Genome wide association analysis of root hair traits in rice reveals novel genomic regions controlling epidermal cell differentiation

Abstract

Background: Genome wide association (GWA) studies demonstrate linkages between genetic variants and traits of interest. Here, we tested associations between single nucleotide polymorphisms (SNPs) in rice (Oryza sativa) and two root hair traits, root hair length (RHL) and root hair density (RHD). Root hairs are outgrowths of single cells on the root epidermis that aid in nutrient and water acquisition and have also served as a model system to study cell differentiation and tip growth. Using lines from the Rice Diversity Panel-1, we explored the diversity of root hair length and density across four subpopulations of rice (aus, indica, temperate japonica, and tropical japonica). GWA analysis was completed using the high-density rice array (HDRA) and the rice reference panel (RICE-RP) SNP sets.

Results: We identified 18 genomic regions related to root hair traits, 14 of which related to RHD and four to RHL. No genomic regions were significantly associated with both traits. Two regions overlapped with previously identified quantitative trait loci (QTL) associated with root hair density in rice. We identified candidate genes in these regions and present those with previously published expression data relevant to root hair development. We re-phenotyped a subset of lines with extreme RHD phenotypes and found that the variation in RHD was due to differences in cell differentiation, not cell size, indicating genes in an associated genomic region may influence root hair cell fate. The candidate genes that we identified showed little overlap with previously characterized genes in rice and Arabidopsis.

Conclusions: Root hair length and density are quantitative traits with complex and independent genetic control in rice. The genomic regions described here could be used as the basis for QTL development and further analysis of the genetic control of root hair length and density. We present a list of candidate genes involved in root hair formation and growth in rice, many of which have not been previously identified as having a relation to root hair growth. Since little is known about root hair growth in grasses, these provide a guide for further research and crop improvement.

Keywords: Candidate gene; GWAS; Rice; Root biology; Root hairs; Subpopulation.

Fig. 1

RH traits of all lines. A RHD expressed as hairs per mm². The group of lines in “other” includes admixed lines and aromatic lines. Significance is indicated with the displayed p value obtained after a Dunn Test. B RHL expressed as length in mm. C Sample images of four genotypes with their average RHD and RHL across all samples. Scale bar is 1 mm. D Correlation between RHD and RHL for all genotypes

Fig. 2

Region L1 on chromosome 5 related to RHL in ALL. A Chromosome-wide Manhattan plots of genome-wide HDRA (GW HDRA) SNPs (top), a pruned set of genome-wide RICE-RP SNPs (GW pruned SNPs, middle), and all RICE-RP SNPs in a chromosome-wide scan (CW All SNPs, bottom). The grey region indicates the genomic region. B Comparison of the RHL phenotype between lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69], orange indicates no previous expression evidence, and white indicates transposable elements

Fig. 3

Region L2 on chromosome 6 related to RHL in ALL and JAPONICA. A Chromosome-wide Manhattan plots of HDRA genome-wide analysis (GW HDRA, left) and all RICE-RP SNPs in a chromosome-wide scan (CW RICE-RP SNPs, right). The grey region indicates the genomic region. B Comparison of the RHL phenotype between lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69], orange indicates no previous expression evidence, and white indicates transposable elements. Haplotypes are presented below the Manhattan plot, with blue representing reference alleles and yellow, alternate alleles, across these regions. D Comparison of the phenotypes of the haplotypes of panel C with significance indicated as a BH-adjusted, Kruskal-Wallace test with a Dunn posthoc test

Fig. 4

Regions L3 and L4 on chromosome 8 associated with RHL in ALL. A Chromosome-wide Manhattan plots of HDRA SNPs (top) and all RICE-RP SNPs in a chromosome-wide scan (bottom). The grey region indicates the genomic regions. B Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region L4. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69], orange indicates no previous expression evidence, and white indicates transposable elements. Below, haplotypes 1–9 are displayed with blue representing the reference identity and yellow, alternate, across this region. C Comparison of the RHL phenotype between ALL lines carrying the alternate and reference allele at the MS-SNP for L3 (top) and L4 (bottom). Significance is indicated by a t-test. D Phenotypes of the haplotypes with significance indicated as a BH-adjusted, Kruskal-Wallace test with a Dunn posthoc test

Fig. 5

Region D2 on chromosome 1 related to RHD in ALL A Chromosome-wide Manhattan plots of HDRA SNPs (left) and all RICE-RP SNPs in a chromosome-wide scan (right). The grey region indicates the genomic region. B Comparison of the RHD phenotype between ALL lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69], orange indicates no previous expression evidence, and white indicates transposable elements. Below, haplotypes 1–6 are displayed with blue representing the reference identity and yellow, alternate, across this region. D Phenotypes of the haplotypes with significance indicated as a BH-adjusted, Kruskal-Wallace test with a Dunn-posthoc test

Fig. 6

Region D3 on chromosome 1 related to RHD in ind. A Chromosome-wide Manhattan plots of HDRA SNPs (left) and all RICE-RP SNPs in a chromosome-wide scan (right). The grey region indicates the genomic region. B Comparison of the RHD phenotype between ind lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69], orange indicates no previous expression evidence, and white indicates transposable elements. Below, haplotypes 1–6 are displayed with blue representing the reference identity and yellow, alternate, across this region. D Phenotypes of the haplotype with p values from a Dunn posthoc test

Fig. 7

Region D5 on chromosome 6 related to RHD in ALL A Chromosome-wide Manhattan plots of HDRA SNPs (left) and all RICE-RP SNPs in a chromosome-wide scan (right). The grey region indicates the genomic region. B Comparison of the RHD phenotype between ALL lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69], orange indicates no previous expression evidence, and white indicates transposable elements. Below, haplotypes 1–8 are displayed with blue representing the reference identity and yellow, alternate, across this region. D Phenotypes of the haplotypes with significance indicated as a BH-adjusted, Kruskal-Wallace test with a Dunn posthoc test

Fig. 8

Region D12 on chromosome 10 related to RHD in ALL A Chromosome-wide Manhattan plots of HDRA SNPs (left) and all RICE-RP SNPs in a chromosome-wide scan (right). The grey region indicates the genomic region. B Comparison of the RHD phenotype between ALL lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69] and orange indicates no previous expression evidence

Fig. 9

Region D6 on chromosome 7 related to RHD in ind A Chromosome-wide Manhattan plots of HDRA SNPs (left) and all RICE-RP SNPs in a chromosome-wide scan (right). The grey region indicates the genomic region. B Comparison of the RHD phenotype between ind lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Orange genes are not expressed in root hairs or root-hair forming cells according to Huang et al. [69]. Below, haplotypes 1–5 are displayed with blue representing the reference identity and yellow, alternate, across this region. D Phenotypes of the haplotypes with significance indicated as a BH-adjusted, Kruskal-Wallace test with a Dunn posthoc test

Fig. 10

Region D8 on chromosome 8 related to RHD in ind A Chromosome-wide Manhattan plots of HDRA SNPs (left) and all RICE-RP SNPs in a chromosome-wide scan (right). The grey region indicates the genomic region. B Comparison of the RHD phenotype between ind lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69], orange indicates no previous expression evidence, and white indicates transposable elements. Below, haplotypes 1–4 are displayed with blue representing the reference identity and yellow, alternate, across this region. D Phenotypes of the haplotypes with significance indicated as a BH-adjusted, Kruskal-Wallace test with a Dunn posthoc test

Fig. 11

Region D11 on chromosome 9 related to RHD in ind A Chromosome-wide Manhattan plots of HDRA SNPs (left) and all RICE-RP SNPs in a chromosome-wide scan (right). The grey region indicates the genomic region. B Comparison of the RHD phenotype between ind lines carrying the alternate and reference allele at the MS-SNP. Significance is indicated by a t-test. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Genes are overlaid on this plot and colored as follows. Green genes are expressed in root hairs or root-hair forming cells according to Huang et al. [69], orange indicates no previous expression evidence, and white indicates transposable elements. Below, haplotypes are displayed with blue representing the reference identity and yellow, alternate, across this region. D Phenotypes of the haplotypes with significance indicated as a BH-adjusted, Kruskal-Wallace test with a Dunn posthoc test

Fig. 12

Region D13 on chromosome 12 related to RHD in ind A Chromosome-wide Manhattan plots of HDRA SNPs (left) and all RICE-RP SNPs in a chromosome-wide scan (right). The grey region indicates the genomic region. B r2 based linkage analysis of the SNPs in this region. SNPs were randomly thinned and included in linkage analysis. Stronger linkage is represented by red coloring. A linkage block around the MS-SNP is outlined in green. C Magnification of the RICE-RP chromosome-wide Manhattan plot of the genomic region. Below, haplotypes are displayed with blue representing the reference alleles and yellow, alternate, across this region. Heterozygotes or missing reads are displayed as white

Fig. 13

Phenotyping of a select set of lines to investigate controls of RHD. A Significant SNPs from our analyses if ind lines across chromosomes 1, 5, 7, 8, 9, and 12. The lines we selected to phenotype further carried either the allele identity of reference (blue), alternate (yellow), or indeterminate (white). B Lines differ in density between the original (yellow, nodal roots, 8-week-old plants) and rephenotyped data sets (purple, seminal roots, 3-week-old plants). C Micrograph of the surface of a rice root with processed image highlighting hair (blue) and non-hair cells (pink). Scale bar represents 1 mm. D Lines do differ in the percent of cells that differentiate into hair forming cells. Significance was determined by a Tukey HSD test p < 0.05