Genetic structure of coastal and inland populations of the annual halophyte Suaeda maritima (L.) dumort. in Central Europe, inferred from amplified fragment length polymorphism markers.

Naturally occurring inland salt habitats are highly threatened due to increasing fragmentation and area reduction, while the surroundings of former potash mining dumps have experienced a massive invasion by halophytes over the last 20 years. We reconstructed colonisation patterns of these purely anthropogenic inland salt sites using molecular markers in the obligate halophyte Suaeda maritima (L.) dumort. (Chenopodiaceae), a typical plant in such areas. In the present study, 120 individual plants from 40 coastal and inland populations in Central Europe were subjected to AFLP analysis with nine primer combinations. A total of 243 AFLP band positions were scored as presence/absence characters. Genetic diversity values were not significantly different in populations from natural and anthropogenic inland salt sites as compared to coastal habitats. Results from principal coordinate analysis, neighbour-joining analysis and analysis of molecular variance (amova) all indicated that most of the genetic variation is preserved within populations, while genetic differentiation among populations is comparatively low. We conclude that S. maritima has repeatedly and independently colonised the surroundings of former potash mining dumps in Central Germany. However, the absence of founder effects and the lack of phylogeographic structure prevented us from identifying putative donor populations.

Application of bovine microsatellite markers for genetic diversity analysis of European bison (Bison bonasus).

In this study, the cross-amplification of a commercial multiplex set of 11 cattle (Bos taurus) microsatellites was tested on a panel of 35 European bison (Bison bonasus) individuals. After polymerase chain reaction optimization, all loci cross-amplified successfully in investigated bisons. Number of alleles and observed and expected heterozygosity per locus are in the range of 2-4, 0.086-0.629 and 0.288-0.621 respectively. The availability of a heterologous set of multiplexed microsatellite markers derived from cattle opens an avenue for collecting profound genetic data for efficient conservation management strategies of the European bison.

Molecular analysis of phylogenetic relationships among Myrmecophytic macaranga species (Euphorbiaceae).

Many species of the paleotropical pioneer tree genus Macaranga Thou. (Euphorbiaceae) live in association with ants. Various types of mutualistic interactions exist, ranging from the attraction of unspecific ant visitors to obligate myrmecophytism. In the latter, nesting space and food bodies are exchanged for protection by highly specific ant partners (mainly species of the myrmicine genus Crematogaster). As a first step toward elucidating the coevolution of ant-plant interactions in the Macaranga-Crematogaster system, we have initiated a molecular investigation of the plant partners' phylogeny. Nuclear ribosomal DNA internal transcribed spacer (ITS) sequences were analyzed for 73 accessions from 47 Macaranga species, representing 17 sections or informally described species groups. Three accessions from the putative sister taxon Mallotus Lour, were included as outgroups. Cladograms of the ITS data revealed Macaranga to be nested within Mallotus. ITS sequences are highly similar within section Pachystemon s.str., suggesting a relatively recent and rapid radiation of obligate myrmecophytes within this section. Forty-three accessions, mainly of ant-inhabited species, were additionally investigated by random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MP-PCR) techniques. Phenetic analysis of RAPD and MP-PCR banding profiles generally confirmed the ITS results. Best resolutions for individual clades were obtained when ITS and RAPD/MP-PCR data were combined into a single matrix and analyzed phenetically. The combined analysis suggests multiple (four) rather than a single evolutionary origin of myrmecophytism, at least one reversal from obligate myrmecophytism to nonmyrmecophytism, and one loss of mutualistic specifity.

Sequence-tagged microsatellite site markers for chickpea (Cicer arietinum L.).

Two small-insert genomic libraries of chickpea (Cicer arietinum L.) were screened with a set of microsatellite-specific oligonucleotide probes. A total of 121 positive clones were identified among 13,000 plated colonies. Thirty-nine clones were recognized by (TAA)5, 26 by (GA)8, 18 by (GT)8, 27 by a pool of AT-rich trinucleotide repeats [(CAA)5, (CAT)5, and (GAA)5], and 11 by a pool of GC-rich trinucleotides [(TCC)5, (CAC)5, (CAG)5, and (CGA)5]. Of 53 clones selected for sequencing, 43 carried a microsatellite. Flanking primer pairs were designed for 28 loci, and used on a small test-set comprising one C. reticulatum and four C. arietinum accessions. Separation of the PCR products on agarose or polyacrylamide gels revealed single bands of the expected size with 22 of the primer pairs. Sixteen of these "Cicer arietinum sequence-tagged microsatellite site" (CaSTMS) markers were polymorphic at an intraspecific level, detecting 2-4 alleles within the four accessions examined. Primer pairs CaSTMS10 and CaSTMS15 revealed 25 and 16 alleles among 63 C. arietinum accessions from different geographic locations, reflecting gene diversity values of 0.937 and 0.922, respectively. Mendelian inheritance of CaSTMS markers was demonstrated using a set of recombinant inbred lines and their parents.

A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms.

Short runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice, and pine, PCR (polymerase chain reaction) with flanking primers has shown that the numbers of A or T residues in such repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)n and (T)n repeats (n > or = 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39 regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed and used to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCR products from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species. Levels of interspecific polymorphism within the genera Nicotiana, Lycopersicon (both Solanaceae), and Actinidia (Actinidiaceae) proved to be high, while intraspecific variation in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis was limited. Sequence analysis of PCR products from three primer pairs revealed variable numbers of A, G, and T residues in mononucleotide arrays as the major cause of polymorphism in Actinidia. Our results suggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplast variation in angiosperms.

Chromosomal localization and distribution of simple sequence repeats and the Arabidopsis-type telomere sequence in the genome of Cicer arietinum L.

We used fluorescence in situ hybridization to probe the physical organization of five simple sequence repeat motifs and the Arabidopsis-type telomeric repeat in metaphase chromosomes and interphase nuclei of chickpea (Cicer arietinum L.). Hybridization signals were observed with the whole set of probes and on all chromosomes, but the distribution and intensity of signals varied depending on the motif. On root-tip metaphase chromosomes, CA and GATA repeats were mainly restricted to centromeric areas, with additional GATA signals along some chromosomes. TA, A and AAC repeats were organized in a more dispersed manner, with centromeric regions being largely excluded. In interphase nuclei of the inner integument, CA and GATA signals predominantly occurred in the heterochromatic endochromocentres, whereas the other motifs were found both in eu- and heterochromatin. The distribution of the Arabidopsis-type telomeric repeat (TTTAGGG)n on metaphase chromosomes was found to be quite exceptional. One major cluster of repeats was spread along the short arm of chromosome B, whereas a second, weaker signal occurred interstitially on chromosome A. Only faint and inconsistent hybridization signals were visualized with the same probe at the chromosomal termini.

Allelic variation at a hypervariable compound microsatellite locus in the ascomycete Ascochyta rabiei.

The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat units (CATTT)n, (CATTA)n, (CATATC-ATTT)n and (TATTT)n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus-specific microsatellite amplification and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational mechanisms have contribute to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic F1 progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)53 to (TATTT)65], while the remainder of the sequence was unchanged.

Increased informativeness of RAPD analysis by detection of microsatellite motifs.

The recently developed random-amplified microsatellite polymorphism (RAMPO) technique detects second-level amplification products that are useful as molecular markers. In the first step of the procedure, genomic DNA is amplified with a single arbitrary or microsatellite-complementary primer. PCR products are then electrophoretically separated, photographed, blotted and hybridized to a 32P-labeled microsatellite probe. Autoradiography reveals highly reproducible, polymorphic, probe-dependent fingerprints, which are different from the ethidium bromide staining patterns. In this paper, we report the successful application of various mono-, tri- and tetranucleotide repeat motifs as RAMPO probes. We also compare the efficiency of arbitrary vs. microsatellite primers for the generation of RAMPO patterns. Repeated rehybridization to different probes has expanded the information contained in a single random-amplified polymorphic DNA (RAPD) gel at least fivefold. Pattern complexity varies with the length and sequence of the probe. Application of the technique to a genetic relatedness study in the genus Dioscorea (yam) yielded highly informative markers, mainly at an interspecific level.

Molecular marker based taxonomy and phylogeny of Guinea yam (Dioscorea rotundata - D. cayenensis).

Four different molecular techniques were used to assess relationships among 21 accessions of Guinea yam (Dioscorea rotundata and Dioscorea cayenensis) and 21 accessions belonging to seven putative progenitor species. Random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MP-PCR) analysis yielded 246 informative characters that were transformed into a matrix of pairwise distances and analyzed by neighbor joining or split decomposition. Both methods gave congruent results. Well-separated groups were formed that corresponded to their species designation. Dioscorea rotundata and D. cayenensis accessions were clearly separated from each other, supporting the concept that both are distinct species. Two morphological intermediates grouped together with D. rotundata. All investigated species fell into two main clusters, one comprising D. rotundata, D. cayenensis, Dioscorea abyssinica, Dioscorea liebrechtsiana, and Dioscorea praehensilis, the other comprising Dioscorea smilacifolia, Dioscorea minutiflora, Dioscorea burkilliana, and Dioscorea togoensis. The same grouping was also obtained by comparative sequence analysis of chloroplast DNA, which supports earlier studies of nuclear rDNA variation and chloroplast restriction fragment length polymorphisms. We also analyzed the same set of Dioscorea samples with the recently developed random amplified microsatellite polymorphism (RAMPO) technique. A series of diagnostic RAMPO bands was identified that clearly distinguished between D. rotundata and D. cayenensis. Some of these bands could also be traced back to the putative progenitors of both species. The evolutionary origin of Guinea yam is discussed in light of the present results.

Northern blot analysis of simple repetitive sequence transcription in plants.

The presence of simple repetitive sequence motifs in RNA from various plant species was probed by Northern blot analysis. Hybridization of total, poly(A)(+)- and poly(A)(-)-RNA with microsatellite-complementary oligonucleotide probes revealed distinct bands with most but not all probe/species combinations, demonstrating the presence of di-, tri- and tetranucleotide repeat motifs in plant transcripts. Only trinucleotide repeat-derived hybridization signals were found to be enriched in the poly(A)(+)-fraction. The quality of Northern blot signals proved to be highly dependent on hybridization stringency. Thus, under the stringency conditions usually applied for oligonucleotide hybridization, some probes [(GT)8, (CAC)5, (TCC)5, and (CCTA)4] cross-hybridized to bands corresponding in size to 18S and/or 26S rRNA. Cross-hybridization to rRNA was significantly reduced at higher stringencies. These results stress the importance of carefully adjusting the hybridization conditions in Northern blot analysis of simple sequence transcripts.

Genomic variation and relationships in aerial yam (Dioscorea bulbifera L.) detected by random amplified polymorphic DNA.

Random amplified polymorphic DNA (RAPD) markers were used to assess intraspecific variability and relationships in aerial yam (Dioscorea bulbifera L.). A total of 23 accessions from different geographic locations in Africa, Asia, and Polynesia were analyzed by 10 arbitrarily chosen GC-rich decamer primers. Using cesium chloride purified genomic template DNA, highly reproducible polymorphic fingerprints were generated by all 10 primers, resulting in a total of 375 informative characters. Only eight bands were monomorphic among all investigated accessions. A binary character matrix was generated by scoring for presence/absence of a band at a particular position, transformed into a matrix of pairwise distances using either the Jaccard or a simple matching coefficient, and analyzed by neighbour joining, UPGMA (unweighted pair group method with arithmetic averaging) cluster analysis, or split decomposition. All methods of data evaluation resulted in similar groupings that reflected the geographical origin of the samples. The African accessions formed a distinct isolated group, whereas Asian and Polynesian accessions proved to be more heterogeneous. With two exceptions (var. suavior and var. sativa), the RAPD data supported previous varietal classification based on morphological characters. Stepwise reduction of the number of evaluated characters did not affect branching patterns of the trees above a minimum threshold of 150. Key words : Dioscorea bulbifera, random amplified polymorphic DNA (RAPD), genetic variation, genetic relatedness.

Oligonucleotide fingerprinting and RAPD analysis of Achillea species: Characterization and long-term monitoring of micropropagated clones.

Two different DNA fingerprinting techniques were applied to a set of Achillea samples (Asteraceae), comprising ten taxa of the medicinally important A. millefolium group and six related species. Field-grown as well as in vitro-micropropagated plants were individually screened for abundance and polymorphism of target sequences recognized by oligonucleotide fingerprinting with 13 different microsatellite-complementary probes. While most probes revealed a high level of intra- and interspecific variability, fingerprints proved to be somatically stable in vegetatively propagated plant material. Analysis of the same samples by polymerase chain reaction with arbitrary 10-mer primers yielded less polymorphic patterns. Because of its higher discriminatory ability, oligonucleotide fingerprinting offers itself as the method of choice for the identification and discrimination of A. asplenifolia and A. roseoalba clones, as well as for monitoring their stability during micropropagation.

Natural genetic engineering of plant cells: the molecular biology of crown gall and hairy root disease.

During the past decade, the molecular mechanisms of crown gall and hairy root development have been elucidated in considerable detail. It now appears that the genetic 'colonization' of plant cells by Agrobacterium evolved by continual adaptation of groups of genes that existed long before the evolution of this plant-microbe association. This is most evident for the signal transduction system leading to vir gene induction, and for the early steps of T-DNA transfer to plant cells which have probably evolved from the bacterial conjugation and protein export machinery. However, the later steps, i.e. nuclear targeting of the T-DNA-protein complex, and integration into the host genome by illegitimate recombination are reminiscent of viral infection, where the T-complex resembles a viral particle. The present article reviews the current knowledge of the molecular basis of crown gall and hairy root tumorigenesis, with some emphasis on the mechanisms of signal exchange between plants and bacteria, as well as of T-DNA excision, transfer, integration and expression.

The potential of microsatellites for hybridization- and polymerase chain reaction-based DNA fingerprinting of chickpea (Cicer arietinum L.) and related species.

The genetic variability in agronomically important chickpea accessions (Cicer arietinum L.) as detected by single-locus restriction fragment length polymorphism (RFLP) probes, random amplified polymorphic DNA (RAPD) and isoenzyme markers, is rather low. Recently, highly polymorphic microsatellites became the markers of choice for linkage mapping and population studies. We are currently following two main strategies to exploit the variability of microsatellites and adjacent sequences for genetic studies in chickpea. (i) In an approach referred to as oligonucleotide fingerprinting, microsatellite-complementary oligonucleotides were employed as multilocus probes for in-gel hybridization. A total of 38 different probes representing di-, tri- and tetranucleotide repeats were used to analyze variability between and within four accessions of C. arietinum. Hybridization signals were obtained with 35 probes. While the abundance and level of polymorphism of different target sequences varied considerably, distinct, intraspecifically informative banding patterns were obtained with the majority of probes and all restriction enzymes tested. No obvious correlation existed between abundance, fingerprint quality, and sequence characteristics of a particular motif. (ii) In a recently developed strategy called microsatellite primed polymerase chain reaction (MP-PCR), microsatellite-complementary oligonucleotides serve as single PCR primers for genomic DNA templates. We tested the general applicability of MP-PCR by amplifying DNA samples from tomato, chickpea and two related annual Cicer species with a variety of di-, tri- and tetranucleotide repeat primers. Most but not all primers generated distinct fingerprint-like banding patterns after agarose gel electrophoresis and ethidium bromide staining of the amplification products. Since the method proved to be sensitive to reaction conditions in a way similar to RAPD analysis, we increased the PCR specificity by the introduction of a modified "touch-down" protocol. In chickpea, touch-down MP-PCR generated highly reproducible banding patterns which predominantly revealed interspecific polymorphisms. The potential of different microsatellite-based strategies for genome analysis in chickpea is discussed.

Genomic fingerprinting by microsatellite-primed PCR: a critical evaluation.

Single PCR primers complementary to microsatellite repeats were used to amplify genomic DNA samples from various plant species, as well as from human, yeast, and Escherichia coli DNA. Most primers generated distinct amplification products, resulting in fingerprint-like banding patterns after agarose gel electrophoresis and ethidium bromide staining. These fingerprints allowed distinction among different plant taxa at an interspecific as well as intraspecific level. Unexpectedly, some of the primers produced bands with the E. coli template DNA as well. A detailed examination of the influence of PCR conditions, especially the annealing temperature, on the quality of banding patterns suggested that the majority of bands were generated by mismatch priming in a way similar to random amplified polymorphic DNAs (RAPDs).

Abundance and polymorphism of di-, tri-and tetra-nucleotide tandem repeats in chickpea (Cicer arietinum L.).

The abundance and polymorphism of 38 different simple-sequence repeat motifs was studied in four accessions of cultivated chickpea (Cicer arietinum L.) by in-gel hybridization of synthetic oligonucleotides to genomic DNA digested with 14 different restriction enzymes. Among 38 probes tested, 35 yielded detectable hybridization signals. The abundance and level of polymorphism of the target sequences varied considerably. The probes fell into three broad categories: (1) probes yielding distinct, polymorphic banding patterns; (2) probes yielding distinct, monomorphic banding patterns, and (3) probes yielding blurred patterns, or diffused bands superimposed on a high in lane background. No obvious correlation existed between abundance, fingerprint quality, and the sequence characteristics of a particular motif. Digestion with methyl-sensitive enzymes revealed that simple-sequence motifs are enriched in highly methylated genomic regions. The high level of intraspecific polymorphism detected by oligonucleotide fingerprinting suggests the suitability of simple-sequence repeat probes as molecular markers for genome mapping.