Background: Map based cloning in Arabidopsis thaliana can be a difficult and time-consuming process,specifically if the phenotype is subtle and scoring labour intensive. An alternative to map basedcloning would be to directly sequence the whole genome of a mutant to uncover the mutationresponsible for the phenotype.
Results: Here, we have re-sequenced the 120 Mb genome of a novel Arabidopsis clock mutant earlybird (ebi-1), using massively parallel sequencing by ligation. This process was further complicated by the fact that ebi-1 is in Wassilewskija (Ws-2), not the reference accession ofArabidopsis. The approach reveals evidence of DNA strand bias in the ethyl methanesulfonate(EMS) mutation process. We have demonstrated the utility of sequencing a backcrossed line andusing gene expression data to limit the number of SNP considered. Using new SNP informationwe have excluded a previously identified clock gene, PRR7. Finally, we have identified a SNPin the gene AtNFXL-2 as the likely cause of the ebi-1 phenotype and validated this bycharacterising a further allele.
Conclusion: In Arabidopsis, as in other organisms, the (EMS) mutation load can be high. Here wedescribe how sequencing a backcrossed line, using functional genomics and analysing new SNPinformation can be used to reduce the number EMS mutations for consideration. Moreover, theapproach we describe here does not require out-crossing and scoring F2 mapping populations, anapproach which can be compromised by background effects. The strategy has broad utility andwill be an extremely useful tool to identify causative SNP in other organisms.
2011. Vol. 12, R28- p.