Molecular cytogenetic characterisation of Salix viminalis L. using repetitive DNA sequences

Abstract Salix viminalis L. (2n=38) is a diploid dicot species belonging to the Salix genus of the Salicaceae family. This short-rotation woody crop is one of the most important renewable bioenergy resources worldwide. In breeding for high biomass productivity, limited knowledge is available on the molecular cytogenetics of willow, which could be combined with genetic linkage mapping. The present paper describes the adaptation of a fluorescence in situ hybridisation (FISH) protocol as a new approach to analyse the genomic constitution of Salix viminalis using the heterologous DNA clones pSc119.2, pTa71, pTa794, pAs1, Afafamily, pAl1, HT100.3, ZCF1 and the GAA microsatellite marker. Three of the nine probes showed unambiguous signals on the metaphase chromosomes. FISH analysis with the pTa71 probe detected one major 18S-5.8S-26S rDNA locus on the short arm of one chromosome pair; however, the pTa794 rDNA site was not visible. One chromosome pair showed a distinct signal around the centromeric region after FISH with the telomere-specific DNA clone HT100.3. Two chromosome pairs were found to have pAs1 FISH signals, which represent a D-genome-specific insert from Aegilops tauschii. Based on the FISH study, a set of chromosomes with characteristic patterns is presented, which could be used to establish the karyotype of willow species

Kései szárazságtűrésben szerepet játszó génjelöltek asszociációs vizsgálata árpában EcoTILLING módszerrel = Association testing of barley candidate genes for terminal drought tolerance using EcoTILLING technology

Célunk a szárazságtűrésben szerepet játszó kandidátus gének genetikai variabilitásának feltárása volt, melyhez az EcoTILLING módszert alkalmaztuk. Vizsgálatainkhoz a világ számos pontjáról gyűjtött 96 genotípust tartalmazó, szárazságtűrés szempontjából variábilis árpa kollekciót állítottunk össze. Az alkalmazott technológia segítségével mintegy 1,5 millió bázispárnyi szekvencia vizsgálata nyomán 94 egyedi allélvariánst különítettünk el a 9 génre tervezett 18 amplikon elemzése útján. Egy bázispárnyi eltérést (SNP) 185, inszerció/deléció-t (INDEL) pedig 46 esetben azonosítottunk. A haplotípus-szekvenciák birtokában 4 kandidátus gén esetében olyan informatív polimorfizmusokat konvertáltunk át genetikai markerekké, melyek által lehetővé vált a valószínűsíthetően funkcionális allélvariánsok elkülönítése. A szárazságtolerancia mértékének komplex stressz diagnosztikai rendszerben történő teszteléséhez összeállítottunk egy 25 genotípust tartalmazó árpa törzskollekciót. A genotípusok szárazság toleranciájának szintjét szántóföldi körülmények között is meghatároztuk. A gyökérnövekedési paraméterek nyomonkövetésére kidolgoztunk egy kísérleti rendszert. Az osztódásban lévő sejtek arányát 5-etinil-2-deoxiuridin (EdU)-re alapozott fluoreszcens mikroszkópiával határoztuk meg. Egy ellenálló és egy érzékeny genotípus esetében a génexpressziós mintázatokat is összehasonlítjuk a gyökérspecifikus és a sejtciklusban szereplő gének vizsgálata útján. | We aim for exploring genetic variability of drought tolerance related candidate genes we have applied a high throughput and relatively inexpensive method namely EcoTILLING as a polymorphism discovery tool. We have established a set of 96 barley genotypes, which contains drought tolerant and sensitive genotypes collected worldwide. By using this method approximately 1.5 million basepairs in barley a total number of 94 verified unique haplotypes were identified in 18 amplicons designed for 9 genes. Overall, 185 single nucleotide polymorphisms (SNPs) and 46 insertions/deletions (INDELs) were detected. Based on overlapping haplotype sequences, of four candidate genes informative poly-morphisms were converted into genetic markers allowing the detection of the potential functional haplotypes. To test drought tolerance and agronomic parameters we have used a complex stress diagnostic system for characterization a subcollection of 25 barley genotypes. The drought tolerance of these genotypes was also tested under field conditions. The two test system provided overlapping ranks of genotypes in drought response. We have developed an experimental system for the detection of changes of root growth parameters of barley seedlings under water deficit. The frequency of S-phase cells was detected by 5-ethynil-2-deoxiuridine (EdU) based fluorescent microscopy. By studies on tolerant and sensitive genotypes we compare gene expression profiles for root specific and cell cycle genes

Monitoring drought responses of barley genotypes with semi-robotic phenotyping platform and association analysis between recorded traits and allelic variants of some stress genes

Genetic improvement of complex traits such as drought adaptation can be advanced by the combination of genomic and phenomic approaches. Semi-robotic phenotyping platform was used for computer-controlled watering, digital and thermal imaging of barley plants grown in greenhouse. The tested barley variants showed 0–76% reduction in green pixel-based shoot surface area in soil with 20% water content, compared to well-watered plants grown in soil with 60% water content. The barley HvA1 gene encoding the group 3 LEA (Late Embryogenesis Abundant) protein exhibited four (A–D) haplotypes as identified by the EcoTILLING and subsequent DNA sequencing. The green pixel mean value of genotypes with haplotype D was higher than the mean value of the remaining haplotypes, indicating a pivotal role of haplotype D in optimizing the green biomass production under drought condition. In water limitation, the canopy temperature of a highly sensitive genotype was 18.0°C, as opposed to 16.9°C of leaves from a tolerant genotype as measured by thermal imaging. Drought-induced changes in leaf temperature showed moderate correlation with the water use efficiency (r2 = 0.431). The haplotype/trait association analysis based on the t-test has revealed a positive effect of a haplotype B (SNPs:GCCCCTGC) in a gene encoding the barley fungal pathogen induced mRNA for pathogen-related protein (HvPPRPX), on harvest index, thousand grain weight, water use efficiency and grain yield. The presented pilot study established a basic methodology for the integrated use of phenotyping and haplotyping data in characterization of genotype-dependent drought responses in barley

Activation of embryogenic cell division in leaf protoplast-derived alfalfa cells: the role of auxin and stress

Leaf protoplast-derive d cells of the embryogenic alfalfa genotype (Medicago sativa ssp. varia A2) follow different fate if cultured in the presence of 0.25, 1 or 10 uM 2,4 - dichlorophenoxyacetic acid (2,4-D). Cells grow n in the presence of the highest auxin (2,4-D) concentration become embryogenic and can develop into somatic embryos if subcultured into fresh medium with the lower 2,4- D level. Cells cultured at the lower auxin concentrations from the beginning develop into elongated differentiated cells. In order to reveal physiological changes that characterize the reactivation of cell division in resting cells as well as the transition of somatic plant cells to an embryogenic state, morphological, cell division, intracellular pH and stress-related parameters have been determined during the first five days of parallel cultures at the above 2,4-D concentrations in combination with stress treatments

A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

<p>Abstract</p> <p>Background</p> <p>Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here.</p> <p>Results</p> <p>Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice.</p> <p>Conclusions</p> <p>In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.</p

Experimental system for studying long-term drought stress adaptation of wheat cultivars

Water limitation is a well-known problem for plants. Lack of water affects their biomass, their yield that is the most conspicuous in case of crops causing severe uncertainty of agricultural productivity. Under drought stress, plants generally display many physiological responses such as stomata closure, decreased/stopped photosynthetic activity, increased root/shoot ratio, reduced growth of vegetative parts. Many of the physiological changes are caused by underlying transcriptional alterations of high number of genes in many cases. One of the most studied phenomenon is the accumulation of proline as an osmoprotectant. Proline biosynthesis is increased by water deficit due to increased expression of the key enzyme, namely Δ1-pyrroline-5-carboxylate synthetase (P5CS). In our experiments, we applied P5CS as a positive control to evaluate our new experimental system, which will allow to follow transcriptional changes in shoots, as well as in roots during drought adaptation. Our alternative approach allows greenhouse or growth chamber experiments that are more similar to natural conditions than the widely used experimental systems based on osmotic agents such as polyethylene glycol (PEG)