Comprehensive analysis of Arabidopsis expression level polymorphisms with simple inheritance

• Published in: Molecular systems biology Vol. 5; no. 1; pp. 242 - n/a
• Main Authors: Goldstein, Darlene R, Hardtke, Christian S, Weber, Johann, Nussbaumer, Cindy, Harshman, Keith, Plantegenet, Stephanie, Weigel, Detlef, Zeller, Georg, Thomas, Jérôme
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 17-02-2009; John Wiley & Sons, Ltd; EMBO Press; Nature Publishing Group; Springer Nature
• Subjects: Arabidopsis; Arabidopsis - genetics; Arabidopsis thaliana; Base Sequence; Cloning; Deoxyribonucleic acid; DNA; EMBO09; EMBO30; eQTL; expression level polymorphism; Gene expression; Gene Expression Profiling; Gene Expression Regulation, Plant; Gene mapping; Genes; Genetic variability; Genetic Variation; Genomes; Heredity; heritability of expression; Mutation; Nucleotide sequence; Phenotypic variations; Polymorphism, Genetic; QTL; Quantitative genetics; Quantitative Trait Loci; Regulatory sequences; Regulatory Sequences, Nucleic Acid; Software; structural gene variation; Tiling; Transcription; Netherlands; QTL; structural gene variation; eQTL; expression level polymorphism; Arabidopsis; heritability of expression
• ISSN: 1744-4292; 1744-4292
• DOI: 10.1038/msb.2008.79

In
Arabidopsis thaliana
, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome‐wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil‐0 and Lc‐0 accessions. Compared with non‐variable controls, 5′ regulatory sequence variation in the corresponding genes is indeed increased. However, ∼42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a >4‐fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay‐0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL.
Synopsis
Microarray technologies have had a major impact on quantitative genetic analyses, enabling, for instance, large‐scale discovery of genetically controlled gene expression level differences. This has led to the identification of numerous expression quantitative trait loci (eQTL) in various organisms (Brem
et al
,
2002
; Morley
et al
,
2004
; Doss
et al
,
2005
; Li
et al
,
2006
; West
et al
,
2007
; Stranger
et al
,
2007b
; Potokina
et al
,
2008
). In this study, we focused on expression level polymorphisms (ELPs) that are observed between parents and display simple, single locus inheritance. Such loci constitute a highly heritable subset of
cis
‐acting eQTL (Doss
et al
,
2005
; Petretto
et al
,
2006
; West
et al
,
2006
,
2007
; Keurentjes
et al
,
2007
; Stranger
et al
,
2007b
; Potokina
et al
,
2008
) and are strong quantitative trait locus (QTL) candidates to explain phenotypic variation between parental lines. Despite the abundance of ELPs with simple inheritance, little is known about their molecular basis. Generally, however, they are assumed to reflect
cis
‐acting sequence polymorphisms in regulatory elements of the corresponding genes (Jansen and Nap,
2001
; Cowles
et al
,
2002
; Schadt
et al
,
2003
; Pastinen and Hudson,
2004
; Ronald
et al
,
2005
; Williams
et al
,
2007
), although few studies have addressed this issue systematically (Cowles
et al
,
2002
; GuhaThakurta
et al
,
2006
).
Counter to the idea that regulatory polymorphisms are major determinants of phenotypic variability, QTL cloning in
Arabidopsis thaliana
has mostly identified knockout mutations as the underlying molecular cause (e.g. Aukerman
et al
,
1997
; Grant
et al
,
1995
; Johanson
et al
,
2000
; Kliebenstein
et al
,
2001
; Kroymann
et al
,
2003
; Kroymann
et al
,
2001
; Mouchel
et al
,
2004
; Werner
et al
,
2005
). Even if many of these loci represent ELPs, generally, a preponderance of indels is observed among these drastic mutations (Koornneef
et al
,
2004
). However, because structural changes are easier to discover, successful reports of QTL isolation might reflect a technical bias towards knockout alleles. Indeed, studies of recombinant inbred line (RIL) populations created from Arabidopsis accessions have identified numerous eQTL (Keurentjes
et al
,
2007
; West
et al
,
2007
), including a sizable fraction of loci representing parental ELPs with simple inheritance. In this study, we analyzed the molecular basis of such ELPs in greater detail, by comprehensive comparison of gene expression, sequence variation and gene structure.
We identified parental ELPs in 480 genes through microarray analyses of the Arabidopsis accessions Eil‐0 and Lc‐0, a number comparable with studies of other accessions (West
et al
,
2006
; Keurentjes
et al
,
2007
). Among them, ELPs with simple inheritance were determined by comparison between single nucleotide polymorphism (SNP)‐genotyped RILs derived from the two accessions and their two parents in microarray analyses. Approximately 20% of parental ELPs displayed simple
cis
‐inheritance; taking into account technical limitations (such as arbitrary thresholds for differential expression), this group is very likely to be even bigger (∼44%).
To determine whether these ELPs with simple inheritance are associated with increased regulatory sequence variation in the corresponding genes, we compared their promoter sequences with control genes that displayed very low variability across all microarray experiments. Indeed, sequence diversity between Eil‐0 and Lc‐0 was considerably higher in the ELP sample, supporting the regulatory hypothesis. However, parallel analyses of genomic Eil‐0 and Lc‐0 DNA using Arabidopsis tiling arrays, empirically calibrated for indel detection using the sequence data (Figure
3
), revealed numerous indels of various sizes in both Eil‐0 and Lc‐0. Such indels were particularly abundant in genes representing ELPs with simple inheritance and, unlike in controls, were generally larger and frequently affected exons. Thus, uni‐parental indels that likely impair or even abolish gene function appear to be much more frequent in genes representing ELPs with simple inheritance than in controls. In the vast majority of cases, the allele that carried deletions was expressed at lower level, consistent with the idea that the majority of deletions negatively affect gene function and lead to loss of selection on gene maintenance and consequently expression. Supporting this notion, parental ELPs that carried indels in their coding region also displayed increased regulatory sequence variation. Notably, the observation that alleles carrying deletions were expressed at lower levels could simply reflect decreased hybridization signal if the deletion overlapped with the probe. However, except for a minority of loci with large deletions, this was generally not evident from the expression array analyses.
To independently corroborate our findings, we analyzed reported ELPs with simple inheritance that were found between the Arabidopsis Bay‐0 and Sha accessions (West
et al
,
2006
,
2007
), using a different microarray platform and a different conceptual approach to extract heritable ELPs. Again, in genome tiling arrays, we observed a strong preponderance of indels in the ELPs with simple inheritance (>6‐fold enrichment) as compared with controls. Similar to our results for Eil‐0 and Lc‐0, their majority occurred at the level of exons (∼33% of loci) or genes (18%).
These findings were corroborated by a recently developed algorithm (Zeller
et al
,
2008
) that identified reduced or absent hybridization signal over extended tracts in an oligonucleotide array‐based re‐sequencing effort of the Bay‐0 and Sha genomes. Matching our tiling array analysis, in nearly all cases, stretches of reduced hybridization matched the presence of deletions as detected by the tiling array approach (Figure
6
).
In summary, our data suggest that ELPs with simple inheritance in Arabidopsis primarily reflect the consequences of structural differences in the corresponding genes, rather than variation in regulatory elements, even if such a variation is observed. Thus, although functional variation in
ci
s‐regulatory elements contributes clearly to phenotypic variation (Bentsink
et al
,
2006
; Rus
et al
,
2006
; Sibout
et al
,
2008
), large‐effect changes that impact the integrity of transcribed regions should be considered as an equally valid explanation for expression variation. Indeed, the prevalence of indels in ELPs with simple inheritance mirrors the preponderance of indels with drastic effect on gene integrity underlying cloned QTL, suggesting that the latter do not reflect a technical bias in the ease of detection. Thus, Arabidopsis QTL representing more subtle regulatory polymorphisms might be less common than anticipated.
Heritable gene expression level polymorphisms (ELPs) between natural strains are strong candidates for quantitative trait loci (QTL) that could explain intra‐specific phenotypic variation.
Here we test the assumption that ELPs with simple, single locus inheritance primarily represent sequence variation in the corresponding regulatory sequences in Arabidopsis thaliana, through comprehensive genome‐wide analyses linking variation in expression level, regulatory sequence and gene structure.
We find that a large fraction of genes representing ELPs with simple inheritance carry uni‐parental indels that likely impair gene function. Thus, these ELPs primarily appear to reflect the consequences of structural differences in the corresponding genes, rather than variation in regulatory elements, even if such variation is observed.
Our results are in line with the experimentally observed preponderance of indels with drastic effects on gene integrity in cloned Arabidopsis QTL, suggesting that they do not reflect a technical bias and that Arabidopsis QTL representing more subtle regulatory polymorphisms might be less common than anticipated.