SSR-based molecular analysis of economically important Turkish apricot cultivars.

Turkey is not only the main apricot (Prunus armeniaca) producer and exporter in the world, but it also has a wide variety of apricot germplasms, owing to its close proximity to the centers of apricot origin. However, there is little or no genetic information on many apricot cultivars that are extensively cultivated in Turkey. We examined the genetic relatedness of 25 Turkish and four exotic apricot cultivars using SSR (simple sequence repeat) markers that were either previously developed for apricot, or for peach (P. persica), a close relative of apricot. Allele diversity (with an average allele number of 6.37) at the SSR loci and the heterozygosity rates (with an average Ho value of 0.648) of these cultivars were found to be higher than in previous studies that used the same loci for apricot. This fact might be attributed to the analysis of different numbers of accessions in the different studies. No correlations were found between the genetic relatedness and the geographical distributions of these cultivars. The data reported here will assist in the prevention of confusions in the apricot propagation and breeding in Turkey. The findings can also be directly compared with other studies that used the same SSR markers on apricot.

Genetic characterization of green bean (Phaseolus vulgaris) genotypes from eastern Turkey.

Green bean genotypes collected from eastern Turkey were characterized using simple sequence repeat (SSR) markers and morphological traits. Among 12 SSR markers, 10 produced successful amplifications and revealed DNA polymorphisms that were subsequently used to assess genetic relatedness of the genotypes. Based on the number of alleles generated and the probability of identity values, the most informative SSR loci were PVGLND5, PVMEIG, PV-ag001, and PV-ag004. Probably, due to the inbreeding nature of beans, the heterozygosity observed within genotypes was low at most of the SSR loci. The UPGMA dendrogram constructed based on the SSR data yielded two major clusters. The overall genetic distance was around 98%, among the genotypes. This information can be used to help select Turkish green bean lines.

Identification of plant defence genes in canola using Arabidopsis cDNA microarrays.

We report the identification of novel defence genes in canola by using a cDNA microarray from Arabidopsis. We examined changes that occur in the abundance of transcripts corresponding to 2375 Arabidopsis expressed sequence tags (selected for defence gene identification) following inoculation of canola plants with the fungal necrotrophic leaf pathogen, Alternaria brassicicola. Microarray data obtained from this cross-hybridisation experiment were compared to expression profiles previously obtained from the equivalent Arabidopsis experiment. Homology searches using a canola expressed sequence tag database with approximately 6000 unique clones led to identification of canola defence genes. Pathogen-responsive transcripts included those associated to known defence genes, reactive oxygen species metabolism, disease resistance and regulatory genes, and cell maintenance/metabolism genes. Using specific primers for quantitative real-time reverse transcriptase PCR, gene expression profiles in canola were obtained that demonstrated coordinated defence responses, including systemic responses in distal tissue and salicylic acid- and methyl jasmonate-mediated signalling against A. brassicicola.

AFLP marker linked to water-stress-tolerant bulks in barley (Hordeum vulgare L)

The amplified fragment length polymorphism (AFLP) assay is an efficient method for the identification of molecular markers, useful in the improvement of numerous crop species. Bulked Segregant Analysis (BSA) was used to identify AFLP markers associated with water-stress tolerance in barley, as this would permit rapid selection of water-stress tolerant genotypes in breeding programs. AFLP markers linked to water-stress tolerance was identified in two DNA pools (tolerant and sensitive), which were established using selected F-2 individuals resulting from a cross between water-stress-tolerant and sensitive barley parental genotypes, based on their paraquat (PQ) tolerance, leaf size, and relative water content (RWC). All these three traits were previously shown to be associated with water-stress tolerance in segregating F-2 progeny of the barley cross used in a previous study. AFLP analysis was then performed on these DNA pools, using 40 primer pairs to detect AFLP fragments that are present/absent, respectively, in the two pools and their parental lines. One separate AFLP fragment, which was present in the tolerant parent and in the tolerant bulk, but absent in the sensitive parent and in the sensitive bulk, was identified. Polymorphism of the AFLP marker was tested among tolerant and sensitive F-2 individuals. The presence of this marker that is associated with water-stress tolerance will greatly enhance selection for paraquat and water-stress tolerant genotypes in future breeding programs

DNA microarrays: new tools in the analysis of plant defence responses.

Summary Large-scale DNA sequencing is providing information on the number and organization of genes and genomes of plant species and their pathogens. The next phase is to identify gene functions and gene networks with key roles in compatible and incompatible plant-pathogen interactions. DNA microarrays can provide information on the expression patterns of thousands of genes in parallel. The application of this technology is already revealing new features of plant-pathogen interactions and will be a key tool for a wide range of experiments in molecular plant pathology.

Coordinated plant defense responses in Arabidopsis revealed by microarray analysis.

Disease resistance is associated with a plant defense response that involves an integrated set of signal transduction pathways. Changes in the expression patterns of 2,375 selected genes were examined simultaneously by cDNA microarray analysis in Arabidopsis thaliana after inoculation with an incompatible fungal pathogen Alternaria brassicicola or treatment with the defense-related signaling molecules salicylic acid (SA), methyl jasmonate (MJ), or ethylene. Substantial changes (up- and down-regulation) in the steady-state abundance of 705 mRNAs were observed in response to one or more of the treatments, including known and putative defense-related genes and 106 genes with no previously described function or homology. In leaf tissue inoculated with A. brassicicola, the abundance of 168 mRNAs was increased more than 2.5-fold, whereas that of 39 mRNAs was reduced. Similarly, the abundance of 192, 221, and 55 mRNAs was highly (>2.5-fold) increased after treatment with SA, MJ, and ethylene, respectively. Data analysis revealed a surprising level of coordinated defense responses, including 169 mRNAs regulated by multiple treatments/defense pathways. The largest number of genes coinduced (one of four induced genes) and corepressed was found after treatments with SA and MJ. In addition, 50% of the genes induced by ethylene treatment were also induced by MJ treatment. These results indicated the existence of a substantial network of regulatory interactions and coordination occurring during plant defense among the different defense signaling pathways, notably between the salicylate and jasmonate pathways that were previously thought to act in an antagonistic fashion.

Expression of the Shpx2 peroxidase gene of Stylosanthes humilis in transgenic tobacco leads to enhanced resistance to Phytophthora parasitica pv. nicotianae and Cercospora nicotianae.

Abstract Previous research indicated that the constitutive expression of a pathogen-inducible peroxidase gene (Shpx6a) from Stylosanthes humilis in transgenic plants resulted in enhanced resistance to fungal pathogens ( Kazan, K., Goulter, K.C., Way, H.M. and Manners, J.M. (1998) Expression of a pathogenesis-related peroxidase of Stylosanthes humilis in transgenic tobacco and canola and its effect on disease development. Plant Sci. 136, 207-217). We have now investigated another pathogen-inducible peroxidase gene of S. humilis, termed Shpx2, which is highly divergent from Shpx6a. Constitutive expression of the Shpx2 cDNA was obtained in tobacco using the 35S CaMV promoter, and up to a 12-fold increase in total peroxidase activity was observed in the leaves of transgenic plants compared to nontransgenic controls. Disease development was evaluated after inoculations in T(1) and T(2) transgenic lines expressing Shpx2. Lesion expansion was significantly (P < 0.05) reduced by up to 25% and 50% on leaves and stems, respectively, of transgenic plants expressing high levels of peroxidase compared to nontransgenic controls, following inoculation with Phytophthora parasitica pv. nicotianae, the cause of black shank disease. In addition, plant survival and recovery were greatly enhanced in transgenic plants following stem inoculations of plants with this plant pathogen. A significant (55%, P < 0.05) reduction in lesion number was observed in transgenic plants with high levels of peroxidase activity following inoculation with the fungus Cercospora nicotianae, the cause of frog-eye disease. No significant differences in disease development were observed between the lines expressing Shpx2 and controls following inoculation with the bacterium Pseudomonas syringae pv. tabaci, the cause of wildfire disease. These results provide evidence for a role for this peroxidase gene in plant defence, and suggest that diverse peroxidase genes may be employed as components of strategies aimed at the engineering of disease resistance.

The promoter of the plant defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal pathogens and responds to methyl jasmonate but not to salicylic acid.

The plant defensin PDF1.2 has previously been shown to accumulate systemically via a salicylic acid-independent pathway in leaves of Arabidopsis upon challenge by fungal pathogens. To further investigate the signalling and transcriptional processes underlying plant defensin induction, a DNA fragment containing 1184 bp and 1232 bp upstream of the transcriptional and translational start sites, respectively, was cloned by inverse PCR. To test for promoter activity this DNA fragment was linked to the beta-glucuronidase (GUS)-encoding region of the UidA gene as a translational fusion and introduced into Arabidopsis ecotype C-24. Challenge of the transgenic plants with the fungal pathogens Alternaria brassicicola and Botrytis cinerea resulted in both local and systemic induction of the reporter gene. Wounding of the transgenic plants had no effect on GUS activity. Treatment of the transgenic plants with either jasmonates or the active oxygen generating compound paraquat strongly induced the reporter gene. In contrast, neither salicylate nor its functional analogues 2,6-dichloroisonicotinic acid and 1,2,3-benzothiodiazole-7-carbothioic acid S-methyl ester resulted in reporter gene induction. These results are consistent with the existence of a salicylic acid-independent signalling pathway, possibly involving jasmonates as regulators, that is triggered by pathogen challenge but not by wounding. The transgenic plants containing the PDF1.2-based promoter-reporter construct will provide useful tools for future genetic dissection of this novel systemic signalling pathway.

Genetic relationships and variation in the Stylosanthes guianensis species complex assessed by random amplified polymorphic DNA.

Genetic variation in the five taxonomic groups of the Stylosanthes guianensis (Aubl.) Sw. complex was investigated using random amplified polymorphic DNA markers (RAPDs). DNA samples from four plants of each of 45 accessions within the S. guianensis species complex were analyzed using 20 oligonucleotides of random sequence. Little variation was found within each of the 18 accessions (1-7% of total RAPD bands in pairwise comparisons) and none within each of the other 27 accessions. However, higher levels of polymorphisms were observed both within (index of genetic distance = 1 - F = 0.16-0.248) and between (1 - F = 0.254-0.408) the five taxa. This level of differentiation at the DNA level supported an earlier classification of the taxa as distinct species. A phenogram based on band sharing was constructed to show genetic relationships among the taxa studied. This phenogram corroborated the description of relationships based on morphological-agronomic characteristics, seed protein patterns, rhizobial affinities, crossability, and pollen stainability of the hybrids. In this phenogram, the most similar species were S. grandiflora and S. hippocampoides (1 - F = 0.264), with S. acuminata also showing closest similarity to these two species (1 - F = 0.277 and 0.283, respectively). Stylosanthes gracilis accessions showed the closest similarity (1 - F = 0.296) to S. guianensis ssp. guianensis accessions. Lowest similarity values (1 - F = 0.335-0.411) were found between these two species and S. grandiflora, S. acuminata, and S. hippocampoides.

Inheritance of random amplified polymorphic DNA markers in an interspecific cross in the genus Stylosanthes.

The inheritance of random amplified polymorphic DNA (RAPD) markers generated via the polymerase chain reaction amplification of genomic DNA sequences in an F2 family of an interspecific cross between Stylosanthes hamata and S. scabra was investigated. An initial comparison between the parental species, S. hamata cv. Verano and S. scabra cv. Fitzroy, demonstrated that 34% of detected RAPD bands were polymorphic. Of 90 primers tested, 35 showed relatively simple and reliably scorable polymorphisms and were used for segregation analysis. Sixty F2 individuals were scored for the segregation of 73 RAPD markers and 55 of these markers fit a 3:1 ratio. Segregation of eight other RAPD markers deviated significantly from a 3:1 ratio. There was no bias in the inheritance of RAPD markers regarding parental origin of the segregating RAPD markers. Linkage analysis revealed 10 linkage groups containing a total of 44 RAPD loci. Another 10 RAPD markers (7 of maternal origin) that were polymorphic between the parents did not segregate in the F2 population. One of the maternally inherited RAPD bands hybridized to chloroplast DNA. Analysis of RAPD loci by DNA hybridization indicated that mainly repeated sequences were amplified. These data indicate that RAPDs are useful genetic markers in Stylosanthes spp. and they may be suitable for genetic mapping.

Inheritance and linkage relationships of morphological and isozyme loci in chickpea (Cicer arietinum L.).

Inheritance and linkage relationships of several morphological and isozyme loci are described in chickpea (Cicer arietinum L.). Segregation data obtained from several F2 families confirmed the previously observed mode of inheritance for most of the morphological loci. Additional morphological markers in chickpea are also described. Most of the isozyme loci studied showed codominant expression and fit expected Mendelian segregation ratios. However, distorted ratios were also observed for some loci. Linkage was found betweenPgd-c, the locus encoding the cytosolic form of 6-phosphogluconate dehydrogenase, andHg, the locus controlling plant growth habit. These 2 loci were separated by approximately 18 recombinational map units. A similar linkage between comparable loci was previously reported in pea (Pisum sativum L.) (Weeden and Wolko 1990). Linkage was also detected among 3 isozyme loci; the cytosolic form of phosphoglucomutase (Pgm-c), glucose-1-phosphate transferase (Gpt1), and the plastid specific form of 6-phosphogluconate dehydrogenase (Pgd-p). The linkage of 2 loci (Pgm-c andPgd-p) in this cluster is also conserved in pea and lentil (Lens Miller). The linkage between an acid phosphatase locus (Acp3) and the locus specifying the cytosolic form of glucosephosphate isomerase (Gpi-c) in chickpea suggested another linkage group in common with pea. Additionally, other linkages that were not previously observed in chickpea or related genera included the linkage of the cytosolic form of aconitase (Aco-c) with adenylate kinase (Adk1) and fructokinase (Fk3), and the linkage of a locus encoding the mitochondrial specific aconitase (Aco-m) with a seed protein locus (Spr1). The loci determining flower color (P), epicotyl color (Gst), seed coat color (T (3)), and seed surface (Rs) were associated with the locus encoding glucose-1-phosphate transferase (Gpt2). These results, along with previous studies, suggest that pea, lentil and chickpea have several common linkage groups consisting of homologous genes. This also indicates that linkages found in one genus can be used to predict similar linkages in related genera in the development of linkage maps.

Genetic variation in agronomically important species of Stylosanthes determined using random amplified polymorphic DNA markers.

Random amplified polymorphic DNA (RAPD) markers were generated from 20 cultivars and accessions representing four agronomically important species of Stylosanthes, S. scabra, S. hamata, S. guianensis, and S. humilis. Approximately 200 fragments generated by 22 primers of arbitrary sequence were used to assess the level of DNA variation. Relatively low levels of polymorphism (0-16% of total bands in pairwise comparisons) were found within each species, while polymorphisms between the species were much higher (up to 46%). Very few polymorphisms (0-2%) were detected between the individuals of the same cultivar or accession. A phenogram of relationships among the species was constructed based on band sharing. Four main clusters corresponding to each species were readily distinguished on this phenogram. The allotetraploid species S. hamata and its putative diploid progenitor, S. humilis, were more similar to each other than to S. scabra and S. guianensis. No variation in RAPD markers was found between the two commercial S. hamata cvs 'Verano' and 'Amiga'. Cultivar 'Oxley' in S. guianensis was considerably different from the other cultivars and accessions of this species. The phylogenetic distinctions obtained with RAPDs were in agreement with other studies from morphology, cytology, and enzyme electrophoresis. The low level of polymorphisms observed within each species suggested that interspecific crosses may be a better vehicle for the construction of RAPD linkage maps in Stylosanthes.