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

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

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

RING-Type E3 Ubiqitin Ligase BarleyGenes(HvYrg1–2) Control Characteristics of Both Vegetative Organs and Seeds as Yield Components

Previously, studies on RING-type E3 ubiquitin ligases in cereals were preferentially focused on GW2 genes primarily controlling seed parameters in rice and wheat. Here we report cloning two HvYrg genes from barley that share significant homology with rice GW2 gene. In antisense genotypes efficiency of gene silencing varied between genes and transgenic lines: ASHvYrg1: 30-50% and ASHvYrg2: 20-27%. Reduced activity of both genes altered shoot system with increasing number of side shoots. Changes in leaf width, weight, or plant weight and height reached significant levels in some transgenic lines. Lowering expression of the two barley HvYrg genes caused opposite responses in spike development. Plants with ASHvYrg1 gene construct showed earlier heading time and prolonged grain-filling period, while plants from ASHvYrg2 genotype flowered in delay. Digital imaging of root development revealed that down-regulation of HvYrg1 gene variant stimulated root growth, while ASHvYrg2 plants developed reduced root system. Comparison of seed parameters indicated an increase in thousand grain weight accompanied with longer and wider seed morphology. In summary we conclude that in contrast to inhibition of GW2 genes in rice and wheat plants, down-regulation of the barely HvYrg genes caused substantial changes in vegetative organs in addition to alteration of seed parameters

Triploid Hybrid Vigor in Above-Ground Growth and Methane Fermentation Efficiency of Energy Willow

Hybrid vigor and polyploidy are genetic events widely utilized to increase the productivity of crops. Given that bioenergy usage needs to be expanded, we investigated triploid hybrid vigor in terms of the biology of biomass-related willow traits and their relevance to the control of biomethane production. To produce triploid hybrid genotypes, we crossed two female diploid Swedish cultivars (Inger, Tordis) with two male autotetraploid willow (Salix viminalis) variants (PP-E7, PP-E15). Field studies at two locations and in two successive years recorded considerable midparent heterosis (MPH%) in early shoot length that ranged between 11.14 and 68.85% and in the growth rate between 34.12 and 97.18%. The three triploid hybrids (THs) developed larger leaves than their parental cultivars, and the MPH% for their CO(2) assimilation rate varied between 0.84 and 25.30%. The impact of hybrid vigor on the concentrations of plant hormones in these TH genotypes reflected essentially different hormonal statuses that depended preferentially on maternal parents. Hybrid vigor was evinced by an elevated concentration of jasmonic acid in shoot meristems of all the three THs (MPH:29.73; 67.08; 91.91%). Heterosis in auxin-type hormones, such as indole-3-acetic acid (MPH:207.49%), phenylacetic acid (MPH:223.51%), and salicylic acid (MPH:27.72%) and benzoic acid (MPH:85.75%), was detectable in the shoots of TH21/2 plants. These hormones also accumulated in their maternal Inger plants. Heterosis in cytokinin-type hormones characterized the shoots of TH3/12 and TH17/17 genotypes having Tordis as their maternal parent. Unexpectedly, we detected abscisic acid as a positive factor in the growth of TH17/17 plants with negative MPH percentages in stomatal conductance and a lower CO(2) assimilation rate. During anaerobic digestion, wood raw materials from the triploid willow hybrids that provided positive MPH% in biomethane yield (6.38 and 27.87%) showed negative MPH in their acid detergent lignin contents (from –8.01 to –14.36%). Altogether, these insights into controlling factors of above-ground growth parameters of willow genotypes support the utilization of triploid hybrid vigor in willow breeding to expand the cultivation of short rotation energy trees for renewable energy production