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. 2009;5:242.
doi: 10.1038/msb.2008.79. Epub 2009 Feb 17.

Comprehensive Analysis of Arabidopsis Expression Level Polymorphisms With Simple Inheritance

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Free PMC article

Comprehensive Analysis of Arabidopsis Expression Level Polymorphisms With Simple Inheritance

Stephanie Plantegenet et al. Mol Syst Biol. .
Free PMC article

Abstract

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, approximately 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.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Assessment of ELP heritability by microarray analyses. RIL from a cross between Eil-0 and Lc-0 were genotyped with a set of 79 genome-wide SNP markers (Warthmann et al, 2007), defining the parental origin of chromosome segments. (A) Principles for the assessment of the heritability of ELPs observed between the Eil-0 and Lc-0 parents. Genotyped RIL from the S6 generation were compared with both parents in dye swap replicates. Based on the RIL genotype for a particular chromosome segment as determined by the flanking SNP markers, differential expression of a parental ELP locus on this chromosome segment was not expected in hybridizations of the RIL against the parent from whom the segment was inherited. However, differential expression (>2-fold) was expected in hybridizations against the other parent. ELPs located in regions of ambiguous genotype, i.e. heterozygous regions or segments spanning recombination breakpoints, were omitted from the analysis of that particular RIL. (B) Summary of parental ELP behavior in the hybridizations of the seven RILs (EL lines) against the two parent lines based on the principles outlined in (A). (C) Percentage of parental ELPs matching predictions across all RIL-parent hybridizations at a given frequency (100 or the 10% intervals below).
Figure 2
Figure 2
Sequence analysis of regulatory regions of a sample of 61 genes representing parental ELPs with simple inheritance and a control group of 85 genes, which displayed very low variability and differential expression (see Supplementary Materials and methods) in the array experiments (‘controls'). For the ELPs with simple inheritance, only genes which perfectly matched predictions (see Figure 1C), and for which at least 10 precise predictions could be made (i.e. loci located in unambiguous chromosome segments in at least five RIL) were included. (A) Summary of sequence analyses of regulatory regions from 61 ELPs with simple inheritance and 85 control genes. Observed total absolute values (tot. line), per gene average values (av. line) and median values (me. line) are indicated. Note that numbers for promoter sequences and 5′ leader sequences do not add up to the total, because leader sequences were not defined for all genes investigated. (B) Relative abundance of SNPs (based on total sequence investigated). (C) Relative amount of bp affected by indels (based on total sequence investigated). Asterisks indicate t-test significance between the ELPs with simple inheritance and the control group (*P<0.05; **P<0.01; NS, not significant).
Figure 3
Figure 3
Genomic tiling array analysis of the Eil-0 and Lc-0 genomic DNA hybridized against a tile of the Col-0 genome. Two independent hybridizations were performed for each genotype. For classification of deletions, thresholds were determined by an empirical approach based on the promoter sequencing data described in Figure 2. The deduced settings of a signal drop below 2.8-fold (–1.5 on log2 scale), a minimum run >35 and for maximum gap ⩽150 allowed detection of indels >30 bp, but detected neither smaller indels nor SNPs. Examples are shown for tiles of individual sequenced regions. (A) Promoter region of At1g29030. No polymorphisms were observed among Lc-0 or Eil-0 as compared with Col-0 or each other. (B) Promoter region of At1g05830. Sequencing revealed a few dispersed small indels and SNPs between the three genotypes. (C) Promoter region of At1g13650. Sequencing revealed an extended stretch of many small indels and SNPs. (D) Promoter region of At1g33480. Sequencing revealed several small indels and SNPs. Only a 39 bp deletion in Eil-0 is picked up as a positive (red horizontal bars) by our settings. Gene structure is shown at the bottom of each panel (thick yellow blocks, exons; small yellow blocks, UTRs; yellow lines, introns). Difference in hybridization signal between Lc-0 or Eil-0 versus Col-0 along the oligonucleotides representing the tiling path is shown as white vertical bars. Upward deviation from the base line indicates positive hybridization signal, downward deviation negative hybridization signal.
Figure 4
Figure 4
Indel analysis of the Eil-0 and Lc-0 genomes using genome tiling arrays. Genomic DNA of genotypes was hybridized against a tile of the Col-0 genome. Two independent hybridizations were performed for each genotype. Indels were deduced using threshold settings (signal drop ⩽2.8-fold, min run >35, max gap ⩽150) determined empirically as described in Figure 3. Examples are shown for tiles of individual genes. (A) At1g59900. No polymorphisms were observed in Lc-0 or Eil-0 as compared with Col-0 or each other. (B) At1g63900. Various deletions were detected in Eil-0 as compared with Lc-0 and Col-0. (C) At1g12220. A large-scale deletion likely covering the whole gene as indicated by a continuous detection bar was observed in Lc-0.
Figure 5
Figure 5
Summary of indel analyses. (A, B) Indel analysis of genes representing parental ELPs between the Eil-0 and Lc-0 accessions. (A) Correlation between strict ELP heritability (matching of hybridization predictions, see Figure 1C) and presence of deletions in the corresponding genes in one of the parents. Percentage of genes in each class displaying structural changes between Eil-0 and Lc-0 (‘indels') or not (‘similar'). Controls represent an extended group of 97 genes as described in Figure 2. (B) Detailed classification of the parental ELPs and controls shown in (A). None: no indels detected in Eil-0 as compared with Lc-0; introns: indel(s) detected in intron(s) of one parent as compared with the other; UTRs: indel(s) detected in UTR(s) or UTR(s) and intron(s) of one parent as compared with the other; exons: indel(s) detected in exon(s) or exons, UTR(s) and/or intron(s) of one parent as compared with the other; whole gene: >50% of gene deleted or duplicated in one parent as compared with the other. (C) Correlation between the presence of indels in the coding region and increased sequence variation in the corresponding 5′ regulatory regions in the parental ELP genes. The quartiles as well as the average (wider line) are indicated. The distribution between the two groups is statistically significant (P<0.0390, t-test). (D) Expression microarray hybridization signal distribution of all genes in the Eil-0 versus Lc-0 parent comparison. (E) As in (D), shown for the parental ELPs with simple inheritance.
Figure 6
Figure 6
Indel analysis and polymorphic region prediction (PRP) analysis of genes representing ELPs with simple inheritance and controls between the Bay-0 and Sha accessions. (A) Percentage of genes representing ELPs with simple inheritance or controls (same group Figure 5) that carry indels in one parent as compared with the other or that display similar gene structure. (B) Detailed classification of genes shown in (A), categories similar to Figure 5B. (C, D) PRP predictions. The graphs (logarithmic scale) represent total PRP size observed in a given gene (in bp, equaling sum of all individual PRPs with respect to the gene model Atxgyyyyy.1, TAIR 7.0 annotation) detected in one accession plotted against the same value for the other accession. Classification of genes is similar to Figure 5B. (C) Genes representing ELPs with simple inheritance. (D) Control genes.

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