Joint QTL Mapping and Transcriptome Sequencing Analysis Reveal Candidate Genes for Salinity Tolerance in Oryza sativa L. ssp. Japonica Seedlings

Abstract

Salinity stress is one of the major abiotic stresses affecting crop growth and production. Rice is an important food crop in the world, but also a salt-sensitive crop, and the rice seedling stage is the most sensitive to salt stress, which directly affects the final yield formation. In this study, two RIL populations derived from the crosses of CD (salt-sensitive)/WD (salt-tolerant) and KY131 (salt-sensitive)/XBJZ (salt-tolerant) were used as experimental materials, and the score of salinity toxicity (SST), the relative shoot length (RSL), the relative shoot fresh weight (RSFW), and the relative shoot dry weight (RSDW) were used for evaluating the degree of tolerance under salt stress in different lines. The genetic linkage map containing 978 and 527 bin markers were constructed in two RIL populations. A total of 14 QTLs were detected on chromosomes 1, 2, 3, 4, 7, 9, 10, 11, and 12. Among them, qSST12-1, qSST12-2, and qRSL12 were co-localized in a 140-kb overlap interval on chromosome 12, which containing 16 candidate genes. Furthermore, transcriptome sequencing and qRT-PCR were analyzed in CD and WD under normal and 120 mM NaCl stress. LOC_Os12g29330, LOC_Os12g29350, LOC_Os12g29390, and LOC_Os12g29400 were significantly induced by salt stress in both CD and WD. Sequence analysis showed that LOC_Os12g29400 in the salt-sensitive parents CD and KY131 was consistent with the reference sequence (Nipponbare), whereas the salt-tolerant parents WD and XBJZ differed significantly from the reference sequence both in the promoter and exon regions. The salt-tolerant phenotype was identified by using two T₃ homozygous mutant plants of LOC_Os12g29400; the results showed that the score of salinity toxicity (SST) of the mutant plants (CR-3 and CR-5) was significantly lower than that of the wild type, and the seedling survival rate (SSR) was significantly higher than that of the wild type, which indicated that LOC_Os12g29400 could negatively regulate the salinity tolerance of rice at the seedling stage. The results lay a foundation for the analysis of the molecular mechanism of rice salinity tolerance and the cultivation of new rice varieties.

Keywords: QTL; candidate gene; rice; salinity tolerance; transcriptome sequencing.

Figure 1

Phenotypic variation in the SST, RSL, RSFW, and RSDW in two RIL populations. (A–D) represent the SST, RSL, RSFW, and RSDW of RIL populations derived from CD/WD. (E–H) represent the SST, RSL, RSFW, and RSDW of RIL populations derived from KY131/XBJZ.

Figure 2

Identification of candidate genes by two RIL populations. (A) Salinity tolerance-related QTLs were detected in 189 RILs derived from CD/WD and mapped to the interval between markers C12_17379052 and C12_17572391 by linkage mapping. (B) Salinity tolerance-related QTLs were detected in 195 RILs derived from KY131/XBJZ and mapped to the interval between markers C12_16988462 and C12_17519826 by linkage mapping. (C) The two RIL populations co-located intervals. (D) The 140-kb region contained 16 genes.

Figure 3

The differentially expressed genes (DEGs) in shoots. (A) Venn diagram of DEGs between two comparative groups. (B,C) Volcano plot of DEGs among different samples. X-axis and Y-axis present the value of log₂^(ratio) and −log₁₀^(FDR) of two comparative groups, respectively. Red (up-regulated) and blue (down-regulated) dots indicated that the genes have significant expression difference. CD_T: The samples of CD treated with 120 mM NaCl treatment for 12 h. CD_CK: The samples of CD treated with nutrient solution. WD_T: The samples of WD treated with 120 mM NaCl treatment for 12 h. WD_CK: The samples of WD treated with nutrient solution.

Figure 4

GO enrichment of differentially expressed genes in different comparison groups. (A) CD_T VS CD_CK. (B) WD_T VS WD_CK.

Figure 5

KEGG enrichment of differentially expressed genes in different comparison groups. (A) CD_T VS CD_CK. (B) WD_T VS WD_CK.

Figure 6

Heatmap of gene expression within candidate intervals. (A) The heatmap of expression of 12 genes in the interval with normal and salt treatment of CD; (B) The heatmap of expression of 12 genes in the interval with normal and salt treatment of WD; CK: control group (untreated); T: test group (salt-treated).

Figure 7

Expression patterns of the four genes under normal growth conditions and salinity stress. (A–D) represent the gene expression of LOC_Os12g29330, LOC_Os12g29350, LOC_Os12g29390, and LOC_Os12g29400 under normal growth conditions and salinity stress. (* p < 0.05,** p < 0.01, Students’ t-test).

Figure 8

Target sequences of WT (Nipponbare) and knockout mutant plants (CR-3, CR-5).

Figure 9

Salinity tolerance phenotype of WT (Nipponbare) and knockout mutant plants (CR-3, CR-5). (A) Phenotype of the control group; (B) Phenotypes of the salt-treated group; (C) Survival statistics of WT (Nipponbare), CR-3, and CR-5 in the salt treatment group; (D) The SST of WT (Nipponbare), CR-3, and CR-5 in the salt treatment group. The red line distinguishes wild-type material from transgenic material. (** p < 0.01; Students’ t-test).