A Genome-Wide Association Analysis Reveals Epistatic Cancellation of Additive Genetic Variance for Root Length in Arabidopsis thaliana

Efforts to identify loci underlying complex traits generally assume that most genetic variance is additive. Here, we examined the genetics of Arabidopsis thaliana root length and found that the genomic narrow-sense heritability for this trait in the examined population was statistically zero. The low amount of additive genetic variance that could be captured by the genome-wide genotypes likely explains why no associations to root length could be found using standard additive-model-based genome-wide association (GWA) approaches. However, as the broad-sense heritability for root length was significantly larger, and primarily due to epistasis, we also performed an epistatic GWA analysis to map loci contributing to the epistatic genetic variance. Four interacting pairs of loci were revealed, involving seven chromosomal loci that passed a standard multiple-testing corrected significance threshold. The genotype-phenotype maps for these pairs revealed epistasis that cancelled out the additive genetic variance, explaining why these loci were not detected in the additive GWA analysis. Small population sizes, such as in our experiment, increase the risk of identifying false epistatic interactions due to testing for associations with very large numbers of multi-marker genotypes in few phenotyped individuals. Therefore, we estimated the false-positive risk using a new statistical approach that suggested half of the associated pairs to be true positive associations. Our experimental evaluation of candidate genes within the seven associated loci suggests that this estimate is conservative; we identified functional candidate genes that affected root development in four loci that were part of three of the pairs. The statistical epistatic analyses were thus indispensable for confirming known, and identifying new, candidate genes for root length in this population of wild-collected A. thaliana accessions. We also illustrate how epistatic cancellation of the additive genetic variance explains the insignificant narrow-sense and significant broad-sense heritability by using a combination of careful statistical epistatic analyses and functional genetic experiments.鉴定复杂性状相关基因座（loci）的研究通常假定，绝大多数遗传方差为加性遗传方差。本研究以拟南芥（Arabidopsis thaliana）根长为研究对象，发现所分析群体中该性状的基因组狭义遗传力（narrow-sense heritability）在统计学上为零。通过全基因组基因型分型所能捕获的加性遗传方差极低，这可解释为何采用基于标准加性模型的全基因组关联分析（GWA）方法，未能检测到与根长相关的关联位点。然而，根长的广义遗传力（broad-sense heritability）显著更高，且该遗传力主要源于上位性（epistasis）效应。为此，我们开展了上位性全基因组关联分析（epistatic GWA），以定位贡献上位性遗传方差的基因座。最终鉴定出4对互作基因座，涉及7个通过标准多重检验校正显著性阈值的染色体基因座。上述基因座对的基因型-表型图谱（genotype-phenotype maps）显示，其上位性效应抵消了加性遗传方差，这也解释了为何这些位点在加性全基因组关联分析中未被检出。诸如本实验这类小型群体，由于在少量表型观测个体中针对极大量多标记基因型组合开展关联检验，会提升检出假阳性上位性互作的风险。为此，我们采用一种新型统计方法估算假阳性风险（false-positive risk），结果显示约半数关联位点对属于真阳性关联。我们对7个关联位点内的候选基因开展实验验证，结果表明该假阳性风险估算值偏保守；我们在其中4个位点（对应3对互作基因座）中鉴定出影响根发育的功能候选基因。因此，统计上位性分析对于在该野生采集拟南芥生态型（accessions）群体中，确认根长相关已知候选基因并挖掘新候选基因而言不可或缺。此外，我们通过结合严谨的统计上位性分析与功能遗传学实验，阐明了上位性对加性遗传方差的抵消效应如何导致该性状狭义遗传力不显著，而广义遗传力显著的现象。