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.

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.

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.

Oligonucleotide fingerprinting detects genetic diversity among Ascochyta rabiei isolates from a single chickpea field in Tunisia.

Fifty isolates of Ascochyta rabiei (Pass.) Labr. were hierarchically sampled from four well-separated locations of a single chickpea field in Beja (Tunisia), and single-spored. DNA was isolated from in-vitro-grown mycelia, digested with HinfI or RsaI, and hybridized to a set of synthetic oligonucleotides complementary to simple repetitive sequences. According to the fingerprint patterns derived from the probes (CA)8, (CAA)5, (CAT)5 and (GATA)4, 12 different fungal haplotypes were found at various frequencies within the investigated field. Seven haplotypes were confined to one location only, four occurred at two, one at three, and none at all four locations. Most of the genetic variability originated from diversity within, rather than between, locations. In some cases, more than one haplotype was isolated from the same lesion of a single host plant. Genetic distances between isolates, as calculated from band-sharing data, varied between 0.05 and 0.22. Relatedness between the different haplotypes was evaluated by cluster analysis using UPGMA.

Oligonucleotide fingerprinting of plant and fungal genomes: a comparison of radioactive, colorigenic and chemiluminescent detection methods.

Digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences were introduced into nonradioactive fingerprint analysis of plant and fungal DNA. The fragment patterns, obtained by blot hybridization of TaqI-restricted DNA from chickpea (Cicer arietinum) and its fungal pathogen Ascochyta rabiei with digoxigenated probes and either a colorigenic or a chemiluminescent detection method, were compared to those obtained with 32P-labeled probes. In combination with alkaline phosphatase and its chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) digoxigenated oligonucleotides yielded clear-cut fingerprints with high signal-to-background ratios within several minutes of exposure to X-ray films. The chemiluminescence reaction remained stable for at least two weeks. A comparison of banding patterns obtained by radioactive versus digoxigenin-based hybridization and detection techniques revealed substantial differences in the relative signal intensities of bands. Both nonradioactive techniques show a tendency to "equalize" band intensity differences. Whereas 32P-labeled oligonucleotides are also applicable to in situ hybridization with DNA immobilized in dried agarose gels, gel hybridization did not work efficiently with digoxigenated probes and either substrate.