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In a screen designed to identify genes in the specification of epidermal cell fate in Arabidopsis primary roots we have isolated 8 new mutants that fall into 6 complementation groups corresponding to the ‘root hairless’ genes RHL1, RHL2 and RHL3 and the ‘ectopic root hair’ genes ERH1, ERH2 and ERH3. The erh2 mutant is allelic to pom1, a conditional root expansion mutant, and reveals a possible link between epidermal root hair initiation and radial cell expansion. Apart from erhl the mutants also show defects in shoot development, indicating a complex role for the affected genes. Mutant phenotypes in the patterning and shape of leaf trichomes in rh11, rhl2, rhl3 and erh3 were particularly obvious. The root hairless mutants are only partly responsive to increased ethylene concentrations, while the ectopic root hair mutants are fully responsive to reduced concentrations of ethylene, a permissive regulator of root hair initiation. This result and the analysis of double mutants suggest a complex pathway leading to root hair initiation that requires the RHL and ERH genes for correct differentiation.

A panel of 13 sugar beet lines and one genotype each of the Beta vulgaris cultivars red beet and Swiss chard, and B. vulgaris ssp. maritima were used to identify polymorphisms in alignments of genomic DNA sequences derived from 315 EST- and 43 non-coding RFLP-derived loci. In sugar beet lines, loci of expressed genes showed an average SNP frequency of 1/72 bp, 1 in 58 bp in non-coding sequences, increasing to 1/47 bp upon the addition of the remaining genotypes. Within analysed DNA fragments, alleles at different SNP positions displayed linkage disequilibrium indicative of haplotype structures. On average 2.7 haplotypes were found in sugar beet lines, and haplotype conservation in expressed genes appeared to exceed 500 bp in length. Seven different genotyping techniques including SNP detection by MALDI-TOF mass spectrometry, pyrosequencing and fluorescence scanning of labelled nucleotides were employed to perform 712 segregation analyses for 538 markers in three F(2) populations. Functions were predicted for 492 mapped sequences. Genetic maps comprised 305 loci covering 599.8 cM in population K1, 241 loci distributed over 636.6 cM in population D2, and 166 loci over 507.1 cM in population K2, respectively. Based on 156 markers common to more than one population an integrated map was constructed with 524 loci covering 664.3 cM. For 377 loci the genome positions of the most similar sequences from A. thaliana were identified, but little evidence for previously presented ancestral genome structures was found.

A set of single nucleotide polymorphism (SNP) markers has been developed for each of the nine linkage groups of sugar beet. Each set can monitor the polymorphic state at five to six linked marker loci. In each set, the loci selected for marker development are first amplified in a multiplexed reaction. These amplification products are the basis for sequence-specific elongation of primers adjacent to SNP positions. The extension step revealing SNP loci is based on fluorescently labelled nucleotides. In each set, primers developed to reveal SNP alleles differ in length to allow clear peak resolution in capillary electrophoresis. The nine linkage group (LG) - specific sets provide information on the polymorphism at a total of 52 SNP marker loci. Using the SNP-based tool, groups of concerned loci have been anchored to three different linkage maps of sugar beet. In a second experiment, sugar beet breeding lines have been fingerprinted. The use of the nine sets of LG-specific markers in sugar beet genetics and breeding is discussed. The information necessary to specify the 52 marker loci, as well as their map location, and all details concerning SNP assays, including allele type and nature of mutation, are reported.

Sequence conservation among resistance genes (R genes) was exploited to identify 47 R gene analogues (RGAs) from sugar beet (Beta vulgaris L.). Using degenerate primers, 11 RGAs were amplified from genomic DNA and 7 from leaf or beet cDNA. Twenty-nine were selected from an EST sequencing program. Twenty-one RGAs contained structures similar to the nucleotide binding site (NBS) – leucine rich repeat (LRR) domain, a motif commonly found in several R genes. Among the remaining RGAs, 19 revealed similarity to the serine (threonine) protein kinase domain of R genes, 4 showed features related to the LRR region of the rice disease resistance gene Xa21, 1 RGA resembled the sugar beet nematode resistance gene Hs1pro-1, and 2 had homologies to other gene products associated with disease resistance. For 20 EST-derived RGAs, transcript levels were compared in leaf and root tissue revealing organ-specific transcription in 7 cases. Thirty-three RGAs were spread over all nine sugar beet chromosomes, except for a cluster of nine closely linked RGAs on chromosome 7. The analysis of linkage between RGAs and loci for rhizomania and Cercospora resistance identified alleles associated with resistance in both cases.Key words: RGA, Beta vulgaris, NBS–LRR, genetic linkage map, molecular marker.

Based on EST sequences, fragments of 37 genes have been amplified and sequenced in two inbred lines of sugar beet. The rate of single nucleotide polymorphisms (SNP) corresponded to 1 every 130 bp, with an average π (nucleotide diversity) value of 7.6×10−3. When extrapolated to the whole sugar beet genome, randomly compared lines differ at 5.4×106 SNPs in the genetic pool considered. In a wider search for SNP-related polymorphisms, 96 fragments of expressed genes were scanned with SSCP (single-strand conformation polymorphism) and heteroduplex (HA) analyses in 8 inbred lines. One SSCP or HA polymorphism was found every 1,470 bp of amplified DNA, corresponding to 5×105 SSCP or HA loci in the whole genome. This frequency, 11 times lower than the SNP rate, was attributed to the high frequency of base pair substitution along the amplified fragment analysed electrophoretically. Therefore nucleotide variability was further studied by sequencing fragments of 10 genes in the same 8 lines. The results indicate that sugar beet alleles of expressed genes are very frequently organized as robust intragene haplotypes. In the 8 lines analysed, two haplotypes were identified for each of three gene fragments, three haplotypes for six gene fragments and four haplotypes for one gene fragment which is in good correspondence with the number of alleles detected by SSCP and HA analysis. In a cross between two lines, SSCP or HA alleles of expressed genes have 54% probability to be different.

To analyse genetic factors that potentially affect sugar quality and yield in Beta vulgaris, we designed primers based on 18 homologous ESTs and conserved regions of 32 heterologous ESTs encoding gene products that act in the Calvin cycle, the oxidative pentose phosphate cycle, photorespiration, synthesis, transport and degradation of sucrose, glycolysis, the citric acid cycle, nitrogen metabolism and osmoprotection. Data on the amplification of 54 gene homologues from B. vulgaris are presented. Among these are 35 homologues for which DNA sequence information from B. vulgaris is now available for the first time. For genetic mapping a PCR-based strategy using CAPS (cleaved amplified polymorphic sequence), DFLP (DNA fragment length polymorphism), SSCP (single-strand conformation polymorphism) and HD (heteroduplex) analysis was adopted. RFLP analysis was also used in some cases. The different techniques used for the detection of polymorphisms are evaluated with respect to their sensitivity and versatility. In all, 42 functional genes have been assigned to the nine linkage groups of sugar beet.

Leaves of resurrection plants tolerate desiccation as do embryos of many higher plants. From the resurrection plant Craterostigma plantagineum a number of desiccation-related transcripts have recently been cloned; they are abundantly expressed in dried leaves and abscisic acid-treated dried callus (D Bartels, K Schneider, G Terstappen, D Piatkowski, F Salamini [1990] Planta 18: 27-34). Five distinct cDNA clones representing low copy number genes were selected for further characterization. Their nucleotide sequences were determined and proteins were predicted with a molecular mass between 16 and 34 kilodaltons. Three of these proteins have unusual amino acid compositions and extreme hydrophilic characters. Two of them contain a cluster of contiguous serine residues and lysine-rich repeats. These sequence motifs display homologies to desiccation-related genes expressed in embryos or dehydrated seedlings of several plants. A third cDNA clone contains tracts of sequences which are related to a cotton Lea (late embryogenesis abundant) gene (JC Baker, C Steele, L Dure III [1988] Plant Mol Biol II: 277-291). Secondary structure predictions are discussed and suggest that the deduced proteins could play a role in protecting core cell structures in a dehydrated cell. It is concluded that at least in part the gene products involved in the desiccation-induced pathways are common to leaves of resurrection plants and embryos. Two cDNA clones appear to code for Craterostigma-specific mRNAs. The expression patterns of all five transcripts were studied in comparison to desiccated leaves in dehydrated roots, in wound-stressed leaves and in salt-stressed callus. The data obtained point to the possibility that not only specificity of induction but also the expression level of specific gene products may be of importance for osmoprotection.

Plants are the basis for the survival of all "higher" organisms on Earth. Development of molecular genetics tools has allowed analysis of the structure, evolution, and function of whole plant genomes, rather than individual genes. DNA-based markers were instrumental in constructing detailed genetic maps of model plants and all major crop species. These molecular maps were the basis of physical maps and the first plant whole genome sequences. Comparative analysis based on genetic, cytogenetic, and physical maps and DNA sequence information provided new insights into the evolution of plant nuclear and organellar genomes. Mapping factors controlling Mendelian and quantitative traits made possible the cloning and functional characterization of novel genes, which function in plant development, adaptation to biotic and abiotic stress, or in the formation of other agronomic characters. The parallel analysis of all transcripts, proteins, and metabolites present in plant cells or tissues has generated information that may lead to a better integrated understanding of genome function. Postfunctional analysis of natural variation of gene function and its effects on phenotype is envisaged to provide new diagnostic and therapeutic molecular tools for applications in plant breeding, adaptation, and ecology.

Three cDNAs encoding desiccation-induced proteins from the resurrection plant Craterostigma plantagineum were each ligated to a triplicated CaMV 35S promoter and a nopaline synthase 3′-flanking region in an Agrobacterium vector and introduced into tobacco. Transgenic plants expressed the encoded Craterostigma proteins at high levels. This did not lead to changes in the phenotype, in the growth habit or in basic photosynthetic parameters. In tobacco, one protein was targeted to the chloroplast stroma which is its normal location in Craterostigma. These desiccation-related proteins are not sufficient per se to increase drought tolerance as measured by ion-leakage tests.

Leaves of the resurrection plant Craterostigma plantagineum Hochst, can be desiccated up to 1% relative water content and are still viable after rehydration. To clone genes related to this extreme desiccation tolerance, an in-vitro system was first developed which allows the induction of the same resurrection response in callus tissue upon treatment with abscisic acid (ABA). Several proteins and in-vitro-synthesized polypeptides were then identified which can be induced both in desiccation-tolerant, naturally dried leaves and in ABA-treated calli surviving after rehydration. Complementary-DNA clones corresponding to mRNAs expressed only in desiccation-tolerant tissues were obtained and classified into several gene families. In hybrid-selected translation experiments, representative cDNA clones were associated with water stress and ABA-inducible polypeptides abundantly expressed in dried leaves and ABA-treated calli. The expression pattern of several of these abundant transcripts was analyzed in RNA-hybridization experiments. Upon stress or ABA treatment the transcription levels increased rapidly, but they declined after relief from the stress state. This, together with data on genomic copy numbers indicated that a set of abundantly expressed genes are involved in the desiccation process of resurrection plants. Data on endogenous ABA contents before and after stress applications and on the physiological effects of exogenous ABA treatments indicate that in Craterostigma plantagineum the induction of an extreme desiccation tolerance is mediated by this plant hormone.

The epidermis ofArabidopsiswild-type primary roots, in which some cells grow hairs and others remain hairless in a position-dependent manner, has become an established model system to study cell differentiation. Here we present a molecular analysis of theRHL1(ROOTHAIRLESS1) gene that, if mutated, prevents the formation of hairs on primary roots and causes a seedling lethal phenotype. We have cloned theRHL1gene by use of a T-DNA-tagged mutant and found that it encodes a protein that appears to be plant specific. The predictedRHL1gene product is a small hydrophilic protein (38.9 kD) containing putative nuclear localization signals and shows no significant homology to any known amino acid sequence. We demonstrate that a 78-amino-acid sequence at its amino terminus is capable of directing an RHL1–GFP fusion protein to the nucleus. TheRHL1transcript is present throughout the wild-type plant and in suspension culture cells, but in very low amounts, suggesting a regulatory function for theRHL1protein. Structural evidence suggests a role for theRHL1gene product in the nucleolus. We have examined the genetic relationship betweenRHL1andGL2,an inhibitor of root hair initiation in non-hair cells. Our molecular and genetic data with double mutants, together with the expression analysis of aGL2promoter–GUS reporter gene construct, indicate that theRHL1gene acts independently ofGL2.