Dynamic genome-wide association analysis and identification of candidate genes involved in anaerobic germination tolerance in rice

Abstract

Background: In Asian rice production, an increasing number of countries now choose the direct seeding mode because of rising costs, labour shortages and water shortages. The ability of rice seeds to undergo anaerobic germination (AG) plays an important role in the success of direct seeding.

Results: In this study, we used 2,123,725 single nucleotide polymorphism (SNP) markers based on resequencing to conduct a dynamic genome-wide association study (GWAS) of coleoptile length (CL) and coleoptile diameter (CD) in 209 natural rice populations. A total of 26 SNP loci were detected in these two phenotypes, of which 5 overlapped with previously reported loci (S1_ 39674301, S6_ 20797781, S7_ 18722403, S8_ 9946213, S11_ 19165397), and two sites were detected repeatedly at different time points (S3_ 24689629 and S5_ 27918754). We suggest that these 7 loci (-log₁₀ (P) value > 7.3271) are the key sites that affect AG tolerance. To screen the candidate genes more effectively, we sequenced the transcriptome of the flooding-tolerant variety R151 in six key stages, including anaerobic (AN) and the oxygen conversion point (AN-A), and obtained high-quality differential expression profiles. Four reliable candidate genes were identified: Os01g0911700 (OsVP1), Os05g0560900 (OsGA2ox8), Os05g0562200 (OsDi19-1) and Os06g0548200. Then qRT-PCR and LC-MS/ MS targeting metabolite detection technology were used to further verify that the up-regulated expression of these four candidate genes was closely related to AG.

Conclusion: The four novel candidate genes were associated with gibberellin (GA) and abscisic acid (ABA) regulation and cell wall metabolism under oxygen-deficiency conditions and promoted coleoptile elongation while avoiding adverse effects, allowing the coleoptile to obtain oxygen, escape the low-oxygen environment and germinate rapidly. The results of this study improve our understanding of the genetic basis of AG in rice seeds, which is conducive to the selection of flooding-tolerant varieties suitable for direct seeding.

Keywords: Anaerobic germination tolerance; Candidate gene; Coleoptile; Dynamic GWAS; RNA-seq; Rice.

Fig. 1

Phenotypic variation of coleoptile in different rice germplasm at different submerging time points. AN is for anaerobic environment. Scale = 1 cm

Fig. 2

Frequency distribution histogram of each phenotype of coleoptile at different submergence time points. Note: CL, coleoptile length; CSA, coleoptile surface area; CD, coleoptile diameter; CV, coleoptile volume

Fig. 3

Genetic evolution of natural populations of indica and japonica rice. a Phylogenetic tree, each branch is a rice germplasm. b Principal component analysis on 2.12 million SNPs of 209 rice accession. PC 1 and PC 2 refer to the first and second principal components, respectively. The numbers in parentheses refer to the proportion of variance explained by the corresponding axes. Red points represent each variety in 209 rice accession. Each point represented a rice germplasm accession. The closer the distance between the points, the closer the relationship was. c Cluster analysis results of population genotypes, in which each color represents a group and each row represents a group value. d Cross validation error rate for each k value. Among them, K is the smallest when k is 5

Fig. 4

Manhattan map of SNPs associated with coleoptile length at different time points under anaerobic germination. The red horizontal line indicates the significance threshold of multiple comparisons (P < 0.1). The genomic regions detected in our GWAS and previous parental mapping studies were labeled as published QTLs with blue text. Orange blocks represented two co-localized genomic regions under anoxic pressure for different lengths of time in our GWAS study

Fig. 5

Manhattan map of SNPs associated with coleoptile diameter at different time points under anaerobic germination. The red horizontal line indicates the significance threshold of multiple comparisons (P < 0.1). The genomic regions detected in our GWAS and previous parental mapping studies were labeled as published QTLs with blue text. Orange blocks represented two co-localized genomic regions under anoxic pressure for different lengths of time in our GWAS study

Fig. 6

Heat map of the fold changes of the 106 candidate differentially expressed genes (DEGs)

Fig. 7

qRT-PCR expression profile of four most promising candidate genes in different oxygen environments. Values are the means ± standard errors (n = 3). A is for aerobic environment, AN is for anaerobic environment, AN3dA1d is for anaerobic environment and A3dAN1d is for aerobic environment and anaerobic environment

Fig. 8

Sequence analysis of four candidate genes in rice with high and low AGs. a The positions of four candidate genes Os01g0911700, Os05g0560900, Os05g0562200 and Os06g0548200 on chromosomes. b Sequence analysis of candidate gene Os01g0911700. c Sequence analysis of candidate gene Os05g0560900. d Sequence analysis of candidate gene Os05g0562200. e Sequence analysis of candidate gene Os06g0548200. Notes: (i) the gene structure of four candidate genes is shown respectively, the blue region represents 5′ or 3′ UTR, the green region represents exon, the straight line represents intron, and the arrow shows gene direction. (ii) the detailed sequence changes of four candidate genes in rice materials with very high and very low AGs are shown, where the position number represents the position related to the “ATG” initiation codon; “-” represents the upstream of “ATG”; and “+” represents the downstream of “ATG”. “--” means that the gene is missing 1 bp. (iii) The phenotypic values of each haplotype

Fig. 9

Proposed mechanism of four candidate genes function in enhancing anaerobic germination. Abbreviations are as follows: G6P, Glucose-6-phosphate; F6P, Fructose-6-phosphate; PEP, phosphoenolpyruvic acid; Phe, phenylalanine; TCA, tricarboxylic acid cycle

Fig. 10

a The relative content of gas and ABA in YZX seeds at different submerging time points. b The relative contents of three kinds of GA: GA₉/GA₁₅/GA₂₀ and ABA under the condition of anaerobic germination. AN is for anaerobic environment