Genetic variation, phylogenetic relationship and spatial distribution of 'Candidatus Phytoplasma ulmi' strains in Germany.

A recent survey in Germany revealed the wide presence of 'Candidatus Phytoplasma ulmi' in native elm stands. Accessions were studied for their genetic variability and phylogenetic relationship based on the conserved groEL and the variable imp gene. While the groEL sequences revealed a high intraspecific homology of more than 99%, the homology of the imp gene dropped to 71% between distantly related sequences. Twenty-nine groEL and 74 imp genotypes were distinguished based on polymorphic sites. Phylogenetic analysis of the groEL gene clustered all 'Ca. P. ulmi' strains and separated them from related phytoplasmas of the 16SrV group. The inferred phylogeny of the imp gene resulted in a different tree topology and separated the 'Ca. P. ulmi' genotypes into two clusters, one closely related to the flavescence dorée phytoplasma strain FD-D (16SrV-D), the other affiliated with the flavescence dorée phytoplasma strains FD-C and FD70 and the alder yellows phytoplasma (16SrV-C). In both phylograms, 'Ca. P. ulmi' genotypes from Scots elm trees formed a coherent cluster, while genotypes from European white elms and field elms grouped less strictly. The regional distribution pattern was congruent for some of the groEL and imp genotypes, but a strict linkage for all genotypes was not apparent.

Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome.

A size-selected genomic library comprising 280,000 colonies and representing approximately 18% of the chickpea genome, was screened for (GA)n, (GAA)n and (TAA)n microsatellite-containing clones, of which 389 were sequenced. The majority (approximately 75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%-9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613 cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P > or = 0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.

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.

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.

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.