Linkage analysis, GWAS, transcriptome analysis to identify candidate genes for rice seedlings in response to high temperature stress

Abstract

Background: Rice plants suffer from the rising temperature which is becoming more and more prominent. Mining heat-resistant genes and applying them to rice breeding is a feasible and effective way to solve the problem.

Result: Three main biomass traits, including shoot length, dry weight, and fresh weight, changed after abnormally high-temperature treatment in the rice seedling stage of a recombinant inbred lines and the natural indica germplasm population. Based on a comparison of the results of linkage analysis and genome-wide association analysis, two loci with lengths of 57 kb and 69 kb in qDW7 and qFW6, respectively, were associated with the rice response to abnormally high temperatures at the seedling stage. Meanwhile, based on integrated transcriptome analysis, some genes are considered as important candidate genes. Combining with known genes and analysis of homologous genes, it was found that there are eight genes in candidate intervals that need to be focused on in subsequent research.

Conclusions: The results indicated several relevant loci, which would help researchers to further discover beneficial heat-resistant genes that can be applied to rice heat-resistant breeding.

Keywords: GWAS; High-temperature-mediated growth response; Linkage analysis; Rice seedling; Transcriptome analysis.

Fig. 1

The phenotypic characters of RIL and the natural population. a Phenotypic characters of the RIL used for linkage analysis. b Phenotypic characters of the natural rice population used for association analysis. SL, FW, DW indicate shoot length, fresh weight, and dry weight of the seedlings

Fig. 2

Correlation analysis among biomass traits of RIL and the natural population. a Recombinant inbred lines. b Natural rice population. The color indicates the correlation coefficient. CSL, CFW, and CDW indicate shoot length, fresh weight, and dry weight of the seedlings in control groups, respectively. SL, FW, and DW indicate the respective shoot length, fresh weight, and dry weight of the seedling in treatment groups, respectively. SR indicates survival rate of the treatment

Fig. 3

Genome-wide association analysis of biomass traits under heat stress. Manhattan plots of a Dry weight of control groups. b Dry weight of treatment groups. c Fresh weight of control groups. d Fresh weight of treatment groups. e Shoot length of control groups. f Shoot length of treatment groups. CSL, CFW, and CDW indicate shoot length, fresh weight, and dry weight of the seedlings in control groups, respectively. SL, FW, and DW indicate the respective shoot length, fresh weight, and dry weight of the seedling in treatment groups, respectively

Fig. 4

Correlation analysis of the survival rate of the natural rice population and analysis of known gene haplotypes. a Manhattan plot of survival rate (SR). b QQplot of survival rate. c Scatter plot of Ugp1. The arrow indicates that the site is the approximate site of Ugp1. d Gene structures (left) and biomass traits of different haplotypes (right) of Ugp1. Red colored numbers indicate the key SNPs among major haplotypes

Fig. 5

Manhattan plot (top) and LD heatmap (bottom) surrounding the peak of the candidate interval. Interval of linkage analysis and genome-wide association analysis in a qDW7. b qFW6. Dashed lines indicate the candidate region of the peak

Fig. 6

Genome-wide transcriptome analysis of heat stress between heat-tolerant and heat-sensitive rice varieties. a The PCA of Hierarchical clustering of all the samples after normalisation. b GO enrichment analysis of differential genes. The threshold of FDR < 0.05 was selected to identify significant enriched GO terms. c Venn diagrams showing the differential genes in response to heat stress. d genes up-regulated by redox-related pathways. e Redox-related pathways down-regulate genes. HR1 and HR2 are varieties with extremely high survival rate. HS1 and HS2 are varieties with extremely low survival rate

Fig. 7

Validation of RNA-seq with qRT-PCR. a The figure shows the multiple relationship of the gene expression of the two groups of extreme materials after heat treatment, including the transcriptome FPKM and qRT-PCR quantitative results. a shows the processing of HR1 and HS1. b HR1 and HS2. c HR2 and HS1. d HR2 and HS2. HR1 and HR2 are varieties with extremely high survival rate; .HS1 and HS2 are varieties with extremely low survival rate