Temperature-Caused Changes in Raman Pattern and Protein Profiles of Winter Triticale (x Triticosecale, Wittm.) Field-Grown Seedlings.

Climate change, which causes periods with relatively high temperatures in winter in Poland, can lead to a shortening or interruption of the cold hardening of crops. Previous research indicates that cold acclimation is of key importance in the process of acquiring cereal tolerance to stress factors. The objective of this work was to verify the hypothesis that both natural temperature fluctuations and the plant genotype influence the content of metabolites as well as proteins, including antioxidant enzymes and photosystem proteins. The research material involved four winter triticale genotypes, differing in their tolerance to stress under controlled conditions. The values of chlorophyll a fluorescence parameters and antioxidant activity were measured in their seedlings. Subsequently, the contribution of selected proteins was verified using specific antibodies. In parallel, the profiling of the contents of chlorophylls, carotenoids, phenolic compounds, and proteins was carried out by Raman spectroscopy. The obtained results indicate that a better PSII performance along with a higher photosystem II proteins content and thioredoxin reductase abundance were accompanied by a higher antioxidant activity in the field-grown triticale seedlings. The Raman studies showed that the cold hardening led to a variation in photosynthetic dyes and an increase in the phenolic to carotenoids ratio in all DH lines.

Investigation of chromosomal genetic characteristics and identification of structural variation in the offspring of hexaploid triticale×hexaploid wheat.

Hexaploid triticale is an important genetic resource for genetic improvement of common wheat, which can broaden the genetic basis of wheat. In order to lay a foundation for the subsequent research and utilization of triticale germplasm materials, the chromosomal genetic characteristics of cross and backcross offspring of hexaploid triticale×hexaploid wheat were investigated in the process of transferring rye chromatin from hexaploid triticale to hexaploid wheat. Hybrid and backcross combinations were prepared with hexaploid triticale 16yin171 as the maternal parent and hexaploid wheat Chuanmai62 as the paternal parent. The chromosomes in root tip cells of F1, BC1F1 and BC1F2 plants were traced and identified non-denaturing florescence in situ hybridization (ND-FISH). The results indicated that the backcross setting rate of hybrid F1 was 2.61%. The transmission frequency of 2R chromosome was the highest in BC1F1 plants while the transmissibility of rye chromosome in BC1F2 plant was 6R>4R>2R, and the 5B-7B wheat translocation in BC1F2 plants showed severe segregation. A total of 24 structural variant chromosomes were observed both in BC1F1 and BC1F2 plants, including chromosome fragments, isochromosomes, translocations, and dicentric chromosomes. In addition, the seed length and 1000-grain weight of some BC1F2 plants were better than that of the hexaploid wheat parent Chuanmai 62. Therefore, multiple backcrosses should be adopted as far as possible to make the rapid recovery of group D chromosomes, ensuring the recovery of fertility in offspring, when hexaploid tritriale is used as a bridge to introduce rye genetic material into common wheat. At the same time, the potential application value of chromosomal structural variation materials should be also concerned.

Isolation and Sequencing of Chromosome Arm 7RS of Rye, Secale cereale.

Rye (Secale cereale) is a climate-resilient cereal grown extensively as grain or forage crop in Northern and Eastern Europe. In addition to being an important crop, it has been used to improve wheat through introgression of genomic regions for improved yield and disease resistance. Understanding the genomic diversity of rye will assist both the improvement of this crop and facilitate the introgression of more valuable traits into wheat. Here, we isolated and sequenced the short arm of rye chromosome 7 (7RS) from Triticale 380SD using flow cytometry and compared it to the public Lo7 rye whole genome reference assembly. We identify 2747 Lo7 genes present on the isolated chromosome arm and two clusters containing seven and sixty-five genes that are present on Triticale 380SD 7RS, but absent from Lo7 7RS. We identified 29 genes that are not assigned to chromosomal locations in the Lo7 assembly but are present on Triticale 380SD 7RS, suggesting a chromosome arm location for these genes. Our study supports the Lo7 reference assembly and provides a repertoire of genes on Triticale 7RS.

S-Adenosyl-L-Methionine and Cu(II) Impact Green Plant Regeneration Efficiency.

The biological improvement of triticale, a cereal of increasing importance in agriculture, may be accelerated via the production of doubled haploid lines using in vitro culture. Among the relevant factors affecting the culture efficiency are Cu(II) or Ag(I) acting, e.g., as cofactors of enzymes. The copper ions are known to positively affect green plant regeneration efficiency. However, the biochemical basis, mainly its role in the generation of in vitro-induced genetic and epigenetic variation and green plant regeneration efficiency, is not well understood. Here, we employed structural equation modeling to evaluate the relationship between de novo DNA methylation affecting the asymmetric context of CHH sequences, the methylation-sensitive Amplified Fragment Length Polymorphism related sequence variation, and the concentration of Cu(II) and Ag(I) ions in induction media, as well as their effect on S-adenosyl-L-methionine perturbations, observed using FTIR spectroscopy, and the green plant regeneration efficiency. Our results allowed the construction of a theory-based model reflecting the biological phenomena associated with green plant regeneration efficiency. Furthermore, it is shown that Cu(II) ions in induction media affect plant regeneration, and by manipulating their concentration, the regeneration efficiency can be altered. Additionally, S-adenosyl-L-methionine is involved in the efficiency of green plant regeneration through methylation of the asymmetric CHH sequence related to de novo methylation. This shows that the Yang cycle may impact the production of green regenerants.

Evaluation of grain yield stability of tritipyrum as a novel cereal in comparison with triticale lines and bread wheat varieties through univariate and multivariate parametric methods.

Salinity is a major abiotic stress affecting cereal production. Thus, tritipyrum (x. Tritipyrum), a potential novel salt-tolerant cereal, was introduced as an appropriate alternative for cereal production. The purposes of this study were to evaluate agronomic traits, yield, and yield stability of eight primary tritipyrum lines, five promising triticale lines, and four bread wheat varieties and to screen a stable yielding line. The experiments were conducted in randomized complete block designs with three replicates in three locations during four growing seasons. Analysis of variance in each environment and Bartlett's test for the variance homogeneity of experimental errors were made. Subsequently, separate experiments were analyzed as a combined experiment. The stability of grain yield was analyzed according to Eberhart and Russell's regression method, environmental variance, Wrick's ecovalance, Shokla's stability variance, AMMI, and Tai methods. Genotype × environment interactions (GEI) and environments were significant for the agronomic traits. Stability analysis revealed that combined primary tritipyrum line (Ka/b)(Cr/b)-5 and triticale 4115, 4108, and M45 lines had good adaptability in all environments. The results of the AMMI3 model and pattern analysis showed that the new cereal, tritipyrum, had the most stable response in various environments. The tritipyrum line (Ka/b)(Cr/b)-5 had the best yield performance and general adaptability. Based on Tai's method, the contribution of spike number to the stability of grain yield over different environments was higher than that of other yield components. Also, tritipyrum lines demonstrated higher stability compared with wheat and triticale. Totally, M45 triticale and tritipyrum (Ka/b)(Cr/b)-5 lines were the most stable genotypes with high grain yield. Complementary agronomic experiments may then release a new grain crop of triticale and a new pasture line of combined primary tritipyrum for grain and forage. Moreover, the combined tritipyrum line can be used in bread wheat breeding programs for producing salt-tolerant wheat cultivars.

Triticale doubled haploid plant regeneration factors linked by structural equation modeling.

Triticale regeneration via anther culture faces many difficulties, e.g., a low percentage of regenerated plants and the presence of albinos. Plant regeneration may be affected by abiotic stresses and by ingredients added to the induction medium. The latter influences biochemical pathways and plant regeneration efficiency. Among such ingredients, copper and silver ions acting as cofactors for enzymatic reactions are of interest. However, their role in plant tissue cultures and relationships with biochemical pathways has not been studied yet.The study evaluated relationships between DNA methylation, changes in DNA sequence variation, and green plant regeneration efficiency influenced by copper and silver ions during triticale plant regeneration. For this purpose, a biological model based on donor plants and their regenerants, a methylation-sensitive amplified fragment length polymorphism, and structural equation modeling were employed.The green plant regeneration efficiency varied from 0.71 to 6.06 green plants per 100 plated anthers. The values for the components of tissue culture-induced variation related to cytosine methylation in a CHH sequence context (where H is A, C, or T) were 8.65% for sequence variation, 0.76% for DNA demethylation, and 0.58% for de novo methylation. The proposed model states that copper ions affect the regeneration efficiency through cytosine methylation and may induce mutations through, e.g., oxidative processes, which may interfere with the green plant regeneration efficiency. The linear regression confirms that the plant regeneration efficiency rises with increasing copper ion concentration in the absence of Ag ions in the induction medium. The least absolute shrinkage and selection operator regression shows that de novo methylation, demethylation, and copper ions may be involved in the green plant regeneration efficiency. According to structural equation modeling, copper ions play a central role in the model determining the regeneration efficiency.

Effect of copper and silver ions on sequence and DNA methylation changes in triticale regenerants gained via somatic embryogenesis.

Somatic embryogenesis is a plant regeneration method that can be exploited in tissue culture systems for a variety of tasks, such as genetic modification or the selection of somaclones with advantageous characteristics. Therefore, it is crucial to create efficient regeneration procedures and comprehend how medium components affect regeneration effectiveness or the degree of variation created in plant tissue cultures. The level of tissue culture-induced variation in triticale regenerants was examined in the current study in relation to the concentration of copper and silver ions in the induction media as well as the length of time immature zygotic embryo explants were incubated on these media. The high degree of variation (45%) revealed by the methylation-sensitive amplified fragment length polymorphism approach for estimating variation included 38% DNA sequence alterations, 6% DNA demethylation, and 1% de novo DNA methylation. Different levels of variance were found in relation to various DNA sequence settings. The CHG context had the most alterations, whereas CG experienced the fewest; sequence variation predominated in each sequence context. Lower copper ion concentrations showed the most variance. However, it could not be connected to the duration of in vitro culture or the effect of silver ions. Accordingly, we think that altering the concentration of copper ions in the induction medium may throw off the equilibrium of the metabolic processes in which copper is involved, resulting in tissue culture-induced variation.

Cold-modulated leaf compounds in winter triticale DH lines tolerant to freezing and Microdochium nivale infection: LC-MS and Raman study.

Tolerance to freezing and seedling diseases caused by Microdochium spp. is an essential trait for the wintering of triticale (×Triticosecale Wittmack) and other cereals. Preceding multi-year studies indicate that after long-term exposure to the low temperature, cereal seedlings acquire a genotype-dependent cross-tolerance to other subsequent stresses. This paper presents the first non-gel protein profiling performed via high performance liquid chromatography coupled with Mass Spectrometry as well as Fourier Transform-Raman spectroscopy measurements performed directly on leaves of triticale seedlings growing under different conditions. The research used doubled haploid lines selected from the mapping population, with extreme tolerance/susceptibility to freezing and M. nivale infection. These non-targeted methods led to the detection of twenty two proteins cold-accumulated in the most tolerant seedlings in relation to susceptible ones, classified as involved in protein biosynthesis, response to different stimuli, energy balancing, oxidative stress response, protein modification, membrane structure and anthocyanin synthesis. Additionally, in seedlings of the most freezing- and M. nivale -tolerant line, cold-hardening caused decrease of the carotenoid and chlorophyll content. Moreover, a decrease in the band intensity typical for carbohydrates as well as an increase in the band intensity characteristic for protein compounds were detected. Both studied lines revealed a different answer to stress in the characteristics of phenolic components.

Genotyping-by-sequencing and genome-wide association study reveal genetic diversity and loci controlling agronomic traits in triticale.

KEY MESSAGE: The genetic diversity and loci underlying agronomic traits were analyzed by the reads coverage and genome-wide association study based genotyping-by-sequencing in a diverse population consisting of 199 accessions. Triticale (× Triticosecale Wittmack) is an economically important grain forage and energy crop planted worldwide for its high biomass. Little is known about the genetic diversity and loci underlying agronomic traits in triticale. We performed genotyping-by-sequencing of 199 cultivars and mapped reads to the A, B, D, and R genomes for karyotype analysis. These cultivars could mostly be grouped into five types. Some chromosome abnormalities occurred with high frequency, such as 2D (2R) substitution, deletion of the long arm of chromosome 2D or the short arm of 5R, and translocation of the long arms of 7D/7A, the short arms of 6D/6A, or the long arms of 1D/1A. We chose only widely planted hexaploid triticale cultivars (153) for genome-wide association study. These cultivars could be divided into nine distinct groups, and the linkage disequilibrium decay was 25.4 kb in this population. We identified 253 significant marker-trait associations (MTAs) on 20 chromosomes, except 7R. Twenty-one reliable MTAs were identified repeatedly over two environments. We predicted 16 putative candidate genes involved in plant growth and development using the genome sequences of wheat and rye. These results provide a basis for understanding the genetic mechanisms of agronomic traits and will benefit the breeding of improved hexaploid triticale.

Quantitative trait loci and candidate genes associated with freezing tolerance of winter triticale (× Triticosecale Wittmack).

Freezing tolerance of triticale is a major trait contributing to its winter hardiness. The identification of genomic regions - quantitative trait loci (QTL) and molecular markers associated with freezing tolerance in winter hexaploid triticale - was the aim of this study. For that purpose, a new genetic linkage map was developed for the population of 92 doubled haploid lines derived from 'Hewo' × 'Magnat' F1 hybrid. Those lines, together with parents were subjected to freezing tolerance test three times during two winter seasons. Plants were grown and cold-hardened under natural fall/winter conditions and then subjected to freezing in controlled conditions. Freezing tolerance was assessed as the plants recovery (REC), the electrolyte leakage (EL) from leaves and chlorophyll fluorescence parameters (JIP) after freezing. Three consistent QTL for several fluorescence parameters, electrolyte leakage, and the percentage of the survived plants were identified with composite interval mapping (CIM) and single marker analysis (SMA). The first locus Qfr.hm-7A.1 explained 9% of variation of both electrolyte leakage and plants recovery after freezing. Two QTL explaining up to 12% of variation in plants recovery and shared by selected chlorophyll fluorescence parameters were found on 4R and 5R chromosomes. Finally, main locus Qchl.hm-5A.1 was detected for chlorophyll fluorescence parameters that explained up to 19.6% of phenotypic variation. The co-located QTL on chromosomes 7A.1, 4R and 5R, clearly indicated physiological and genetic relationship of the plant survival after freezing with the ability to maintain optimal photochemical activity of the photosystem II and preservation of the cell membranes integrity. The genes located in silico within the identified QTL include those encoding BTR1-like protein, transmembrane helix proteins like potassium channel, and phosphoric ester hydrolase involved in response to osmotic stress as well as proteins involved in the regulation of the gene expression, chloroplast RNA processing, and pyrimidine salvage pathway. Additionally, our results confirm that the JIP test is a valuable tool to evaluate freezing tolerance of triticale under unstable winter environments.

Genotyping-by-sequencing and genomic selection applications in hexaploid triticale.

Triticale, a hybrid species between wheat and rye, is one of the newest additions to the plant kingdom with a very short history of improvement. It has very limited genomic resources because of its large and complex genome. Objectives of this study were to generate dense marker data, understand genetic diversity, population structure, linkage disequilibrium (LD), and estimate accuracies of commonly used genomic selection (GS) models on forage yield of triticale. Genotyping-by-sequencing (GBS), using PstI and MspI restriction enzymes for reducing genome complexity, was performed on a triticale diversity panel (n = 289). After filtering for biallelic loci with more than 70% genome coverage, and minor allele frequency (MAF) > 0.05, de novo variant calling identified 16,378 single nucleotide polymorphism (SNP) markers. Sequences of these variants were mapped to wheat and rye reference genomes to infer their homologous groups and chromosome positions. About 45% (7430), and 58% (9500) of the de novo identified SNPs were mapped to the wheat and rye reference genomes, respectively. Interestingly, 28.9% (2151) of the 7430 SNPs were mapped to the D genome of hexaploid wheat, indicating substantial substitution of the R genome with D genome in cultivated triticale. About 27% of marker pairs were in significant LD with an average r2 > 0.18 (P < 0.05). Genome-wide LD declined rapidly to r2 < 0.1 beyond 10 kb physical distance. The three sub-genomes (A, B, and R) showed comparable LD decay patterns. Genetic diversity and population structure analyses identified five distinct clusters. Genotype grouping did not follow prior winter vs spring-type classification. However, one of the clusters was largely dominated by winter triticale. GS accuracies were estimated for forage yield using three commonly used models with different training population sizes and marker densities. GS accuracy increased with increasing training population size while gain in accuracy tended to plateau with marker densities of 2000 SNPs or more. Average GS accuracy was about 0.52, indicating the potential of using GS in triticale forage yield improvement.

Metabolomic Changes as Key Factors of Green Plant Regeneration Efficiency of Triticale In Vitro Anther Culture.

Green plant regeneration efficiency (GPRE) via in vitro anther culture results from biochemical pathways and cycle dysfunctions that may affect DNA and histone methylation, with gene expression influencing whole cell functioning. The reprogramming from gametophytic to sporophytic fate is part of the phenomenon. While DNA methylation and sequence changes related to the GPRE have been described, little attention was paid to the biochemical aspects of the phenomenon. Furthermore, only a few theoretical models that describe the complex relationships between biochemical aspects of GPRE and the role of Cu(II) ions in the induction medium and as cofactors of enzymatic reactions have been developed. Still, none of these models are devoted directly to the biochemical level. Fourier transform infrared (FTIR) spectroscopy was used in the current study to analyze triticale regenerants derived under various in vitro tissue culture conditions, including different Cu(II) and Ag(I) ion concentrations in the induction medium and anther culture times. The FTIR spectra of S-adenosyl-L-methionine (SAM), glutathione, and pectins in parallel with the Cu(II) ions, as well as the evaluated GPRE values, were put into the structural equation model (SEM). The data demonstrate the relationships between SAM, glutathione, pectins, and Cu(II) in the induction medium and how they affect GPRE. The SEM reflects the cell functioning under in vitro conditions and varying Cu(II) concentrations. In the presented model, the players are the Krebs and Yang cycles, the transsulfuration pathway controlled by Cu(II) ions acting as cofactors of enzymatic reactions, and the pectins of the primary cell wall.

Proteolytic and Structural Changes in Rye and Triticale Roots under Aluminum Stress.

Proteolysis and structural adjustments are significant for defense against heavy metals. The purpose of this study was to evaluate whether the Al3+ stress alters protease activity and the anatomy of cereale roots. Azocaseinolytic and gelatinolytic measurements, transcript-level analysis of phytocystatins, and observations under microscopes were performed on the roots of Al3+-tolerant rye and tolerant and sensitive triticales exposed to Al3+. In rye and triticales, the azocaseinolytic activity was higher in treated roots. The gelatinolytic activity in the roots of rye was enhanced between 12 and 24 h in treated roots, and decreased at 48 h. The gelatinolytic activity in treated roots of tolerant triticale was the highest at 24 h and the lowest at 12 h, whereas in treated roots of sensitive triticale it was lowest at 12 h but was enhanced at 24 and 48 h. These changes were accompanied by increased transcript levels of phytocystatins in rye and triticale-treated roots. Light microscope analysis of rye roots revealed disintegration of rhizodermis in treated roots at 48 h and indicated the involvement of root border cells in rye defense against Al3+. The ultrastructural analysis showed vacuoles containing electron-dense precipitates. We postulate that proteolytic-antiproteolytic balance and structural acclimation reinforce the fine-tuning to Al3+.

Comparative proteomic analysis provides new insights into regulation of microspore embryogenesis induction in winter triticale (× Triticosecale Wittm.) after 5-azacytidine treatment.

Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.5-10 µM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes (TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE (GSTF2)) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores (TaTPD1-like, SERK and GSTF2).

Long-term breeding progress of yield, yield-related, and disease resistance traits in five cereal crops of German variety trials.

KEY MESSAGE: Considerable breeding progress in cereal and disease resistances, but not in stem stability was found. Ageing effects decreased yield and increased disease susceptibility indicating that new varieties are constantly needed. Plant breeding and improved crop management generated considerable progress in cereal performance over the last decades. Climate change, as well as the political and social demand for more environmentally friendly production, require ongoing breeding progress. This study quantified long-term trends for breeding progress and ageing effects of yield, yield-related traits, and disease resistance traits from German variety trials for five cereal crops with a broad spectrum of genotypes. The varieties were grown over a wide range of environmental conditions during 1988-2019 under two intensity levels, without (I1) and with (I2) fungicides and growth regulators. Breeding progress regarding yield increase was the highest in winter barley followed by winter rye hybrid and the lowest in winter rye population varieties. Yield gaps between I2 and I1 widened for barleys, while they shrank for the other crops. A notable decrease in stem stability became apparent in I1 in most crops, while for diseases generally a decrasing susceptibility was found, especially for mildew, brown rust, scald, and dwarf leaf rust. The reduction in disease susceptibility in I2 (treated) was considerably higher than in I1. Our results revealed that yield performance and disease resistance of varieties were subject to considerable ageing effects, reducing yield and increasing disease susceptibility. Nevertheless, we quantified notable achievements in breeding progress for most disease resistances. This study indicated an urgent and continues need for new improved varieties, not only to combat ageing effects and generate higher yield potential, but also to offset future reduction in plant protection intensity.

Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8'OH1 loci.

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

Triticale Isolated Microspore Culture for Doubled Haploid Production.

Here, we describe a method of triticale isolated microspore culture for production of doubled haploid plants via androgenesis. We use this method routinely because it is highly efficient and works well on different triticale genotypes. To force microspores into becoming embryogenic, we apply a 21-day cold pretreatment. The shock of cold facilitates redirecting microspores from their predestined pollen developmental program into the androgenesis pathway. Ovaries are included in our culture methods to help with embryogenesis, and the histone deacytelase inhibitor Trichostatin A (TSA) is added to further improve androgenesis and increase our ability to recover green doubled haploid plants.

Molecular identification of triticale introgression lines carrying leaf rust resistance genes transferred from Aegilops kotschyi Boiss. and Ae. tauschii Coss.

Triticale (× Triticosecale Wittmack) is a commercial hybrid harboring wheat (Triticum sp.) and rye (Secale cereale L.) genomes. The limited genetic diversity of this crop resulted in the collapse of fungal disease resistance. Leaf rust disease, caused by Puccinia triticina Eriks., is reported to reduce the triticale yield significantly (more than 30%). There is a need to enlarge the genetic variability of this crop including leaf resistance genes. The main aim of this research was to evaluate the leaf rust resistance of the offspring of translocation lines of triticale carrying chromatin of Ae. tauschii and Ae. kotschyi. A reaction of seedlings of 200 plants of two triticale-Aegilops translocation lines (Bogo-2Dt.2R and Sekundo-2Sk.2R) was compared after inoculation with a natural mixture of P. triticina races, specific to triticale in controlled condition. Before inoculation, each plant was screened using molecular cytogenetics and molecular markers linked to leaf rust resistance genes. The presence of Aegilops chromosome segments was confirmed using genomic in situ hybridization (GISH). Lr39 and Lr54 leaf rust resistance genes were identified using Xgdm35 and S14 molecular markers, respectively. After inoculation, a significant improvement of resistance severity was observed in Sekundo-2Sk.2R in comparison with triticale cv. Sekundo plants. The resistance level of Bogo-2Dt.2R did not differ compared with triticale cv. Bogo plants. It was shown that Lr39 gene did not increase the leaf rust resistance level of triticale cv. Bogo.

Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium.

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

Generation and molecular marker and cytological characterization of wheat - Secale strictum subsp. anatolicum derivatives.

Cereal rye and its wild forms are important sources of genetic diversity for wheat breeding due to their resistances to biotic and abiotic stresses. Secale strictum subsp. anatolicum (Boiss.) K. Hammer (SSA) is a weedy relative of cultivated rye, S. cereale. Meiotic chromosome pairing in F1 hybrids of SSA and S. cereale reveals strong genomic affinity between the two genomes. A study of the transferability of S. cereale sequence-based markers to SSA and hexaploid triticale demonstrated their applicability for tracing SSA chromatin in wheat. The transferability of the markers was over 80% from homoeologous groups 1, 2, and 3, and greater than 70% from groups 4 to 7. This study focused on the generation and molecular and cytogenetic characterization of wheat-SSA alien derivatives. Twelve were identified using combinations of non-denaturing fluorescence in situ hybridization (ND-FISH), genomic in situ hybridization (GISH), and molecular marker analysis. All SSA chromosomes, except 3Ra and 6Ra, were transferred to wheat either in the form of monosomic additions (MA), mono-telosomic additions (MtA), double-mono-telosomic additions (dMtA), or double-monosomic additions (dMA). The germplasm developed in this study will help to enhance the genetic base of wheat and facilitate molecular breeding of wheat and triticale.