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Genome-wide Association Study and Candidate Gene Analysis of Alkalinity Tolerance in Japonica Rice Germplasm at the Seedling Stage

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Genome-wide Association Study and Candidate Gene Analysis of Alkalinity Tolerance in Japonica Rice Germplasm at the Seedling Stage

Ning Li et al. Rice (N Y).

Abstract

Background: Salinity-alkalinity stress is one of the major factors limiting rice production. The damage caused by alkaline salt stress to rice growth is more severe than that caused by neutral salt stress. At present, the genetic resources (quantitative trait loci (QTLs) and genes) that can be used by rice breeders to improve alkalinity tolerance are limited. Here, we assessed the alkalinity tolerance of rice at the seedling stage and performed a genome-wide association study (GWAS) based on genotypic data including 788,396 single-nucleotide polymorphisms (SNPs) developed by re-sequencing 295 japonica rice varieties.

Results: We used the score of alkalinity tolerance (SAT), the concentrations of Na+ and K+ in the shoots (SNC and SKC, respectively) and the Na+/K+ ratio of shoots (SNK) as indices to assess alkalinity tolerance at the seedling stage in rice. Based on population structure analysis, the japonica rice panel was divided into three subgroups. Linkage disequilibrium (LD) analysis showed that LD decay occurred at 109.77 kb for the whole genome and varied between 13.79 kb and 415.77 kb across the 12 chromosomes, at which point the pairwise squared correlation coefficient (r2) decreased to half of its maximum value. A total of eight QTLs significantly associated with the SAT, SNC and SNK were identified by genome-wide association mapping. A common QTL associated with the SAT, SNC and SNK on chromosome 3 at the position of 15.0 Mb, which explaining 13.36~13.64% of phenotypic variation, was selected for further analysis. The candidate genes were filtered based on LD decay, Gene Ontology (GO) enrichment, RNA sequencing data, and quantitative real-time PCR (qRT-PCR) analysis. Moreover, sequence analysis revealed one 7-bp insertion/deletion (indel) difference in LOC_Os03g26210 (OsIRO3) between the alkalinity-tolerant and alkalinity-sensitive rice varieties. OsIRO3 encodes a bHLH-type transcription factor and has been shown to be a negative regulator of the Fe-deficiency response in rice.

Conclusion: Based on these results, OsIRO3 maybe a novel functional gene associated with alkalinity tolerance in japonica rice. This study provides resources for improving alkalinity tolerance in rice, and the functional molecular marker could be verified to breed new rice varieties with alkalinity tolerance via marker-assisted selection (MAS).

Keywords: Alkalinity tolerance; Gene; Genome-wide association study (GWAS); Japonica rice.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Phenotypic variation in the SNC, SKC, SNK and SAT in 295 japonica rice varieties. a, The score of alkalinity tolerance (SAT). b, The concentration of Na+ in the shoots (SNC). c, The concentration of K+ in the shoots (SNC). d, The Na+/K+ ratio of shoots (SNK)
Fig. 2
Fig. 2
Population structure of 295 japonica rice varieties. a, The CV error of each K value. b, Subgroups (K = 3) inferred using ADMIXTURE software. c, Principal component analysis of 295 japonica rice varieties. Colors of green, blue, and red represent P1, P2 and P3 in Fig. 2b, respectively. d, Neighbor-joining tree of 295 japonica rice varieties. Colors of green, blue, and red represent P1, P2 and P3 in Fig. 2b, respectively
Fig. 3
Fig. 3
Manhattan plots and quantile-quantile (Q-Q) plots of genome-wide association studies for the SNC, SNK and SAT. a, Manhattan plot for the SNC. b, Q-Q plot for the SNC. c, Manhattan plot for the SNK. d, Q-Q plot for SNK. e, Manhattan plot for the SAT. f, Q-Q plot for the SAT
Fig. 4
Fig. 4
The location of qSNC3 on chromosome 3 and sequence difference analysis of LOC_Os03g26210. a, Colocalization of LOC_Os03g26210 and LOC_Os03g26430 with qSNC3. The arrow indicates the location and direction of LOC_Os03g26210 and LOC_Os03g26430. b, The gene structure of LOC_Os03g26210. c, Sequence differences in LOC_Os03g26210 between ten alkalinity-tolerant varieties (low SAT) and ten alkalinity-sensitive varieties (high SAT). T indicates alkalinity-tolerant varieties, S indicates alkalinity-sensitive varieties, Ref is the reference sequence of Nipponbare genome
Fig. 5
Fig. 5
Expression patterns of LOC_Os03g26210 and LOC_Os03g26430 under normal growth conditions and alkalinity stress conditions. C indicates normal growth conditions, T indicates alkalinity stress conditions. The number preceding C and T indicates the number of rice varieties
Fig. 6
Fig. 6
The distribution of tolerant genotype and sensitive genotype in 126 japonica rice varieties. T (blue box) indicates tolerant genotype without the 7 bp sequence, S (red box) indicates sensitive genotype with the 7 bp sequence

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