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

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

Phenotyping shows improved physiological traits and seed yield of transgenic wheat plants expressing the alfalfa aldose reductase under permanent drought stress

Members of the aldo-keto reductase family including aldose reductases are involved in antioxidant defense by metabolizing a wide range of lipid peroxidation-derived cytotoxic compounds. Therefore, we produced transgenic wheat genotypes over-expressing the cDNA of alfalfa aldose reductase gene. These plants consequently exhibit 1.5-4.3 times higher detoxification activity for the aldehyde substrate. Permanent drought stress was generated in the greenhouse by growing wheat plants in soil with 20 % water capacity. The control and stressed plants were monitored by a semi automatic phenotyping platform providing computer-controlled watering, digital and thermal imaging. Calculation of biomass values was based on the correlation (R2 = 0.7556) between fresh weight and green pixel-based shoot surface area. The green biomass production by plants of the three transgenic lines was 12-26-41 % higher than the non-transgenic plants' grown under water limitation. Thermal imaging of stressed non-transgenic plants indicated an elevation in the leaf temperature. The thermal status of transformants was similar at both normal and suboptimal water regime. In drought, the transgenic plants used more water during the growing season. The described phenotyping platform provided a comprehensive data set demonstrating the improved physiological condition of the drought stressed transgenic wheat plants in the vegetative growth phase. In soil with reduced water capacity two transgenic genotypes showed higher seed weight per plant than the control non-transgenic one. Limitation of greenhouse-based phenotyping in analysis of yield potential is discussed. © 2013 The Author(s)

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis L.

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO(2) uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity

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