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

Lachowiec J, Shen X, Queitsch C, Carlborg Ö - PLoS Genet. (2015)

Bottom Line: 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.Therefore, we estimated the false-positive risk using a new statistical approach that suggested half of the associated pairs to be true positive associations.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America; Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
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.

No MeSH data available.


Summary of combined statistical and functional support for loci underlying root length.Four epistatic pairs, involving seven unique loci (connected boxes), were identified at a genome-wide significance-threshold in a two-dimensional genome-wide GWA scan. Green, yellow, and red lines connect pairs of loci with very low (p = 0.003), intermediate (0.28 < p < 0.37), and high (p = 0.95) risk for the interaction being a false-positive when accounting for population size. The risk of the statistical epistatic association resulting from high-order LD to an unobserved functional variant in the genome (i.e. “apparent epistasis”) is illustrated by arrow color, in which yellow indicates an intermediate risk (0.18 < p < 0.42) and green a very low risk (p = 0.0021). Green boxes indicate loci for which the T-DNA insertion line analyses suggest the named genes to be involved in root development. When considering the joint statistical and functional results, two pairs emerge as highly likely true positive two-locus associations: 3_66596/3_9273674 due to very strong statistical support and one identified functional candidate gene, and 3_10891195/5_1027939 where the identification of functional candidate genes at both loci suggest that the two-locus association in the original genome-wide scan is true despite the lower statistical support in after the conservative statistical correction for sample-size. For the other two pairs, the results are inconclusive. There is strong support for one of the two associated loci (3_66596 from its statistical interaction with 3_9273674 and 1_17257526 by the detection of the a functional candidate gene in the T-DNA analysis), but weaker support for the second locus. Further work is thus needed to conclude whether these pairs represent true positive two-locus associations, or whether they are false-positives due to the small population-size or high-order LD (“apparent epistasis”) to unknown functional variants.
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pgen.1005541.g004: Summary of combined statistical and functional support for loci underlying root length.Four epistatic pairs, involving seven unique loci (connected boxes), were identified at a genome-wide significance-threshold in a two-dimensional genome-wide GWA scan. Green, yellow, and red lines connect pairs of loci with very low (p = 0.003), intermediate (0.28 < p < 0.37), and high (p = 0.95) risk for the interaction being a false-positive when accounting for population size. The risk of the statistical epistatic association resulting from high-order LD to an unobserved functional variant in the genome (i.e. “apparent epistasis”) is illustrated by arrow color, in which yellow indicates an intermediate risk (0.18 < p < 0.42) and green a very low risk (p = 0.0021). Green boxes indicate loci for which the T-DNA insertion line analyses suggest the named genes to be involved in root development. When considering the joint statistical and functional results, two pairs emerge as highly likely true positive two-locus associations: 3_66596/3_9273674 due to very strong statistical support and one identified functional candidate gene, and 3_10891195/5_1027939 where the identification of functional candidate genes at both loci suggest that the two-locus association in the original genome-wide scan is true despite the lower statistical support in after the conservative statistical correction for sample-size. For the other two pairs, the results are inconclusive. There is strong support for one of the two associated loci (3_66596 from its statistical interaction with 3_9273674 and 1_17257526 by the detection of the a functional candidate gene in the T-DNA analysis), but weaker support for the second locus. Further work is thus needed to conclude whether these pairs represent true positive two-locus associations, or whether they are false-positives due to the small population-size or high-order LD (“apparent epistasis”) to unknown functional variants.

Mentions: Two linked SNPs on chromosome 5 (5_15862026 and 5_15862525; Table 2) were detected by their statistical epistatic interaction with an SNP on chromosome 1 (17,257,526 bp—SNP 1_17257526; Table 2). Both of these linked SNPs, as well as the other SNPs in high LD, were intergenic with the nearest gene, NAC6 (At5g39610; Table 5), which is located 1.9 kb away. NAC6 is a transcription factor regulating leaf senescence [46] and is highly expressed in senescing leaves and in maturing seeds [44]. We phenotyped the available At5g39610 insertion mutant for NAC6 and found a significant decrease in mean root length (p = 5.82 × 10−10; Tukey’s post-hoc test; Fig 4), strongly implicating NAC6 in the regulation of root length.


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

Lachowiec J, Shen X, Queitsch C, Carlborg Ö - PLoS Genet. (2015)

Summary of combined statistical and functional support for loci underlying root length.Four epistatic pairs, involving seven unique loci (connected boxes), were identified at a genome-wide significance-threshold in a two-dimensional genome-wide GWA scan. Green, yellow, and red lines connect pairs of loci with very low (p = 0.003), intermediate (0.28 < p < 0.37), and high (p = 0.95) risk for the interaction being a false-positive when accounting for population size. The risk of the statistical epistatic association resulting from high-order LD to an unobserved functional variant in the genome (i.e. “apparent epistasis”) is illustrated by arrow color, in which yellow indicates an intermediate risk (0.18 < p < 0.42) and green a very low risk (p = 0.0021). Green boxes indicate loci for which the T-DNA insertion line analyses suggest the named genes to be involved in root development. When considering the joint statistical and functional results, two pairs emerge as highly likely true positive two-locus associations: 3_66596/3_9273674 due to very strong statistical support and one identified functional candidate gene, and 3_10891195/5_1027939 where the identification of functional candidate genes at both loci suggest that the two-locus association in the original genome-wide scan is true despite the lower statistical support in after the conservative statistical correction for sample-size. For the other two pairs, the results are inconclusive. There is strong support for one of the two associated loci (3_66596 from its statistical interaction with 3_9273674 and 1_17257526 by the detection of the a functional candidate gene in the T-DNA analysis), but weaker support for the second locus. Further work is thus needed to conclude whether these pairs represent true positive two-locus associations, or whether they are false-positives due to the small population-size or high-order LD (“apparent epistasis”) to unknown functional variants.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4580642&req=5

pgen.1005541.g004: Summary of combined statistical and functional support for loci underlying root length.Four epistatic pairs, involving seven unique loci (connected boxes), were identified at a genome-wide significance-threshold in a two-dimensional genome-wide GWA scan. Green, yellow, and red lines connect pairs of loci with very low (p = 0.003), intermediate (0.28 < p < 0.37), and high (p = 0.95) risk for the interaction being a false-positive when accounting for population size. The risk of the statistical epistatic association resulting from high-order LD to an unobserved functional variant in the genome (i.e. “apparent epistasis”) is illustrated by arrow color, in which yellow indicates an intermediate risk (0.18 < p < 0.42) and green a very low risk (p = 0.0021). Green boxes indicate loci for which the T-DNA insertion line analyses suggest the named genes to be involved in root development. When considering the joint statistical and functional results, two pairs emerge as highly likely true positive two-locus associations: 3_66596/3_9273674 due to very strong statistical support and one identified functional candidate gene, and 3_10891195/5_1027939 where the identification of functional candidate genes at both loci suggest that the two-locus association in the original genome-wide scan is true despite the lower statistical support in after the conservative statistical correction for sample-size. For the other two pairs, the results are inconclusive. There is strong support for one of the two associated loci (3_66596 from its statistical interaction with 3_9273674 and 1_17257526 by the detection of the a functional candidate gene in the T-DNA analysis), but weaker support for the second locus. Further work is thus needed to conclude whether these pairs represent true positive two-locus associations, or whether they are false-positives due to the small population-size or high-order LD (“apparent epistasis”) to unknown functional variants.
Mentions: Two linked SNPs on chromosome 5 (5_15862026 and 5_15862525; Table 2) were detected by their statistical epistatic interaction with an SNP on chromosome 1 (17,257,526 bp—SNP 1_17257526; Table 2). Both of these linked SNPs, as well as the other SNPs in high LD, were intergenic with the nearest gene, NAC6 (At5g39610; Table 5), which is located 1.9 kb away. NAC6 is a transcription factor regulating leaf senescence [46] and is highly expressed in senescing leaves and in maturing seeds [44]. We phenotyped the available At5g39610 insertion mutant for NAC6 and found a significant decrease in mean root length (p = 5.82 × 10−10; Tukey’s post-hoc test; Fig 4), strongly implicating NAC6 in the regulation of root length.

Bottom Line: 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.Therefore, we estimated the false-positive risk using a new statistical approach that suggested half of the associated pairs to be true positive associations.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America; Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
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.

No MeSH data available.