Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants
Figure 2
The High Arsenic Content 1 (HAC1) gene controls natural variation in leaf arsenic in A. thaliana.
(A) Genome-wide association analysis of leaf arsenic concentration at 213,497 SNPs across 377 A. thaliana accessions using a mixed model analysis with correction for population structure. (B) A detailed plot of the peak region on chromosome 2 is shown with the location of HAC1 indicated by the vertical red line. (C) DNA microarray-based bulk segregant analysis of the high leaf arsenic phenotype of Kr-0 using phenotyped F2 progeny from a Kr-0×Col-0 cross genotyped using the 256K AtSNPtilling microarray. Lines represent allele frequency differences between high and low leaf arsenic pools of F2 plants at SNPs known to be polymorphic between Kr-0 and Col-0 (Solid line = sense strand probes, dashed line = antisense strand probes). (D) The causal gene was mapped between CAPS makers CS8901K and CS9249K using 315 F2 plants. (E) Fine mapping narrowed hac1 down to a 40 kb interval between markers CS9M and CS9040 using 1,321 F2 plants. Numbers below the horizontal line in (D) and (E) represent the number of recombinants between the indicated marker and hac1. (F) Gene structure of different HAC1 alleles. Arrows indicate T-DNA insertion sites for hac1-1 (GABI_868F11) and hac1-2 (SM_3_38332). Grey boxes indicate exons, and black lines indicate introns. The causal polymorphism in Kr-0 is shown to the right. (G) Leaf arsenic concentrations of different HAC1 alleles and their F1 progenies indicate through deficiency complementation that HAC1 is the causal gene for the high leaf arsenic in Kr-0. (H) Kr-0 was transformed with the Col-0 genomic DNA fragment of HAC1 (including 1.5Kb promoter sequence) and shown to complement the high leaf arsenic of Kr-0 to Col-0 levels in five independent transgenic lines (represented by numbers above the line in the x-axis legend), confirming HAC1 is the causal gene for high leaf arsenic in Kr-0. Data in (G) and (H) represents the means ± S.E. (n = 4–12 independent plants per genotype). Letters above bars indicate statistically different groups using a one-way ANOVA followed by least significant difference (LSD) test at the probability of p<0.05. All leaf arsenic concentration data are accessible using the digital object identifiers (DOIs) 10.4231/T9H41PBV and 10.4231/T9VD6WCJ (see http://dx.doi.org/) and available in Data S2.