The aim of this proposal is to develop a genetic map for the cruciferous plant Arabidopsis thaliana consisting of approximately 3,000 biallelic markers. This proposal builds on our present map of approximately 230 Arabidopsis markers proven to be amenable to genotyping by chip hybridization, as well as our successful effort of constructing a similar map for Saccharomyces cerevisiae. Markers will be generated by comparative sequencing of the Columbia and Landsberg erecta ecotypes of Arabidopsis, which are the most commonly used by the scientific community. However, it is expected that 50% of the markers will also work for other ecotypes, a number sufficient to enable the successful mapping of traits by linkage disequilibrium. Markers will be spaced at an average distance of approx. 60 kb, which translates into a map with a resolution better than 0.5 cM. Biallelic markers will be discovered by sequencing 4,000 approximately 600-bp fragments amplified from noncoding regions of Columbia and Landsberg erecta. Based on current experience approximately 75% of the screened fragments will differ in sequence. Simple sequence polymorphisms will be genotyped by amplification of the genomic region containing the marker. The amplicons will be used as templates in single base extension reactions using chimeric primers with 3'complementarity to specific SSP loci and 5'complementarity to specific probes on a commercially available bar code tag array. The primers are extended with labeled dideoxy NTPs, using a different label for each allele, and hybridized to the tag array. Genotypes are deduced from the fluorescence intensity ratio of the two colors. We will also develop a modification of this protocol to multiplex at least 100 genotyping reactions. Extension reactions will be carried out first on genomic DNA using biotinylated dideoxy NTPs and chimeric primers that contain beside the 3'complementarity to the specific SSP locus a unique bar code bracketed by two 18-mer sequences for amplification of the former. Biotinylated extension products will be captured with magnetic streptavidin beads, followed by amplification of the bar codes with different fluorescent tags for either allele. The labeled amplicons will be hybridized to the bar code chip and the genotypes deduced. We will then map both monogenic and multigenic traits involved in photoreceptor signaling and response. Chip mapping with an expected resolution of 0.5 cM (approximately 75 kb) will be complemented with fine-mapping by DHPLC to obtain a final resolution of <0.2 cM. DHP will be also employed to pinpoint the mutation by screening the open reading frames in the interval. This is accomplished by amplifying the mutant and its wild-type parent separately, mixing, denaturing and reannealing of the two PCR reactions, followed by direct loading onto DHPLC. It is anticipated that experience gained on the mapping of QTLs in Arabidopsis will be very valuable for similar efforts in other eukaryotes, including human.
| Rickert, Andreas M; Premstaller, Andreas; Gebhardt, Christiane et al. (2002) Genotyping of Snps in a polyploid genome by pyrosequencing. Biotechniques 32:592-3, 596-8, 600 passim |
