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Cultivated chickpea is the third most important legume after field bean and garden pea worldwide. Despite considerable breeding towards improved yield and resistance to biotic and abiotic stresses, the production of chickpea remained stagnant, but molecular tools are expected to increase the impact of current improvement programs. As a first step towards this goal, various genetic linkage maps have been established and markers linked to resistance genes been identified. However, until now, only one linkage group (LG) has been assigned to a specific chromosome. In the present work, mitotic chromosomes were sorted using flow cytometry and used as template for PCR with primers designed for genomic regions flanking microsatellites. These primers amplify sequence-tagged microsatellite site markers. This approach confirmed the assignment of LG8 to the smallest chromosome H. For the first time, LG5 was linked to the largest chromosome A, LG4 to a medium-sized chromosome E, while LG3 was anchored to the second largest chromosome B. Chromosomes C and D could not be flow-sorted separately and were jointly associated to LG6 and LG7. By the same token, chromosomes F and G were anchored to LG1 and LG2. To establish a set of preferably diagnostic cytogenetic markers, the genomic distribution of various probes was verified using FISH. Moreover, a partial genomic bacterial artificial chromosome (BAC) library was constructed and putative single/low-copy BAC clones were mapped cytogenetically. As a result, two clones were identified localizing specifically to chromosomes E and H, for which no cytogenetic markers were yet available. [ABSTRACT FROM AUTHOR]

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea ( Cicer arietinum ICC 4958 × C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM—ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2–21 alleles and polymorphic information content value 0.04–0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago. [ABSTRACT FROM AUTHOR]

We developed a new platform for genome-wide gene expression analysis in any eukaryotic organism, which we called SuperSAGE array. The SuperSAGE array is a microarray onto which 26-bp oligonucleotides corresponding to SuperSAGE tag sequences are directly synthesized. A SuperSAGE array combines the advantages of the highly quantitative SuperSAGE expression analysis with the high-throughput microarray technology. We demonstrated highly reproducible gene expression profiling by the SuperSAGE array for 1,000 genes (tags) in rice. We also applied this technology to the detailed study of expressed genes identified by SuperSAGE in Nicotiana benthamiana, an organism for which sufficient genome sequence information is not available. We propose that the SuperSAGE array system represents a new paradigm for microarray construction, as no genomic or cDNA sequence data are required for its preparation. [ABSTRACT FROM AUTHOR]

SAMPL primers designed for genomic profiling in chickpea (Cicer arietinum L.) were tested for their applicability to fingerprinting of DNA of banana cultivars and somaclonal variants. Most of the chickpea primers allowed amplification of genomic DNA of banana and detection of sequence polymorphisms within the Musa acuminata genome (A). Our results demonstrate that the highly resolving SAMPL technique is useful in banana genomics, especially tor the distinction and characterization of commercially important cultivars and promising somaclonal variants containing the A genome. [ABSTRACT FROM AUTHOR]

Degenerate primers deduced from the TPase region of plant En/Spm-like transposons allowed the amplification of similar sequences from various plant species including sugar beet, wheat and pea. These primers are efficient tools for the detection of this family of transposons in many plant genomes irrespective of sequence knowledge or phenotypic pecularities. An efficient PCR assay was therefore developed for these class II transposons, similar to assays already available for Ty1-copia-, Ty3-gypsy- or LINEs. This approach allowed us not only to show the widespread almost-ubiquitous presence of En/Spm-elements in plant genomes, but also to characterize their genomic organization and chromosomal distribution in the genome of chickpea ( Cicer arietinum L.) and its abundance in related Cicer species. This approach can be used for the detection and characterization of endogenous DNA transposable elements in plant species, their complete isolation and evaluation of their use for genome analysis. [ABSTRACT FROM AUTHOR]

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), (CAA), (CAT) and (GATA), 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. [ABSTRACT FROM AUTHOR]

The ascomycete fungus Ascochyta rabiei, an important pathogen of the grain legume crop chickpea ( Cicer arietinum L.) in the Mediterranean region, has not been adequately characterized in molecular terms. We therefore used DNA fingerprinting, with synthetic oligodeoxynucleotides complementary to simple repetitive sequences, to pathotype different isolates of the fungus. Six single-spored A. rabiei isolates were first categorized using a host differential set of nine chickpea genotypes. Seedlings were inoculated under controlled environmental conditions, and disease severity was recorded 9 days after inoculation. DNA was extracted from in vitro-grown mycelia of the six purified fungal isolates, restricted with EcoRI, HinfI, MboII and TaqI, and fingerprinted with radiolabeled (GATA), (GTG), (CA), and (TCC), respectively. High levels of polymorphism were detected with optimal enzyme/probe combinations that allow one to discriminate between the isolates. The potential of DNA fingerprinting with simple repetitive sequences can thus be expanded to the identification of fungal races and pathotypes. The characterization of the geographic distribution and genetic variability of pathotypes will facilitate the selection of suitable host cultivars to be grown in specific regions. [ABSTRACT FROM AUTHOR]

Chromatin-bound, DNA-dependent RNA polymerase (EC 2.7.7.6) activity and chromatin template availability, as measured with saturating amounts of E. coli RNA polymerase, changes rhythmically during the formation, dormancy, and sprouting of potato tubers. Active growth processes coincide with the highest RNA polymerase activity as well as the greatest template accessibility, during tuberization and sprouting. Consequently, chromatin-associated RNA and protein content is highest in young developing tubers and in old tubers at the onset of sprouting. Ribosomal RNA content, in turn, is maximal in small tubers, remains constant during dormancy, and decreases when sprouting begins, probably due to the translocation of rRNA into the sprouts. The nucleolus changes its shape and size concomitantly with the process of tuberization. [ABSTRACT FROM AUTHOR]

The reaction of potato tuber tissue upon wounding and gibberellic acid (GA) treatment is strictly dependent on the tuber age. Young, rapidly growing tubers decline both chromatin-bound, DNA-dependent RNA polymerase (EC 2.7.7.6) activity and template availability as a consequence of wounding and are not responsive toward GA. At the onset of dormancy, ripened tubers do not at all respond to wounding or hormones, but later on develop the ability to increase their transcriptional rate and template accessibility, both after injury and treatment with 10 mol l GA. The size of the nucleolus and the rRNA content of the ribosomal population follows the same pattern. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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The chromatin structure of pea ( Pisum sativum) rbcS genes in inactive (root), potentially active (dark-grown leaf), and active states (light-grown leaf) was analysed using (a) pancreatic DNAseI to detect general DNAseI sensitivity and DNAseI-hypersensitive sites, and (b) methyl-sensitive restriction endonucleases to probe for cytosine methylation within the promoter region. We showed that within the same organ individual members of the pea rbcS multigene family are differentially sensitive to DNAseI suggesting differential protection in nuclei. During light activation general DNAseI sensitivity increases in some genes, especially their 5′ upstream regulatory sequences. DNAseI-hypersensitive sites are constitutively present in 5′ upstream regulatory sequences around positions −335, −465, −650, and −945 (5′ constitutive domain) and in the coding region around position +340, +450, +530, +640, and +810 (3′ constitutive domain). One additional hypersensitive site appears after light induction (inducible site). This region is centred around position −190 and flanked by light-responsive elements (LREs). In spite of changes in the chromatin structure of rbcS genes during their transition from an inactive to an active state, their cytosine methylation at Alu I, Fnu 4HI, Hae III, Sau 3AI and Sau 96I sites in the promoter region remains uniform. [ABSTRACT FROM AUTHOR]

White potato tuber tissue reacts upon wounding with a rapid increase in activity of both chromatin-bound DNA-dependent RNA polymerase I and II as well as protein phosphokinase. This enhancement is more pronounced if 0.1 mM of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is added shortly after wounding. The effect of the hormone on protein kinases becomes evident only after a lag phase of about 10h and lasts throughout the wound-healing period. Different protein kinases with different substrate specificity (i.e. histone, phosvitin, casein phosphokinases) are distinctly more active in auxin-treated tissues. The phosphate is apparently introduced into proteins via seryl and threonyl bonds. Acyl or histidyl phosphates are not involved. The properties of protein phosphokinases are virtually identical in wounded and auxin-treated tissues. However, the pattern of chromosomal proteins and the pattern of their phosphorylation in hormone-treated tissues is different from those in wounded ones. A drastic stimulation of phosphorylation of both high and low-molecular weight chromosomal proteins is characteristic for auxin-treated cells. [ABSTRACT FROM AUTHOR]

Resting plant storage organs, e.g., potato tubers, carrots or sugar beets usually exhibit a very low metabolic activity. Slicing into thin disks and incubation of these tissue fragments in a moist atmosphere, however, leads to a coordinated genetic and metabolic effort to adapt the exposed cells to their new environment. Nearly all reactions occurduring this struggle are dependent on genetic activity: slicing strikingly mimics well known hormone effects in mammalian systems and most certainly evokes an induction similar to that of the hormones (e.g., estrogen on uterine cells, Table VIII). Moreover, the cell controls the magnitude of induced reactions by way of its considerable inductive, repressive or modifying capabilities. However, in sharp contrast to the widely studied hormone systems of the plant and animal kingdoms, plant storage tissue slices and their capabilities have been largely neglected until now or are not known at all. Nevertheless they provide an excellent example of genetic and metabolic regulation occurring side by side and easy-to-handle material for the study of the elementary processes at the gene level following derepression of previously repressed genes in multicellular organisms. [ABSTRACT FROM AUTHOR]