Microsatellite Markers: A Tool to Assess the Genetic Diversity of Yellow Mustard (Sinapis alba L.).

Microsatellite markers were used for the assessment of genetic diversity and genetic structure in a germplasm collection of yellow mustard, Sinapis alba L. The comprehensive collection of genetic resources represented 187 registered varieties, landraces, and breeding materials. Microsatellites generated 44 polymorphic alleles in 15 loci. Eleven of them were medium to highly polymorphic, and the high levels of observed heterozygosity (0.12-0.83) and Nei's gene diversity index (0.11-0.68) indicated a high level of polymorphism. Based on PCoA and neighbor joining analyses, the genetic resources were divided into two groups. The range of genetic dissimilarity in the analysed collection was in the range of 0.00-1.00. The high level of dissimilarity between the accessions was documented by the high WAM value (33.82%). Bayesian clustering algorithms were performed in the STRUCTURE 2.3.4 software. The number of clusters was estimated at K = 2. The accessions were classified according to Q1/Q2 values. The low average values of the parameters Fst_1 (0.3482), Fst_2 (0.1916), and parameter alpha (0.0602) indicated substantial mating barriers between varieties and reproductive isolation due to the limited exchange of genetic resources between breeders. These results demonstrated the importance of extensive collections of genetic resources for the maintenance of genetic diversity and indicated considerable genetic differentiation among accessions.

Evaluation of Resistance of Oilseed Rape Genotypes to Turnip Yellows Virus.

Turnip yellows virus (TuYV), is one of the most important pathogens of oilseed rape, which has caused enormous yield losses in all growing regions of the world in recent years. Therefore, there is a need for resistant varieties for sustainable crop protection. We have investigated the resistance of known varieties and newly developed advanced-breeding lines of oilseed rape to TuYV in greenhouse and field trials. We have analysed the TuYV titre of individual genotypes inoculated with the virus using viruliferous aphids Myzus persicae. The genotypes 'DK Temptation' and 'Rescator' had the lowest and highest virus titres, respectively, and were used as resistant and susceptible models for comparative analyses with other genotypes. In the greenhouse, the best results were obtained with the genotypes 'OP-8143 DH' (2.94 × 105 copies), OP-BN-72 (3.29 × 105 copies), 'Navajo' (3.58 × 105 copies) and 'SG-C 21215' (4.09 × 105 copies), which reached virus titres about 2 times higher than the minimum virus concentration measured in 'DK Temptation' (1.80 × 105 copies). In the field trials, the genotypes 'Navajo' (3.39 × 105 copies), 'OP-8148 DH' (4.44 × 105 copies), 'SG-C 21215' (6.80 × 105 copies) and OP-8480 (7.19 × 105 copies) had the lowest virus titres and reached about 3 times the virus titre of DK Temptation (2.54 × 105 copies). Both trials showed that at least two commercial varieties (e.g., DK Temptation, Navajo) and three advanced breeding lines (e.g., OP-8143 DH, OP-BN-72, SG-C 21215) had low titres of the virus after TuYV infection. This indicates a high level of resistance to TuYV in 'Navajo' or the newly developed breeding lines and the basis of resistance is probably different from R54 (as in 'DK Temptation'). Furthermore, the greenhouse trials together with RT -qPCR-based virus titre analysis could be a cost-effective and efficient method to assess the level of resistance of a given genotype to TuYV infection compared to the field trials. However, further research is needed to identify the underlying mechanisms causing this difference in susceptibility.

Proteomics of wheat and barley cereals in response to environmental stresses: Current state and future challenges.

Wheat and barley genera represent a wide range of genotypes from Triticeae group grown around the globe. The broad plasticity of Triticeae phenotypes mirrors the robustness of their genomes revealing a high level of gene homeology. Publication and annotation of the reference genome sequences for spring barley Morex and Chinese Spring wheat represents an important milestone enabling the researchers to precisely identify and annotate nearly all proteins. Due to the broad range of environments used for wheat and barley cultivation and their economical importance, proteomic studies focused on their responses to environmental stresses including combined stress treatments. Most of the Triticeae stress proteomics studies are comparative ones aimed at determination of differentially abundant proteins (DAPs) between two or more genotypes with contrasting stress tolerance. Studies focused on subcellular fractions and protein posttranslational modifications (PTMs) are still relatively rare although PTMs play a crucial role in modulation of protein biological function. Functional and interactomics studies are needed although gene homeology and the resulting protein functional redundancy practically excludes the utilization of knock-out mutants. The alternatives could represent either gene overexpression in a heterologous system such as A. thaliana or transient posttranscriptional gene silencing using RNAi. Publication of complete reference genome sequences together with novel technological approaches such as pQTL mapping boost the Triticeae proteomics studies not only to provide data but also to contribute to designing novel genotypes with improved adaptations to ever changing environments.

Ergosterol and polyphenol contents as rapid indicators of orchardgrass silage safety.

The ergosterol (ERG) has been proposed as a potential indicator of fungal contamination, along with polyphenol content analysis to predict silage safety. Despite efforts in controlling fungal growth in silage, mycotoxin co-contamination represents a possible risk for animal and human health. Modern analytical techniques determine a multitude of fungal metabolites contaminating feed. Nonetheless, these methods require sometimes arduous sample pre-treatment, long separation times, and expensive standard compounds to identified contaminants. Thus, the goal of this study was to suggest a rapid analysis of ERG and polyphenol contents to assess silage hygienic quality in ten orchardgrass varieties ensiled without and with biological and chemical additives. The determination of ERG on samples was performed by high-performance liquid chromatography using UV detection and UV/Vis spectrophotometry to determine the polyphenol content. Statistically significant differences (P < 0.05) between varieties, years and silage additives were found. Bepro was the unique variety that did not present ERG in the first cut in 2012. ERG content increased in the first cut in 2013 using biological additives as well as ERG and polyphenol contents in the first cut in 2013 using chemical additives compared with untreated silage. In addition, biological and chemical additives used in this study did not satisfactorily reduce the content of ERG and polyphenols in silage grass. Consequently, our results provide fast information about the progressive fungal contamination of grass silage. To our knowledge, it is the first time that the presence of ERG and polyphenols is determined in ten different orchardgrass varieties treated without and with additives. In general, ERG and polyphenol contents showed to be good indicators of orchardgrass silage safety.

Plant Proteoforms Under Environmental Stress: Functional Proteins Arising From a Single Gene.

Proteins are directly involved in plant phenotypic response to ever changing environmental conditions. The ability to produce multiple mature functional proteins, i.e., proteoforms, from a single gene sequence represents an efficient tool ensuring the diversification of protein biological functions underlying the diversity of plant phenotypic responses to environmental stresses. Basically, two major kinds of proteoforms can be distinguished: protein isoforms, i.e., alterations at protein sequence level arising from posttranscriptional modifications of a single pre-mRNA by alternative splicing or editing, and protein posttranslational modifications (PTMs), i.e., enzymatically catalyzed or spontaneous modifications of certain amino acid residues resulting in altered biological functions (or loss of biological functions, such as in non-functional proteins that raised as a product of spontaneous protein modification by reactive molecular species, RMS). Modulation of protein final sequences resulting in different protein isoforms as well as modulation of chemical properties of key amino acid residues by different PTMs (such as phosphorylation, N- and O-glycosylation, methylation, acylation, S-glutathionylation, ubiquitinylation, sumoylation, and modifications by RMS), thus, represents an efficient means to ensure the flexible modulation of protein biological functions in response to ever changing environmental conditions. The aim of this review is to provide a basic overview of the structural and functional diversity of proteoforms derived from a single gene in the context of plant evolutional adaptations underlying plant responses to the variability of environmental stresses, i.e., adverse cues mobilizing plant adaptive mechanisms to diminish their harmful effects.

Relationship between WCS120 Protein Family Accumulation and Frost Tolerance in Wheat Cultivars Grown under Different Temperature Treatments.

Frost tolerance (FT) is generally acquired after exposure of plants to low, but non-freezing temperatures, where it is associated with the accumulation of COR proteins. The aim of the study was to reveal the effect of different temperature treatments (25, 17, 9 and 4 °C) on accumulation of cold-regulated dehydrins, dry weight content, and the development of FT in five wheat cultivars of different frost-tolerances in detail. The levels of cold-regulated dehydrins, WCS120 proteins in wheat were determined by immunoblot analysis, probed with an anti-dehydrin antibody. The lower the growth temperature: the higher the level of frost tolerance, dry weight content, and dehydrin accumulation, in all cultivars. There was a significant correlation between the level of induced FT and the accumulation of WCS120 proteins in cultivars grown at lower temperatures (9 and 4 °C). Moreover, the highly frost-tolerant wheat cultivars (as opposed to the lower-tolerant) accumulated higher levels of WCS120 proteins at 17 °C, a temperature at which it was not possible to differentiate between them via a frost test. Here, we demonstrated the possibility to distinguish differently frost-tolerant cultivars grown at different temperatures by the accumulation of different members of WCS120 family.

The Resistance of Oilseed Rape Microspore-Derived Embryos to Osmotic Stress Is Associated With the Accumulation of Energy Metabolism Proteins, Redox Homeostasis, Higher Abscisic Acid, and Cytokinin Contents.

The present study aims to investigate the response of rapeseed microspore-derived embryos (MDE) to osmotic stress at the proteome level. The PEG-induced osmotic stress was studied in the cotyledonary stage of MDE of two genotypes: Cadeli (D) and Viking (V), previously reported to exhibit contrasting leaf proteome responses under drought. Two-dimensional difference gel electrophoresis (2D-DIGE) revealed 156 representative protein spots that have been selected for MALDI-TOF/TOF analysis. Sixty-three proteins have been successfully identified and divided into eight functional groups. Data are available via ProteomeXchange with identifier PXD024552. Eight selected protein accumulation trends were compared with real-time quantitative PCR (RT-qPCR). Biomass accumulation in treated D was significantly higher (3-fold) than in V, which indicates D is resistant to osmotic stress. Cultivar D displayed resistance strategy by the accumulation of proteins in energy metabolism, redox homeostasis, protein destination, and signaling functional groups, high ABA, and active cytokinins (CKs) contents. In contrast, the V protein profile displayed high requirements of energy and nutrients with a significant number of stress-related proteins and cell structure changes accompanied by quick downregulation of active CKs, as well as salicylic and jasmonic acids. Genes that were suitable for gene-targeting showed significantly higher expression in treated samples and were identified as phospholipase D alpha, peroxiredoxin antioxidant, and lactoylglutathione lyase. The MDE proteome profile has been compared with the leaf proteome evaluated in our previous study. Different mechanisms to cope with osmotic stress were revealed between the genotypes studied. This proteomic study is the first step to validate MDE as a suitable model for follow-up research on the characterization of new crossings and can be used for preselection of resistant genotypes.

COR/LEA Proteins as Indicators of Frost Tolerance in Triticeae: A Comparison of Controlled versus Field Conditions.

Low temperatures in the autumn induce enhanced expression/relative accumulation of several cold-inducible transcripts/proteins with protective functions from Late-embryogenesis-abundant (LEA) superfamily including dehydrins. Several studies dealing with plants grown under controlled conditions revealed a correlation (significant quantitative relationship) between dehydrin transcript/protein relative accumulation and plant frost tolerance. However, to apply these results in breeding, field experiments are necessary. The aim of the review is to provide a summary of the studies dealing with the relationships between plant acquired frost tolerance and COR/LEA transcripts/proteins relative accumulation in cereals grown in controlled and field conditions. The impacts of cold acclimation and vernalisation processes on the ability of winter-type Triticeae to accumulate COR/LEA proteins are discussed. The factors determining dehydrin relative accumulation under controlled cold acclimation treatments versus field trials during winter seasons are discussed. In conclusion, it can be stated that dehydrins could be used as suitable indicators of winter survival in field-grown winter cereals but only in plant prior to the fulfilment of vernalisation requirement.

Relationship Between Dehydrin Accumulation and Winter Survival in Winter Wheat and Barley Grown in the Field.

Low temperatures represent a crucial environmental factor determining winter survival (WS) of barley and wheat winter-type varieties. In laboratory experiments, low temperatures induce an active plant acclimation response, which is associated with an enhanced accumulation of several stress-inducible proteins including dehydrins. Here, dehydrin accumulations in sampled wheat (WCS120 protein family, or WCS120 and WDHN13 transcripts) and barley (DHN5 protein) varieties grown in two locations for two winters were compared with the variety WS evaluated by a provocation wooden-box test. A high correlation between dehydrin transcripts or protein relative accumulation and variety WS score was found only in samples taken prior vernalization fulfillment, when high tolerant varieties accumulated dehydrins earlier and to higher level than less tolerant varieties, and the plants have not yet been vernalized. After vernalization fulfillment, the correlation was weak, and the apical development indicated that plants reached double ridge (DR) in barley or stayed before DR in wheat. Dehydrin proteins and transcripts can be thus used as reliable markers of wheat or barley variety winter hardiness in the field conditions; however, only at the beginning of winter, when the plants have not yet finished vernalization. In wheat, a higher correlation was obtained for the total amount of dehydrins than for the individual dehydrin proteins. HIGHLIGHTS: -More tolerant winter-type wheat and barley plants reveal higher threshold induction temperatures for dehydrin accumulation in comparison to less tolerant varieties. Thus, more tolerant winter cereals have higher dehydrin levels than the less tolerant ones upon the same ambient temperature in November samplings.-A significant correlation between dehydrin transcript/protein accumulation and winter survival was found in both winter wheat and winter barley plants in the field conditions, but only prior to vernalization fulfillment.

Proteomic and physiological approach reveals drought-induced changes in rapeseeds: Water-saver and water-spender strategy.

The cultivar-dependent differences in Brassica napus L. seed yield are significantly affected by drought stress. Here, the response of leaf proteome to long-term drought (28days) was studied in cultivars (cvs): Californium (C), Cadeli (D), Navajo (N), and Viking (V). Analysis of twenty-four 2-D DIGE gels revealed 134 spots quantitatively changed at least 2-fold; from these, 79 proteins were significantly identified by MALDI-TOF/TOF. According to the differences in water use, the cultivars may be assigned to two categories: water-savers or water-spenders. In the water-savers group (cvs C+D), proteins related to nitrogen assimilation, ATP and redox homeostasis were increased under stress, while in the water-spenders category (cvs N+V), carbohydrate/energy, photosynthesis, stress related and rRNA processing proteins were increased upon stress. Taking all data together, we indicated cv C as a drought-adaptable water-saver, cv D as a medium-adaptable water-saver, cv N as a drought-adaptable water-spender, and cv V as a low-adaptable drought sensitive water-spender rapeseed. Proteomic data help to evaluate the impact of drought and the extent of genotype-based adaptability and contribute to the understanding of their plasticity. These results provide new insights into the provenience-based drought acclimation/adaptation strategy of contrasting winter rapeseeds and link data at gasometric, biochemical, and proteome level. SIGNIFICANCE: Soil moisture deficit is a real problem for every crop. The data in this study demonstrates for the first time that in stem-prolongation phase cultivars respond to progressive drought in different ways and at different levels. Analysis of physiological and proteomic data showed two different water regime-related strategies: water-savers and spenders. However, not only water uptake rate itself, but also individual protein abundances, gasometric and biochemical parameters together with final biomass accumulation after stress explained genotype-based responses. Interestingly, under a mixed climate profile, both water-use patterns (savers or spenders) can be appropriate for drought adaptation. These data suggest, than complete "acclimation image" of rapeseeds in stem-prolongation phase under drought could be reached only if these characteristics are taken, explained and understood together.

Biological Networks Underlying Abiotic Stress Tolerance in Temperate Crops--A Proteomic Perspective.

Abiotic stress factors, especially low temperatures, drought, and salinity, represent the major constraints limiting agricultural production in temperate climate. Under the conditions of global climate change, the risk of damaging effects of abiotic stresses on crop production increases. Plant stress response represents an active process aimed at an establishment of novel homeostasis under altered environmental conditions. Proteins play a crucial role in plant stress response since they are directly involved in shaping the final phenotype. In the review, results of proteomic studies focused on stress response of major crops grown in temperate climate including cereals: common wheat (Triticum aestivum), durum wheat (Triticum durum), barley (Hordeum vulgare), maize (Zea mays); leguminous plants: alfalfa (Medicago sativa), soybean (Glycine max), common bean (Phaseolus vulgaris), pea (Pisum sativum); oilseed rape (Brassica napus); potato (Solanum tuberosum); tobacco (Nicotiana tabaccum); tomato (Lycopersicon esculentum); and others, to a wide range of abiotic stresses (cold, drought, salinity, heat, imbalances in mineral nutrition and heavy metals) are summarized. The dynamics of changes in various protein functional groups including signaling and regulatory proteins, transcription factors, proteins involved in protein metabolism, amino acid metabolism, metabolism of several stress-related compounds, proteins with chaperone and protective functions as well as structural proteins (cell wall components, cytoskeleton) are briefly overviewed. Attention is paid to the differences found between differentially tolerant genotypes. In addition, proteomic studies aimed at proteomic investigation of multiple stress factors are discussed. In conclusion, contribution of proteomic studies to understanding the complexity of crop response to abiotic stresses as well as possibilities to identify and utilize protein markers in crop breeding processes are discussed.

Detection of self-incompatible oilseed rape plants (Brassica napus L.) based on molecular markers for identification of the class I S haplotype.

The selection of desirable genotypes with recessive characteristics, such as self-incompatible plants, is often difficult or even impossible and represents a crucial barrier in accelerating the breeding process. Molecular approaches and selection based on molecular markers can allow breeders to overcome this limitation. The use of self-incompatibility is an alternative in hybrid breeding of oilseed rape. Unfortunately, stable self-incompatibility is recessive and phenotype-based selection is very difficult and time-consuming. The development of reliable molecular markers for detecting desirable plants with functional self-incompatible genes is of great importance for breeders and allows selection at early stages of plant growth. Because most of these reliable molecular markers are based on discrimination of class I S-locus genes that are present in self-compatible plants, there is a need to use an internal control in order to detect possible PCR inhibition that gives false results during genotyping. In this study, 269 double haploid F2 oilseed rape plants obtained by microspore embryogenesis were used to verify the applicability of an improved PCR assay based on the detection of the class I SLG gene along with an internal control. Comparative analysis of the PCR genotyping results vs. S phenotype analysis confirmed the applicability of this molecular approach in hybrid breeding programs. This approach allows accurate detection of self-incompatible plants via a different amplification profile.

Significant relationships among frost tolerance and net photosynthetic rate, water use efficiency and dehydrin accumulation in cold-treated winter oilseed rapes.

Five winter oilseed rape cultivars (Benefit, Californium, Cortes, Ladoga, Navajo) were subjected to 30 days of cold treatment (4 °C) to examine the effect of cold on acquired frost tolerance (FT), dehydrin (DHN) content, and photosynthesis-related parameters. The main aim of this study was to determine whether there are relationships between FT (expressed as LT50 values) and the other parameters measured in the cultivars. While the cultivar Benefit accumulated two types of DHNs (D45 and D35), the other cultivars accumulated three additional DHNs (D97, D47, and D37). The similar-sized DHNs (D45 and D47) were the most abundant; the others exhibited significantly lower accumulations. The highest correlations were detected between LT50 and DHN accumulation (r=-0.815), intrinsic water use efficiency (WUEi; r=-0.643), net photosynthetic rate (r=-0.628), stomatal conductance (r=0.511), and intracellular/intercellular CO2 concentration (r=0.505). Those cultivars that exhibited higher Pn rate in cold (and further a significant increase in WUEi) had higher levels of DHNs and also higher FT. No significant correlation was observed between LT50 and E, PRI, or NDVI. Overall, we have shown the selected physiological parameters to be able to distinguish different FT cultivars of winter oilseed rape.