Bertrand Lemieux

Genome-wide mapping with biallelic markers in Arabidopsis thaliana

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Epicuticular waxes of eceriferum mutants of Arabidopsis thaliana

Abstract The principal surface lipids of Arabidopsis are n -nonacosane, 14- and 15-nonacosanol, 15-nonacosanone, C 16 –C 30 free fatty acids, C 26 –C 30 primary alcohols and C 26 –C 30 aldehydes. We have analysed the chemical composition of the epicuticular wax of 10 Arabidopsis thaliana eceriferum ( cer ) mutants. One of the mutants ( cer 2) is blocked in the elongation of octacosanoic acid and accumulates large amounts of primary alcohols and fatty acids in its epicuticular wax. The surface lipid composition of another mutant ( cer 4) appears to be defective in the production of primary alcohols and accumulates elevated levels of epicuticular alkanes. We have also identified a mutant ( cer 1) which accumulates epicuticular aldehydes and is severely deficient in alkanes on its surface. Seven other mutants had only slightly different epicuticular wax compositions compared to those of wild type plants.

Molecular genetics of epicuticular wax biosynthesis

The epicuticular wax (EW) layer that coats the outer surface of plants is composed of a variety of long chain length hydrocarbons. Genetic studies indicate that a very large number of genes are involved in wax production. However, the pathway(s) by which the waxes are synthesized and deposited onto the plant surface remain elusive. Recently, clones of several genes involved in the production of the epicuticular wax layer of Arabidopsis and maize have been isolated through genetic approaches. These clones will provide researchers with an opportunity to develop a better understanding of the developmental and environmental regulation of this pathway, and may help to elucidate the biological function(s) of these surface lipids.

37 Epicuticular Wax and eceriferum Mutants

Like most plants, the aboveground surface of Arabidopsis thaliana is covered by a layer of lipids such as fatty acids, fatty aldehydes, primary alcohols, alkanes, secondary alcohols, ketones, and esters. Figure 1 illustrates the chemical structures of some of the principal epicuticular wax components found on the surface of Arabidopsis. The majority of these wax components can be separated in a single chromatographic separation by capillary gas chromatography with a low polarity solid phase (Yang et al. 1992). The use of mass spectrometry coupled to a gas chromatograph has allowed the identification of the individual compounds that make up the epicuticular wax layer of plants (Walton 1990). As shown in Figure 2, Arabidopsis eceriferum ( cer ) mutants are characterized by a bright green color when compared to wild-type plants because the reduced amount of wax deposition on the stem alters the reflection of light such that mutants can be isolated by a simple visual inspection. Epicuticular wax components are derived from very long chain fatty acids. The existence of a fatty acid elongation activity within the endoplasmic reticulum has been demonstrated by the partial purification of a 500-kD enzyme complex that elongates stearyl-CoA (18:0) to eicosanoyl-CoA (20:0) (Bessoule et al. 1989). Partial purifications of fatty acid reductase activities have shown that a fatty acid reductase and a fatty aldehyde reductase activity can be separated by protein fractionation (Kolattukudy 1971). Experiments with particulate cell wall fractions of pea have demonstrated that a fatty aldehyde decarbonylation activity is presumably responsible for the biosynthesis...