, a well-studied QTL at seedling stage, was shown on chromosome 1

Fig. 4

Map location of salinity-tolerance candidate genes on 7 distinct regions of chromosome 1. Gene RAP locus IDs are in red

Fig. 5

Changes in relative gene expression level of candidate genes at different times after salinity stress in different rice cultivars. a Peroxidase BP1, b PM H⁺-ATPase. T24, T48 and T72: 24, 48 and 72 h after salt stress. CV6 and CV178 (ARC6578 and Shoemed): sensitive cultivars. CV48 (Bombilla): tolerant cultivar