Identification of the Genetic Basis of Response to De-Acclimation in Winter Barley.

Mechanisms involved in the de-acclimation of herbaceous plants caused by warm periods during winter are poorly understood. This study identifies the genes associated with this mechanism in winter barley. Seedlings of eight accessions (four tolerant and four susceptible to de-acclimation cultivars and advanced breeding lines) were cold acclimated for three weeks and de-acclimated at 12 °C/5 °C (day/night) for one week. We performed differential expression analysis using RNA sequencing. In addition, reverse-transcription quantitative real-time PCR and enzyme activity analyses were used to investigate changes in the expression of selected genes. The number of transcripts with accumulation level changed in opposite directions during acclimation and de-acclimation was much lower than the number of transcripts with level changed exclusively during one of these processes. The de-acclimation-susceptible accessions showed changes in the expression of a higher number of functionally diverse genes during de-acclimation. Transcripts associated with stress response, especially oxidoreductases, were the most abundant in this group. The results provide novel evidence for the distinct molecular regulation of cold acclimation and de-acclimation. Upregulation of genes controlling developmental changes, typical for spring de-acclimation, was not observed during mid-winter de-acclimation. Mid-winter de-acclimation seems to be perceived as an opportunity to regenerate after stress. Unfortunately, it is competitive to remain in the cold-acclimated state. This study shows that the response to mid-winter de-acclimation is far more expansive in de-acclimation-susceptible cultivars, suggesting that a reduced response to the rising temperature is crucial for de-acclimation tolerance.

Association mapping of drought tolerance-related traits in barley to complement a traditional biparental QTL mapping study.

KEY MESSAGE: Association mapping of drought-related traits in barley was used to increase the density of existing QTL maps without recreating mapping populations. We used 109 spring barley genotypes exhibiting high or low drought tolerance to elucidate the associations between diversity array technology sequencing (DArTseq) and single nucleotide polymorphism (SNP) markers and various physiological parameters related to plant responses to drought conditions. The study was performed in controlled conditions (growth chambers), drought tolerance was phenotyped in the four-leaf seedlings. We identified 58 associations including 34 new markers (i.e., 16 DArTseq and 18 SNP markers). The results for three markers were consistent with the data obtained in an earlier traditional biparental QTL mapping study. The regions neighboring markers on linkage group 2H contained the highest number of significant marker-trait associations. Five markers related to the photosynthetic activity of photosystem II were detected on chromosome 4H. The lowest number of associations were observed for the sequences neighboring DArT markers on linkage group 6H. A chromosome 3H region related to water use efficiency and net photosynthesis rate in both biparental QTL, and association study, may be particularly valuable, as these parameters correspond to the ability of plants to remain highly productive under water deficit stress. Our findings confirm that association mapping can increase the density of existing QTL maps without recreating mapping populations.

Downregulation of three novel candidate genes is important for freezing tolerance of field and laboratory cold acclimated barley.

Diversity arrays technology (DArT) marker sequences for barley were used for identifying new potential candidate genes for freezing tolerance (FT). We used quantitative trait loci (QTL) genetic linkage maps for FT and photosynthetic acclimation to cold for six- and two-row barley populations, and a set of 20 DArT markers obtained using the association mapping of parameters for photosynthetic acclimation to low temperatures in barley for the bioinformatics analyses. Several nucleotide and amino acid sequence, annotation databases and associated algorithms were used to identify the similarities of six of the marker sequences to potential genes involved in plant low temperature response. Gene ontology (GO) annotations based on similarities to database sequences were assigned to these marker sequences, and indicated potential involvement in signal transduction pathways in response to stress factors and epigenetic processes, as well as auxin transport mechanisms. Furthermore, relative gene expressions for three of six of new identified genes (Hv.ATPase, Hv.DDM1, and Hv.BIG) were assessed within four barley genotypes of different FT. A physiological assessment of FT was conducted based on plant survival rates in two field-laboratory and one laboratory experiments. The results suggested that plant survival rate after freezing but not the degree of freezing-induced leaf damage between the tested accessions can be correlated with the degree of low-temperature downregulation of the studied candidate genes, which encoded proteins involved in the control of plant growth and development. Additionally, candidate genes for qRT-PCR suitable for the analysis of cold acclimation response in barley were suggested after validation.

Water stress-induced flag leaf senescence may be accelerated by rehydration.

The aim of the study was to determine molecular, biochemical and physiological responses of non-fully recovered DH line of triticale exposed to water stress during generative stage. The study involved two DH lines of winter triticale that produce different number of shoots with ears during rehydration. We analyzed the content of proteins associated with the photosynthetic apparatus and plant senescence. We also determined the content of hydrogen peroxide and assimilation pigments and assessed stomatal conductance and activity of the photosynthetic apparatus. Water stress-initiated senescence did not slow down during rehydration in the not fully recovered DH line. This line showed an increase in pheophorbide a oxygenase (PaO), a protein associated with chlorophyll degradation, and a decrease in the proteins related to its synthesis (chlorophyll synthase - ChS, protochlorophilide oxidoreductase - POR). Pheophorbide a oxygenase is a marker of accelerated cell death as it catalyzes opening of the porphyrin ring in the chlorophyll degradation pathway. The level of hydrogen peroxide remained high during rehydration with the photosynthetic apparatus being one of its sources. Lower content of Rieske protein reduced the quantum yield of electron transport (ϕRo) from the primary acceptors QA/QB to the final acceptors in PSI. Intensification of metabolic processes during rehydration resulted in overloading the electron transport chain in PSII and transfer of electrons from the primary acceptors to oxygen molecule. Overproduction of hydrogen peroxide accelerated senescence during rehydration and significantly reduced plant yield.

Genome-Wide Associations of Chlorophyll Fluorescence OJIP Transient Parameters Connected With Soil Drought Response in Barley.

One hundred and nine accessions of spring barley seedlings were phenotyped under soil drought conditions. Chlorophyll fluorescence induction (OJIP) parameters, leaf water content, relative turgidity, net assimilation rate (P N), and water use efficiency (WUE) of plants were measured. All the tested lines were genotyped by means of DArT sequencing (DArTseq) technology. For association mapping a 11,780 polymorphic DArTseq and 4,725 DArTseq SNP markers were used. Our results revealed dissimilar patterns of the relationships between OJIP-parameters under control and drought conditions. A high level of correlation between parameters characterizing Photosystem's II (PSII) energy trapping efficiency (Fv/Fm) and photochemical events downstream of PSII reaction center (e.g., Performance Index-PICSo) was observed only in the case of drought-treated plants. Generally, OJIP parameters were correlated with leaf water content (less in control). This correlation was weaker with WUE, and absent with P N. Under drought stress, 6,252 genotype × phenotype associations, which passed false discovery rate (FDR) verification, were found between all the studied phenotypic characteristics (23, including 19 OJIP parameters) and 2,721 markers. On the other hand, only 282 associations passed FDR test in the control. They comprised 22 phenotypic parameters and 205 markers. Probing for gene annotations of sequences was performed for markers associated with Fv/Fm for both drought and control, markers were associated with studied traits in both control and drought, as well as for markers associated with both OJIP and other physiological parameters in drought. Our work allowed us to conclude that drought treatment differentiates the studied lines through the revealing of relationships between water content and the damages to PSII reaction centers or different components of PSII energy transfer chain. Moreover, the former was not connected with net photosynthesis rate.

Development of DArT-based PCR markers for selecting drought-tolerant spring barley.

The tolerance of spring barley (Hordeum vulgare L.) cultivars to spring drought is an important agronomic trait affecting crop yield and quality in Poland. Therefore, breeders require new molecular markers to select plants with lower spring drought susceptibility. With the advent of genomic selection technology, simple molecular tools may still be applicable to screen material for markers of the most important traits and in-depth genome scanning. In previous studies, diversity arrays technology (DArT)-based genetic maps were constructed for F2 populations of Polish fodder and malt barley elite breeding lines, and 15 and 18 quantitative trait loci (QTLs) related to spring drought tolerance were identified, respectively. In this paper, we show the results of a conversion of 30 DArT markers corresponding to 11 QTLs into simple sequence repeat (SSR) and sequence tagged site (STS) markers. Twenty-two polymorphic markers were obtained, including 13 DArT-based SSRs. Additionally, 31 SSR markers, located in close proximity to the DArT markers, were selected from the GrainGenes database and tested. Further analyses of 24 advanced breeding lines with different drought tolerances confirmed that five out of the 30 converted markers, as well as three out of the 31 additional SSR markers, were effective in marker-assisted selection for drought tolerance. The possible function of clones related to these markers in drought tolerance is discussed.