A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plants.

Plant genes for pathogen resistance can be used to engineer disease resistant crops. Oligonucleotides were designed from sequence motifs conserved between resistance genes of tobacco and Arabidopsis thaliana and used as PCR primers in potato DNA. Amplification products were obtained that were homologous to known resistance genes and linked without recombination with the nematode resistance locus Gro1 and the Phytophthora infestans resistance locus R7 of potato. Map positions of PCR-derived potato gene fragments were also correlated with resistance loci of the related tomato and tobacco genomes. Our results indicate that plant resistance genes that are effective against nematodes, fungi, viruses and bacteria may be isolated based on common sequence motifs and PCR methodology.

Genetic engineering of potato for broad-spectrum protection against virus infection.

Transgenic potato plants expressing mutant alleles of PLRV ORF4, the gene for the movement protein pr17 of this luteovirus, were generated for broad-range protection against virus infection. When tested for protection against infection by PLRV, all transgenic lines showed a significant reduction of virus antigen. Potato lines accumulating N- or C-terminally extended PLRV pr17 mutant proteins were resistant to infection by the unrelated potato viruses PVY and PVX. Transgenic lines that did not express protein despite high transcript levels failed to exhibit virus resistance.

GST-PRIME: a genome-wide primer design software for the generation of gene sequence tags.

The availability of sequenced genomes has generated a need for experimental approaches that allow the simultaneous analysis of large, or even complete, sets of genes. To facilitate such analyses, we have developed GST-PRIME, a software package for retrieving and assembling gene sequences, even from complex genomes, using the NCBI public database, and then designing sets of primer pairs for use in gene amplification. Primers were designed by the program for the direct amplification of gene sequence tags (GSTs) from either genomic DNA or cDNA. Test runs of GST-PRIME on 2000 randomly selected Arabidopsis and Drosophila genes demonstrate that 93 and 88% of resulting GSTs, respectively, fulfilled imposed length criteria. GST-PRIME primer pairs were tested on a set of 1900 Arabidopsis genes coding for chloroplast-targeted proteins: 95% of the primer pairs used in PCRs with genomic DNA generated the correct amplicons. GST-PRIME can thus be reliably used for large-scale or specific amplification of intron-containing genes of multicellular eukaryotes.

Estimation of recombination frequencies and construction of RFLP linkage maps in plants from crosses between heterozygous parents.

The construction of a restriction fragment length polymorphism (RFLP) linkage map is based on the estimation of recombination frequencies between genetic loci and on the determination of the linear order of loci in linkage groups. RFLP loci can be identified as segregations of singular or allelic DNA-restriction fragments. From crosses between heterozygous individuals several allele (fragment) configurations are possible, and this leads to a set of formulas for the evaluation of p, the recombination frequency between two loci. Tables and figures are presented illustrating a general outline of gene mapping using heterozygous populations. The method encompasses as special cases the mapping of loci from segregating populations of pure lines. Formulas for deriving the recombination frequencies and information functions are given for different fragment configurations. Information functions derived for relevant configurations are also compared. A procedure for map construction is presented, as it has been applied to RFLP mapping in an allogamous crop.

Clustering of genes for 20 kd zein subunits in the short arm of maize chromosome 7.

Zein is the major storage protein of the endosperm of maize kernels. When this alcohol-soluble protein is subjected to SDS polyacrylamide gel electrophoresis, it is resolved into four fractions of different molecular weight: 10, 14, 20 and 22 kilodaltons (kd). Each fraction is heterogeneous with respect to isoelectric pH. For example, the 20 kd fraction contains at least seven subfractions as revealed by isoelectric focusing in polyacrylamide gels. In this report, we present evidence that the structural genes coding for the 20 kd proteins are clustered on the short arm of chromosome 7, a region that also bears loci regulating endosperm zein biosynthesis [opaque-2 (02) and defective endosperm-B30 (De(*)-B30)]. The organization of these zein genes suggests that the evolution of at least some of the maize genome has occurred as the result of repeated duplication and divergence of chromosome segments.

Ds-Induced Alleles at the OPAQUE-2 Locus of Maize.

Transposon mutagenesis has been used to isolate mutable alleles at the Opaque-2 (O2) locus of maize. Plants with the Activator-Dissociation (Ac-Ds) system of transposable elements and O2 were crossed as males to a stable o2 tester line. Among a population of 200,000 kernels, 198 exceptional kernels with somatic instability were recovered. In four cases, designated O2-m1, o2-m2, O2-m3 and O2-m4, variegated phenotypes appeared in F(2) and subsequent generations. Genetic analyses indicated that the presence of Ds near or within the O2 gene was responsible for the observed somatic instability at the O2 locus. The phenotypes of the newly induced alleles were of two types. Alleles O2-m1, O2-m3 and O2-m4, in the absence of Ac, were characterized by kernel phenotypes indistinguishable from the wild type; in the presence of Ac they generated kernels with opaque sectors interspersed within a vitreous background. In contrast, the mutable allele o2-m2, in the absence of Ac, was characterized by kernels with a recessive phenotype similar to o2 recessive mutants. In the presence of Ac, it reverted somatically to wild-type-producing kernels with vitreous spots in an o2 background. The association of the Ds element with the O2 locus may prove a valuable tool directed to the isolation of DNA fragments bearing the O2 gene.

An AFLP-based procedure for the efficient mapping of mutations and DNA probes in barley.

A strategy based upon AFLP markers for high-efficiency mapping of morphological mutations and DNA probes to linkage groups in barley is presented. First, 511 AFLP markers were placed on the linkage map derived from the cross Proctor x Nudinka. Second, loci controlling phenotypic traits were assigned to linkage groups by AFLP analysis, using F2 populations consisting of 30-50 mutant plants derived from crosses of the type "mutant x Proctor" and "mutant x Nudinka." To map DNA probes, 67 different wild-type barley lines were selected to generate F2 populations by crossing with Proctor and Nudinka. F2 plants that were polymorphic for a given RFLP fragment were classified into genotypic classes. Linkage of the RFLP polymorphism to 1 of the 511 AFLP loci was indicated by cosegregation. The use of the strategy is exemplified by the mapping of the mutation branched-5 to chromosome 2 and of the DNA probes Bkn2 and BM-7 to chromosomes 5 and 1, respectively. Map expansion and marker order in map regions with dense clustering of markers represented a particular problem. A discussion considering the effect of noncanonical recombinant products on these two parameters is provided.

Quantitative resistance to Phytophthora infestans in potato: a case study for QTL mapping in an allogamous plant species.

Phytophthora infestans is the most important fungal pathogen in the cultivated potato (Solanum tuberosum). Dominant, race-specific resistance alleles and quantitative resistance--the latter being more important for potato breeding--are found in the germplasm of cultivated and wild potato species. Quantitative trait loci (QTLs) for resistance to two races of P. infestans have been mapped in an F1 progeny of a cross between non-inbred diploid potato parents with multiple alleles. Interval mapping methods based on highly informative restriction fragment length polymorphism markers revealed 11 chromosome segments on 9 potato chromosomes showing significant contrasts between marker genotypic classes. Whereas phenotypically no difference in quantitative resistance response was observed between the two fungal races, QTL mapping identified at least one race specific QT locus. Two QT regions coincided with two small segments on chromosomes V and XII to which the dominant alleles R1, conferring race specific resistance to P. infestans, Rx1 and Rx2, both inducing extreme resistance to potato virus X, have been allocated in independent mapping experiments. Some minor QTLs were correlated with genetic loci for specific proteins related to pathogenesis, the expression of which is induced after infection with P. infestans.

Genetics of mutations affecting the development of a barley floral bract.

Two groups of mutants that affect the morphology of the lemma, a floral bract of barley, are described. The first comprises phenotypes associated with mutant alleles of calcaroides loci. On the lemma of these mutants, a well-organized neomorphic structure is formed, termed the sac. We provide a morphological description of wild-type (WT) and mutant lemmas, based on scanning electron microscopy (SEM), showing that both consist of similar tissues, but that the mutant is characterized by reversed growth polarity. The sac is a unique structure among grasses, and it is remarkable that recessive mutations at five different genetic loci lead to the same organ. The second group of mutants carry recessive alleles of two leafy lemma genes, both of which are necessary to cause the transformation of the lemma into a structure having all characteristics of a vegetative leaf, as shown by SEM analysis. The presence of sheath, blade, and ligule in the mutant lemma suggests that wild-type lemma development is interrupted at a leaf-like stage. The genes cal a, b, C, d, 23, lel1, and lel2 have now been mapped at precise positions on linkage groups 2, 7, 7, 3, 7, 5, and 7, respectively. The mutants considered in this article are unaffected in other floral organs. A model for lemma development is suggested.

Molecular Characterization of the Rehydration Process in the Resurrection Plant Craterostigma plantagineum.

The resurrection plant Craterostigma plantagineum is unique among higher plants because it is able to survive a desiccation treatment. For this reason it has been used as a model system for the analysis of the molecular mechanisms of desiccation tolerance. Many transcripts and proteins are expressed de novo during dehydration. This paper describes the molecular events that occur during the rehydration process of dried C. plantagineum plants. Changes in gene expression patterns were first analyzed by in vivo labeling and in vitro translation experiments. In a second set of experiments steady-state mRNA levels were monitored using specific cDNA clones. The experiments indicated three major changes on the molecular level during rehydration: the dehydration-specific gene products disappeared during an early phase of rehydration, a small number of rehydration-specific transcripts were synthesized around 12 to 15 h after the onset of rewatering, and hydration-related gene products appeared concomitantly. The gene expression patterns during rehydration are discussed with respect to the possible roles of the gene products.

Identification of a promoter sequence from the BETL1 gene cluster able to confer transfer-cell-specific expression in transgenic maize

The maize (Zea mays L.) betl1 locus, encoding a basal endosperm transfer layer-specific protein, has been mapped and molecularly cloned in its entirety. The locus is shown to consist of three gene copies in the maize inbred line A69Y. To distinguish the three transcription units from the locus name, we have termed them BETL1a, BETL1b, and BETL1c. Two of the copies are expressed, whereas one is inactive and contains retrotransposon-like insertions in both promoter and intron regions. Based on this information, and a restriction site map covering 17 kb around the BETL1 locus, a DNA fragment putatively containing an active promoter sequence was identified. This fragment was tested for its ability to confer transfer-cell-specific expression in transient and stably transformed maize tissues. The transgenic maize plants obtained showed the predicted cell-type specificity of expression restricted to the basal endosperm transfer cells, although there were minor deviations in promoter strength and timing and accumulation of the transgene product from the corresponding BETL-1 endogene expression pattern.

Mapping QTLs for sucrose content, yield and quality in a sugar beet population fingerprinted by EST-related markers.

Seventy five expressed sequence tags (ESTs) that are associated with functions in carbohydrate and nitrogen metabolism were genotyped in 108 plants of an F2 population of sugar beet ( Beta vulgaris L.) segregating for sugar quality and yield parameters. Supplemented by known RFLP and AFLP markers, the resulting map spans 446 cM of the 758-Mbp genome of sugar beet. F3 test-cross plants were analysed for corrected sugar yield, beet yield, ion balance and the content of sugar, amino nitrogen, potassium and sodium in six locations. Twenty one significant quantitative trait loci (QTLs) were detected using the composite interval mapping approach. Expressed genes flanking the QTLs were identified in all cases. Correlations between QTLs and potential candidate genes are discussed.

Epicuticular waxes of maize as affected by the interaction of mutantgl8 withgl3, gl4 andgl15.

Chemical composition of epicuticular waxes from double mutants of maizegl2gl8, gl3gl8, gl4gl8 andgl15gl8 is compared to that of the wild type and of the single mutants. The wax composition was moderately affected in the double mutants studied. Meanwhile, free fatty acids became a normal class constituent of the waxes. The sites of action of the single mutants as deduced from previous studies are confirmed. The influence ofgl2, gl3 andgl4 on the terminal steps of the chain elongation process ingl2gl8, gl3gl8 andgl4gl8 genotypes is presented and discussed. The study ofgl3gl8 waxes also confirms that the mutantgl3 induces a metabolic defect defiitely different from those ofgl2 andgl4. The pattern of alkanes fromgl15gl8 is unusual, supporting the thesis thatgl15 controls mainly alkane synthesis. However, based on variations induced on wax composition, its manner of action is difficult to account for. Taken together, the available data on single and double mutants affecting wax synthesis in maize suggest that elongases might be heteromeric enzymes.

Are we Genomic Mosaics? Variations of the Genome of Somatic Cells can Contribute to Diversify our Phenotypes.

Theoretical and experimental evidences support the hypothesis that the genomes and the epigenomes may be different in the somatic cells of complex organisms. In the genome, the differences range from single base substitutions to chromosome number; in the epigenome, they entail multiple postsynthetic modifications of the chromatin. Somatic genome variations (SGV) may accumulate during development in response both to genetic programs, which may differ from tissue to tissue, and to environmental stimuli, which are often undetected and generally irreproducible. SGV may jeopardize physiological cellular functions, but also create novel coding and regulatory sequences, to be exposed to intraorganismal Darwinian selection. Genomes acknowledged as comparatively poor in genes, such as humans', could thus increase their pristine informational endowment. A better understanding of SGV will contribute to basic issues such as the "nature vs nurture" dualism and the inheritance of acquired characters. On the applied side, they may explain the low yield of cloning via somatic cell nuclear transfer, provide clues to some of the problems associated with transdifferentiation, and interfere with individual DNA analysis. SGV may be unique in the different cells types and in the different developmental stages, and thus explain the several hundred gaps persisting in the human genomes "completed" so far. They may compound the variations associated to our epigenomes and make of each of us an "(epi)genomic" mosaic. An ensuing paradigm is the possibility that a single genome (the ephemeral one assembled at fertilization) has the capacity to generate several different brains in response to different environments.

Independent wheat B and G genome origins in outcrossing Aegilops progenitor haplotypes.

The origin of modern wheats involved alloploidization among related genomes. To determine if Aegilops speltoides was the donor of the B and G genomes in AABB and AAGG tetraploids, we used a 3-tiered approach. Using 70 amplified fragment length polymorphism (AFLP) loci, we sampled molecular diversity among 480 wheat lines from their natural habitats encompassing all S genome Aegilops, the putative progenitors of wheat B and G genomes. Fifty-nine Aegilops representatives for S genome diversity were compared at 375 AFLP loci with diploid, tetraploid, and 11 nulli-tetrasomic Triticum aestivum wheat lines. B genome-specific markers allowed pinning the origin of the B genome to S chromosomes of A. speltoides, while excluding other lineages. The outbreeding nature of A. speltoides influences its molecular diversity and bears upon inferences of B and G genome origins. Haplotypes at nuclear and chloroplast loci ACC1, G6PDH, GPT, PGK1, Q, VRN1, and ndhF for approximately 70 Aegilops and Triticum lines (0.73 Mb sequenced) reveal both B and G genomes of polyploid wheats as unique samples of A. speltoides haplotype diversity. These have been sequestered by the AABB Triticum dicoccoides and AAGG Triticum araraticum lineages during their independent origins.

Molecular diversity at 18 loci in 321 wild and 92 domesticate lines reveal no reduction of nucleotide diversity during Triticum monococcum (Einkorn) domestication: implications for the origin of agriculture.

The diploid wheat Triticum monococcum L. (einkorn) was among the first crops domesticated by humans in the Fertile Crescent 10,000 years ago. During the last 5,000 years, it was replaced by tetraploid and hexaploid wheats and largely forgotten by modern breeders. Einkorn germplasm is thus devoid of breeding bottlenecks and has therefore preserved in unfiltered form the full spectrum of genetic variation that was present during its domestication. We investigated haplotype variation among >12 million nucleotides sequenced at 18 loci across 321 wild and 92 domesticate T. monococcum lines. In contrast to previous studies of cereal domestication, we sampled hundreds of wild lines, rather than a few dozen. Unexpectedly, our broad sample of wild lines reveals that wild einkorn underwent a process of natural genetic differentiation, most likely an incipient speciation, prior to domestication. That natural differentiation was previously overlooked within wild einkorn, but it bears heavily upon inferences concerning the domestication process because it brought forth 3 genetically, and to some extent morphologically, distinct wild einkorn races that we designate here as alpha, beta, and gamma. Only one of those natural races, beta, was exploited by humans for domestication. Nucleotide diversity and haplotype diversity in domesticate einkorn is higher than in its wild sister group, the einkorn beta race, indicating that einkorn underwent no reduction of diversity during domestication. This is in contrast to findings from previous studies of domestication history among more intensely bred crop species. Taken together with archaeological findings from the Fertile Crescent, the data indicate that a specific wild einkorn race that arose without human intervention was subjected to multiple independent domestication events.

Quantification of genetic relationships among A genomes of wheats.

The genetic relationships of A genomes of Triticum urartu (Au) and Triticum monococcum (Am) in polyploid wheats are explored and quantified by AFLP fingerprinting. Forty-one accessions of A-genome diploid wheats, 3 of AG-genome wheats, 19 of AB-genome wheats, 15 of ABD-genome wheats, and 1 of the D-genome donor Ae. tauschii have been analysed. Based on 7 AFLP primer combinations, 423 bands were identified as potentially A genome specific. The bands were reduced to 239 by eliminating those present in autoradiograms of Ae. tauschii, bands interpreted as common to all wheat genomes. Neighbour-joining analysis separates T. urartu from T. monococcum. Triticum urartu has the closest relationship to polyploid wheats. Triticum turgidum subsp. dicoccum and T. turgidum subsp. durum lines are included in tightly linked clusters. The hexaploid spelts occupy positions in the phylogenetic tree intermediate between bread wheats and T. turgidum. The AG-genome accessions cluster in a position quite distant from both diploid and other polyploid wheats. The estimates of similarity between A genomes of diploid and polyploid wheats indicate that, compared with Am, Au has around 20% higher similarity to the genomes of polyploid wheats. Triticum timo pheevii AG genome is molecularly equidistant from those of Au and Am wheats.

A reconsideration of the domestication geography of tetraploid wheats.

The domestication of tetraploid wheats started from their wild progenitor Triticum dicoccoides. In this paper, the geographical distribution of this progenitor is revised to include more sampling locations. The paper is based on a collection of wild and domesticated lines (226 accessions in total) analyzed by AFLP at 169 polymorphic loci. The collection includes the 69 wild lines considered by Mori et al. (2003) in their study on chloroplast DNA haplotypes of T. dicoccoides. The goal of the experiment was to reconsider which location thought to have generated the domesticated germplasm has the highest chance of being the actual site from which wild progenitors were sampled during domestication. Phylogenetic analysis of the nuclear AFLP databases indicates that two different genetic taxa of T. dicoccoides exist, the western one, colonizing Israel, Syria, Lebanon and Jordan, and the central-eastern one, which has been frequently sampled in Turkey and rarely in Iran and Iraq. It is the central-eastern race that played the role of the progenitor of the domesticated germplasm. This is supported by the cumulative results of the AFLP data from the collections of Ozkan et al. (2002) and of Mori et al. (2003), which indicate that the Turkish Karacadag population, intermixed with some Iraq-Iran lines, has a tree topology consistent with that of the progenitor of domesticated genotypes. The Turkish Kartal population belongs genetically to the central-eastern T. dicoccoides race but at the nuclear DNA level is less related to the domesticated gene pool. A general agreement between published work on tetraploid wheat domestication emerges from these results. A disagreement is nevertheless evident at the local geographical scale; the chloroplast DNA data indicate the Kartal mountains while AFLP fingerprinting points to the Karacadag Range as the putative site of tetraploid wheat domestication.