Full genome re-sequencing reveals a novel circadian clock mutation in Arabidopsis

Abstract

Map based cloning in Arabidopsis thaliana can be a difficult and time-consuming process, specifically if the phenotype is subtle and scoring labour intensive. Here, we have re-sequenced the 120-Mb genome of a novel Arabidopsis clock mutant early bird (ebi-1) in Wassilewskija (Ws-2). We demonstrate the utility of sequencing a backcrossed line in limiting the number of SNPs considered. We identify a SNP in the gene AtNFXL-2 as the likely cause of the ebi-1 phenotype.

Figure 1

ebi-1 causes the circadian clock to oscillate with a short period. (a,b) Transgenic seedlings carrying the LUC reporter gene fused to the CAB2 promoter were entrained under 12-h light/12-h dark cycles for 7 days, after which luminescence was monitored in either constant darkness (a) or constant red light (measured in counts/second, CPS) (b): WT, open squares; ebi-1, closed squares. The plots are representative of multiple experiments and are an average of between 24 and 79 individual seedlings; error bars are standard error of the mean. The inset in (b) is a mathematical analysis of the experiment represented in (b): period estimates for individual seedlings plotted against their relative amplitude errors (R.A.E.). (c) Representative leaf movement plots for WT (open squares) and ebi-1 (closed squares).

Figure 2

Schematic representation of the analysis pathway used in this study. In this two step process, (1) a list of putative SNPs, relative to Col-0, were generated for each genome (ebi-1 and Ws-2) for each of the five possible matching schemas (25_2, 25_3, 35_2, 35_3, and 35_4) used by the Corona_lite software pipeline. Then (2), considering each chromosome (chr1, chr2, chr3, chr4, chr5, mitochondrial chromosome (chrM), and chloroplast (chrC)) in turn, the results of each schema were analyzed and filtered, and finally merged to form a collection of high-confidence SNPS used in the subsequent analysis (summarized in Tables 1 and 2).

Figure 3

Location of ebi-1 SNPs relative to Ws-2. SNPs occurring in either ebi-1 only (blue circles) or Ws only (red squares), relative to Col-0, are plotted at their respective chromosome locations. The overall depth of coverage of unique tags is plotted in grey. Coverage depths of all data are determined from 35_4 schema results.

Figure 4

Alignment of the conserved regions of NFXL proteins across plant taxa. The amino acids were aligned using the ClustalW program using the following sequences: [gi: 168037431], Physcomitrella patens; [gi: 218187558], Oryza sativa; [gi: 224028969], Zea mays; [gi: 242052039], Sorghum bicolor; [gi: 56694214], Solanum lycopersicum; [gi: 145357676], Arabidopsis thalina; [gi: 297810665], Arabidopsis lyrata; [gi: 157351181], Vitis vinifera; [gi:224112501], Populus trichocarpa. Identical and similar amino acid residues are highlighted with blue and light blue, respectively. The location of the V to I SNP within a zinc finger motif is highlighted in red.

Figure 5

A T-DNA allele of ebi-2 that results in a reduction in EBI expression and crossing out the PRR7 SNP result in similar clock phenotypes to ebi-1, supporting that the circadian phenotype of ebi-1 is due to a SNP in At5g05660. Transgenic seedlings carrying the LUC reporter gene fused to the CAB2 promoter were entrained under 12-h light/12-h dark cycles for 7 days, after which luminescence was monitored in either constant darkness or constant red light. (a) Analysis of CAB2 activity under constant red light at 22°C in: ebi-1-clean-1, the ebi-1 mutant with a WT PRR7 gene (closed triangles); the ebi-1 mutant (closed squares); prr7-clean-1, the prr7 mutant with WT ebi-1 (open triangles) and WT Ws-2 (open squares). (b) Analysis of CAB2 activity under constant red light at 22°C in ebi-2 (closed squares) and WT Col-0 (open squares). (c) Analysis of CAB2 activity under constant darkness at 22°C in ebi-2 (closed squares) and WT Col-0 (open squares). (d) EBI expression is reduced in the ebi-2 mutant. RNA expression levels of EBI relative to β-tubulin were measured at either 1 h or 13 h under 12-h light/12-h dark cycles in both WT (white columns) and ebi-2 (gray columns).