Mahinur S. Akkaya

The use of microsatellite DNA markers for soybean genotype identification

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT)n/(TA)n and (ATT)n/(TAA)n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, “n”, results in PCR product length differences. The SSR alleles present at three (AT)n/(TA)n and four (ATT)n/(TAA)n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.

Analysis of genetic relationships among perennial and annual Cicer species growing in Turkey using RAPD markers

Abstract.Random amplified polymorphic DNA (RAPD) fragments were used to assess genetic relationships among Cicer spp. growing in Turkey. Seven 10-mer primers selected from a 50 random oligonucleotide primer set, depending on their ability to amplify genomic DNA in all species, were used to detect RAPD variation in 43 wild and cultivated accessions representing ten species. These primers yielded 95 reproducible amplification products, 92 of which were polymorphic. Pairwise genetic distances of accessions estimated according to Nei and Li (1979) were used to produce a dendrogram using UPGMA. The dendrogram contained two main clusters, one of which comprised accessions of the four perennial species (Cicer montbretii, Cicer isauricum, Cicer anatolicum and Cicer incisum) together with the accessions of the three annual species (Cicer pinnatifidum, Cicer judaicum and Cicer bijugum), and the other cluster included the remaining three annual species (Cicer echinospermum, Cicer reticulatum and Cicer arietinum). Analysis of RAPD variation showed that C. incisum is the most similar perennial species to annuals, and C. reticulatum is the closest annual species to chickpea. These results generally agree with our allozyme study which was carried out using same Cicer collection and previous studies of relationships among annual species. The results also show that RAPD markers can be used to distinguish Cicer species and to survey genetic variation and relationships among taxonomic units in this genus.

Genetic relationships among perennial and annual Cicer species growing in Turkey assessed by AFLP fingerprinting

AFLP markers were used to assess genetic relationships among Cicer species with distribution in Turkey. Genetic distances were computed among 47 Cicer accessions representing four perennial and six annual species including chickpea, using 306 positions on AFLP gels. AFLP-based grouping of species revealed two clusters, one of which includes three perennial species, Cicer montbretii, Cicer isauricum and Cicer anatolicum, while the other cluster consists of two subclusters, one including one perennial, Cicer incisum, along with three annuals from the second crossability group (Cicer pinnatifidum, Cicer judaicum and Cicer bijugum) and the other one comprising three annuals from the first crossability group (Cicer echinospermum, Cicer reticulatum and Cicer arietinum). Consistent with previous relationship studies in the same accession set using allozyme and RAPD markers, in AFLP-based relationships, C. incisum was the closest perennial to nearly all annuals, and C. reticulatum was the closest wild species to C. arietinum. Cluster analysis revealed the grouping of all accessions into their distinct species-clusters except for C. reticulatum accessions, ILWC247, ILWC242 and TR54961; the former was found to be closer to the C. arietinum accessions while the latter two clustered with the C. echinospermum group. Small genetic distance values were detected among C. reticulatum accessions (0.282) and between C. reticulatum and C. arietinum (0.301) indicating a close genetic similarity between these two species. Overall, the AFLP-based genetic relationships among accessions and species were congruous with our previous study of genetic relationships using allozymes. The computed level of AFLP variation and its distribution into within and between Cicer species paralleled the previous report based on RAPD analyses. AFLP analysis also confirmed the presence of the closest wild relatives and previous projections of the origin of chickpea in southern Turkey. Results presented in this report indicate that AFLP analysis is an efficient and reliable marker technology in determination of genetic variation and relationships in the genus Cicer. Obviously, the use of AFLP fingerprinting in constructing a detailed genetic map of chickpea and cloning, and characterizing economically important traits would be promising as well.

Methodology of generation and characteristics of simple sequence repeat DNA markers in avocado (Persea americana M.)

SummaryGeneration of Simple Sequence Repeat (SSR) DNA markers was based on the construction of genomic DNA library of avocado (Persea americana M.). The library was screened with the four dinucleotide probes (AG), (AT), (GC) and (CA). Positive clones were sequenced to validate the presence of simple sequence repeats (SSR) and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the simple sequence repeat. Twenty six different pairs of primers which yield a PCR product in the initial screening were synthesized. The SSR A1E11 was found to have eleven alleles while A3F8 has eight alleles. The SSRs in avocado were found to be inherited in a Mendelian fashion.

Cellular and transcriptional responses of wheat during compatible and incompatible race‐specific interactions with Puccinia striiformis f. sp. tritici

The initial stages of Puccinia striiformis f. sp. tritici (the causal agent of yellow rust in wheat) infection triggered a hypersensitive cell death (HCD) response in both compatible and Yr1-mediated incompatible interactions, although the response was earlier and more extensive in the incompatible interaction. Later stages of fungal development were only associated with an HCD response in the incompatible interaction, the HCD response being effectively suppressed in the compatible interaction. Cell autofluorescence was seen in mesophyll cells in direct contact with fungal infection hyphae (primary HCD) and in adjacent mesophyll cells (secondary HCD), indicating the activation of cell-to-cell signalling. Microarray analysis identified a number of defence-related transcripts implicated in Yr1-mediated resistance, including classical pathogenesis-related (PR) transcripts and genes involved in plant cell defence responses, such as the oxidative burst and cell wall fortification. A quantitative reverse transcriptase-polymerase chain reaction time course analysis identified a number of defence-related genes, including PR2, PR4, PR9, PR10 and WIR1 transcripts, associated with the latter stages of Yr1-mediated resistance. A meta-analysis comparison of the Yr1-regulated transcriptome with the resistance transcriptomes of the race-specific resistance gene Yr5 and the race-nonspecific adult plant resistance gene Yr39 indicated limited transcript commonality. Common transcripts were largely confined to classic PR and defence-related genes.

Virus induced gene silencing in Brachypodium distachyon, a model organism for cereals

Brachypodium distachyon is emerging as a model organism for crops as a better alternative to Oryzae sativa. It shares common characteristics of a model plant with its small genome, small physical plant size, a short lifecycle, and less demanding growth requirements similar to Arabidopsis thaliana. In this study, we are reporting for the first time, an implementation of virus induced gene silencing (VIGS), a powerful method allowing rapid and effective means of loss of gene function through RNA interference. To this end, Phytoene desaturase (Pds) gene, commonly preferred in gene silencing studies as a phenotypic marker, was silenced using Barley Stripe Mosaic Virus (BSMV), the most effectively used virus in monocots for VIGS., a fragment of the ORF of the B. distachyonPds gene was cloned into the BSMV vector having the γ genome proviral DNA. The decreased Pds gene expression was confirmed after plant infection by qRT-PCR. The effectiveness of BSMV infection was also tested with the transcripts of the vector constructed for GFP expression. We believe the demonstration of BSMV mediated VIGS will be an important step for evaluating functions of crop genes in this model organism.

Remarkable cooperative action of two zinc centers in the hydrolysis of plasmid DNA

Abstract A novel binuclear zinc complex has been synthesized. The complex is highly efficient in the hydrolysis of plasmid DNA at pH 7.5. Furthermore, a comparison to a mononuclear complex reveals a high level of cooperativity between the two metal ion centers.

RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment

No amino acid or DNA sequence information in sequence databases was found for a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions in the lactate dehydrogenase enzymes of all taxonomies are found to be betaalphabeta nucleotide binding and substrate binding sites, also catalysis/active site. The conserved regions were selected as PCR primer target regions. The degenerate primers were designed according to the codon usage, determined by analyzing a number of different genes of Rhizopus species. A fragment of the gene (ldh), coding for approximately 72% of the lactate dehydrogenase enzyme from Rhizopus oryzae, was amplified using degenerate primers by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The size of the amplified fragment containing betaalphabeta nucleotide binding site, substrate binding site and catalysis/active site is found to be about 700 bp. The reported degenerate PCR primers and the amplification conditions may lead to the cloning of the lactate dehydrogenase gene of R. oryzae, which is an important organism due to its utilization in lactic acid and enzyme productions in industrial scales.

Remarkable phosphodiester hydrolysis activity of a novel CeIV complex in neutral aqueous solutions

Abstract A novel Ce IV -complex of 1,4,7-tris(carbamoylmethyl)-1,4,7-triazacyclononane has been synthesized. The complex is both stable in aqueous solutions at neutral pH and very efficient in promoting the hydrolysis of a phosphodiester model compound and yeast tRNA phe . The RNA model compound, 2-hydroxypropyl- p -nitrophenylphosphate hydrolysis is accelerated 7400-fold at pH 7.5, as a result of an unprecedented hydrolytic activity.

Light regulation of protein synthesis factor EF-G in pea chloroplasts.

The activity of pea chloroplast elongation factor G (EF-G), a nuclear-coded protein required for the elongation cycle of chloroplast protein synthesis, is regulated in response to light. In pea seedlings germinated and grown under continuous white or red light, EF-G specific activity reaches a maximum between days 10 to 15, and then decreases. EF-G activity is almost undetectable in extracts from dark-grown seedlings. When 13-day dark-grown pea seedlings are transferred to light, EF-G specific activity reaches a higher value after 2 to 3 days than observed in seedlings grown under continuous light. The small and large subunits of ribulose bisphosphate carboxylase continue to accumulate after EF-G specific activity has reached maximum levels. Cytoplasmically synthesized components of the chloroplast protein synthetic apparatus, such as EF-G, may help coordinate cytoplasmic and nuclear events with chloroplast gene expression during light-induced chloroplast differentiation.