Integrated RNA Sequencing and QTL Mapping to Identify Candidate Genes from Oryza rufipogon Associated with Salt Tolerance at the Seedling Stage

Abstract

Soil salinity is a common abiotic stress affecting crop productivity. To identify favorable alleles from wild rice (Oryza rufipogon Griff.) that enhance salinity tolerance of rice (O. sativa L.), a set of introgression lines (ILs) were developed. The ILs were derived from an O. rufipogon accession collected from Chaling (Hunan Province, China) as the donor, and a widely grown O. sativa indica cultivar 93-11 as the recipient. Through evaluating the salt tolerance of 285 ILs at the seedling stage, a total of 10 quantitative trait loci (QTLs) related to salt tolerance were identified on chromosomes 1, 5, 7 and 9-12, with individual QTLs explaining 2-8% of phenotypic variance. The O. rufipogon-derived alleles at four QTLs improved salt tolerance in the 93-11 background. At the same time, a salt-tolerant IL, 9L136, was identified and characterized. Compared with the recipient parent 93-11, a total of 1,391 differentially expressed genes (DEGs) were detected specifically in 9L136 between salt stress and normal condition through genome-wide expression analysis. Of these, four DEGs located in the QTL regions carried by 9L136, suggesting that the four genes might be candidates associated with salt tolerance. Both the highly salt-tolerant ILs and the favorable O. rufipogon-derived QTLs identified in the present study will provide new genetic resources for improving the resistance of cultivated rice against salinity stress using molecular breeding strategies in the future.

Keywords: QTL analysis; RNA-seq; common wild rice; introgression line; salt tolerance; seedling stage.

FIGURE 1

Frequency distribution of salt tolerance score (A) of seedlings and survival rate (B) in introgression lines under 125 mM NaCl treatment for 9 days and recovery for 7 days. The phenotype of the recurrent parent, 93-11, is indicated by arrows.

FIGURE 2

Chromosomal location of salt tolerance-related QTLs (A) and the 9L136 and 93-11 phenotypes treated with 125 mM NaCl for 9 days (B). Symbols on the right of chromosomes indicate the positions of putative QTLs for salt tolerance-related traits. The closed symbols indicate that the O. rufipogon-derived alleles conferred a positive effect on salt tolerance-related traits, and the open symbols indicate a negative effect. The markers related to QTLs for salt tolerance are underlined with black. In the graphical genotypes, the black regions indicate homozygous regions from Chaling common wild rice in 9L136 and the white regions indicate homozygous regions for recurrent parent 93-11. The QTLs enhancing salinity tolerance and located on the introgressed region in 9L136 are underlined with red.

FIGURE 3

Comparison of physiological characteristics between salt-tolerant line 9L136 and recurrent parent 93-11 under salt stress conditions (125 mM NaCl). Physiological characteristics included the activities of catalase (CAT) (A), superoxide dismutase (SOD) (B), peroxidase (POD) (C), and malondialdehyde (MDA) (D), and the contents of soluble sugars (E) and proline (F). Data are means ± standard deviation with three replicates. Asterisks represent significant differences between 93-11 and 9L136 using Student’s t-test: ^∗P < 0.05 and ^∗∗P < 0.01.

FIGURE 4

Identification of candidate genes related to salt tolerance from wild rice. (A) Venn diagram analysis for RNA-seq data showing differentially expressed genes between 9L136-ST/9L136-CK and 93-11-ST/93-11-CK. There were 1,391 genes showed differentially expressed only in 9L136-ST/9L136-CK but not in 93-11-ST/93-11-CK (A), suggesting these genes should be specifically induced in introgression line 9L136. Among the 1,391 genes, four were co-localized with QTLs for salt tolerance (shown in gray circle). Arrows indicated the up-regulated (↑) and down-regulated (↓) genes. (B) Expression profiles of LOC_Os05g31620 and LOC_Os10g34730 detected by qRT-PCR and RNA-seq, respectively.