eXtreme Array Mapping (XAM) for the low leaf Cd phenotype in Arabidopsis thaliana accession CS28181. Arabidopsis thaliana

Understanding the mechanism of cadmium (Cd) accumulation in plants is important to help reduce its potential toxicity to both plants and humans through dietary and environmental exposure. Here, we report on a study to uncover the genetic basis underlying natural variation in Cd accumulation in a world-wide collection of 349 wild collected Arabidopsis thaliana accessions. We identified a 4-fold variation (0.5 – 2 μg Cd g-1 dry weight) in leaf Cd accumulation when these accessions were grown in a controlled common garden. By combining genome-wide association mapping, linkage mapping in an experimental F2 population and transgenic complementation, we reveal that HMA3 is the sole major locus responsible for the variation in leaf Cd accumulation we observe in this diverse population of A. thaliana accessions. Analysis of the predicted amino acid sequence of HMA3 from 149 A. thaliana accessions reveals the existence of 10 major natural protein haplotypes. Association of these haplotypes with leaf Cd accumulation and genetics complementation experiments indicate that 5 of these haplotypes are active and 5 are inactive, and that elevated leaf Cd accumulation is associated with the reduced function of HMA3 caused by a nonsense mutation and polymorphisms that change two specific amino acids. Overall design: Genomic DNA from two F2 progeny pools (low leaf Cd pool and high leaf Cd pool) of CS28181 x Col-0 was labelled and hybridized separately to the Affymetrix SNP-tiling array AtSNPtile. The signal intensity difference between the two pools for all probes were analyzed using R scripts that are available at http://ars.usda.gov/mwa/bsasnp .

解析植物镉（Cd）积累的分子机制，对于通过膳食与环境暴露途径降低镉对植物及人类的潜在毒性具有重要意义。本研究以全球范围内收集的349份野生拟南芥（Arabidopsis thaliana）种质资源为材料，解析其叶片镉积累自然变异的遗传基础。在可控同质园环境下种植该批种质时，我们发现其叶片镉积累量存在4倍差异，范围为0.5~2 μg Cd·g⁻¹干重。通过整合全基因组关联分析、实验性F₂群体连锁定位与转基因互补实验，我们发现HMA3是调控本研究中拟南芥种质群体叶片镉积累变异的唯一主效位点。对149份拟南芥种质的HMA3预测氨基酸序列进行分析，共鉴定出10种主要的天然蛋白质单倍型。将这些单倍型与叶片镉积累量进行关联分析，并结合遗传互补实验结果显示：其中5种单倍型具备功能活性，剩余5种则丧失功能；叶片镉积累量升高与HMA3功能弱化显著相关，而该功能弱化由无义突变以及改变两个特定氨基酸的多态性所导致。实验整体设计：将CS28181与Col-0杂交获得的F₂后代分为两个混池（低叶片镉积累混池与高叶片镉积累混池），提取其基因组DNA并分别进行标记，随后分别与Affymetrix公司的SNP平铺阵列芯片AtSNPtile进行杂交。针对所有探针的信号强度差异，我们采用公开于http://ars.usda.gov/mwa/bsasnp 的R脚本完成分析。