Genetic control of the root system in rice under normal and drought stress conditions by genome-wide association study

Abstract

A variety of adverse conditions including drought stress severely affect rice production. Root system plays a critical role in drought avoidance, which is one of the major mechanisms of drought resistance. In this study, we adopted genome-wide association study (GWAS) to dissect the genetic basis controlling various root traits by using a natural population consisting of 529 representative rice accessions. A total of 413 suggestive associations, containing 143 significant associations, were identified for 21 root traits, such as maximum root length, root volume, and root dry weight under normal and drought stress conditions at the maturation stage. More than 80 percent of the suggestive loci were located in the region of reported QTLs for root traits, while about 20 percent of suggestive loci were novel loci detected in this study. Besides, 11 reported root-related genes, including DRO1, WOX11, and OsPID, were found to co-locate with the association loci. We further proved that the association results can facilitate the efficient identification of causal genes for root traits by the two case studies of Nal1 and OsJAZ1. These loci and their candidate causal genes provide an important basis for the genetic improvement of root traits and drought resistance.

Fig 1. Comparison of significant association loci for four categories of root traits under different conditions.

Significant association loci under normal conditions, drought stress conditions, and normal and drought stress conditions simultaneously, are shown in green, red and blue, respectively.

Fig 2. Root association loci overlapping with reported root QTLs.

Proportion of root association loci overlapping with reported QTLs for any root-related traits (A) and for the four categories of root traits (B), respectively. The black and white portions of each bar indicate the overlapping loci and nonoverlapping loci, respectively, and the overlapping percentage is shown in bracket at the top of each bar.

Fig 3. Genome-wide association results for 8 root traits.

Manhattan plots (left) and quantile-quantile plots (right) are presented for (A) RWDD, (B) RWDN, (C) RVDN, (D) RVTN, (E) DRVD, (F) RWSN, (G) RVDD, and (H) RWTN. For the Manhattan plots, -log₁₀ P-values from a genome-wide scan were plotted against the position of the SNPs on each of 12 chromosomes and the horizontal grey dashed line indicates the suggestive threshold (P = 1.21×10⁻⁶). For the quantile-quantile plots, the horizontal axis indicates the -log₁₀-transformed expected P-values, and the vertical axis indicates the -log₁₀-transformed observed P-values. The names of reported root-related genes near the association signals are indicated.

Fig 4. Sequence, expression and phenotypic analyses of Nal1.

(A) Sequence variations in the promoter (2 kb upstream of the start codon) of Nal1 between ZS97B and IRAT109. (B) Sequence variations in the ORF of Nal1 between ZS97B and IRAT109 (the corresponding amino acid changes are shown in parentheses). (C) Relative expression levels of Nal1 in different tissues of near isogenic lines (NILs) qZS and qIR at different stages. Panicle-B, panicle before heading; Panicle-A, panicle after heading; (D) Southern blot of complementary transgenic plants. Single copy line COM4 and negative control line NC are indicated by dashed boxes. (E) Genotypic identification of the complementary plants and NILs. (F) Root volume of the complementary lines and NILs. Data represent the mean ± SE (n = 10). **P < 0.01, Student’s t-test.

Fig 5. Root phenotypes of Nal1 transgenic plants and association analysis of Nal1.

(A, F) Relative expression level of Nal1 in two overexpression lines (A) and two RNAi lines (F), compared to the corresponding segregated negative control under normal conditions. (B, D) Visual root phenotypes of the Nal1-overexpression (OE6-3(+)) plants and the segregated negative-transgenic control (OE6-13(-)) at the seed maturation stage in PVC tubes under normal (B) and drought stress (D) conditions. (C, E) Root volume and weight of OE6-3(+) plants and OE6-13(-) control at the seed maturation stage in PVC tubes under normal (C) and drought stress (E) conditions. Data represent the mean ± SE (n = 10). **P < 0.01, Student’s t-test. (G, I) Visual root phenotype of the Nal1-RNAi (Ri12-11(+)) plants and the segregated negative-transgenic control (Ri12-5(-)) at the seed maturation stage in PVC tubes under normal (G) and drought stress (I) conditions. (H, J) Root volume and weight of Ri12-11(+) plants and Ri12-5(-) at the seed maturation stage in PVC tubes under normal (H) and drought stress (J) conditions. Data represent the mean ± SE (n = 10). **P < 0.01, Student’s t-test. (K) Association analysis of sequence variations of Nal1 with RVTN and the pattern of pair wise LD of associated SNPs. (L) RVTN of two main haplotypes of Nal1.

Fig 6. Association analysis of OsJAZ1 and the root phenotypes of OsJAZ1-OE plants.

(A) Association analysis of the genetic variation of OsJAZ1 with RWTN and the pattern of pair wise LD of the associated SNPs in OsJAZ1. (B) RWTN of two main haplotypes of OsJAZ1. (C) Relative expression levels of OsJAZ1 in the seedling and root of OsJAZ1-OE plants and wild-type ZH11 at the seedling stage. (D, E) Visual root phenotypes of OE and ZH11 plants at the tillering (D) and the seed maturation (E) stages. (F) Root dry weight of OE and ZH11 plants at the tillering and the seed maturation stages. Data represent the mean ± SE (n = 15). **P < 0.01, Student’s t-test.