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Background: Microspore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation-DM, de novo methylation-DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and β-glucan affect the ability of green plant regeneration efficiency (GPRE)., Results: The experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO 4 : 0.1; 5; 10 µM) and silver salts (AgNO 3 : 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation. Phenotype variability in β-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, β-glucans, Cu(II), GPRE)., Conclusions: We can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE., (© 2024. The Author(s).)

This study aimed to determine whether using DNA-based markers assigned to individual chromosomes would detect the genetic structures of 446 winter triticale forms originating from two breeding companies more effectively than using the entire pool of markers. After filtering for quality control parameters, 6380 codominant single nucleotide polymorphisms (SNPs) markers and 17,490 dominant diversity array technology (silicoDArT) markers were considered for analysis. The mean polymorphic information content (PIC) values varied depending on the chromosomes and ranged from 0.30 (2R) to 0.43 (7A) for the SNPs and from 0.28 (2A) to 0.35 (6R) for the silicoDArTs. The highest correlation of genetic distance (GD) matrices based on SNP markers was observed among the 5B-5R (0.642), 5B-7B (0.626), and 5A-5R (0.605) chromosomes. When silicoDArTs were used for the analysis, the strongest correlations were found between 5B-5R (0.732) and 2B-5B (0.718). A Bayesian analysis showed that SNPs (total marker pool) allowed for the identification of a more complex structure (K = 4, ΔK = 2460.2) than the analysis based on silicoDArTs (K = 2, ΔK = 128). Triticale lines formed into groups, ranging from two (most of the chromosomes) to four (7A) groups depending on the analyzed chromosome when SNP markers were used for analysis. Linkage disequilibrium (LD) varied among individual chromosomes, ranging from 0.031 for 1A to 0.228 for 7R.

Background: The development of the plant in vitro techniques has brought about the variation identified in regenerants known as somaclonal or tissue culture-induced variation (TCIV). S-adenosyl-L-methionine (SAM), glutathione (GSH), low methylated pectins (LMP), and Cu(II) ions may be implicated in green plant regeneration efficiency (GPRE) and TCIV, according to studies in barley (Hordeum vulgare L.) and partially in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). Using structural equation models (SEM), these metabolites have been connected to the metabolic pathways (Krebs and Yang cycles, glycolysis, transsulfuration), but not for triticale. Using metabolomic and (epi)genetic data, the study sought to develop a triticale regeneration efficiency statistical model. The culture's induction medium was supplemented with various quantities of Cu(II) and Ag(I) ions for regeneration. The period of plant regeneration has also changed. The donor plant, anther-derived regenerants, and metAFLP were utilized to analyze TCIV concerning DNA in symmetric (CG, CHG) and asymmetric (CHH) sequence contexts. Attenuated Total Reflectance-Fourier Transfer Infrared (ATR-FTIR) spectroscopy was used to gather the metabolomic information on LMP, SAM, and GSH. To frame the data, a structural equation model was employed., Results: According to metAFLP analysis, the average sequence change in the CHH context was 8.65%, and 0.58% was de novo methylation. Absorbances of FTIR spectra in regions specific for LMP, SAM, and GSH were used as variables values introduced to the SEM model. The average number of green regenerants per 100 plated anthers was 2.55., Conclusions: The amounts of pectin demethylation, SAM, de novo methylation, and GSH are connected in the model to explain GPRE. By altering the concentration of Cu(II) ions in the medium, which influences the amount of pectin, triticale's GPRE can be increased., (© 2024. The Author(s).)

Increased grain yield (GY) is the primary breeding target of wheat breeders. We performed the genome-wide association study (GWAS) on 168 elite winter wheat lines from an ongoing breeding program to identify the main determinants of grain yield. Sequencing of Diversity Array Technology fragments (DArTseq) resulted in 19,350 single-nucleotide polymorphism (SNP) and presence-absence variation (PAV) markers. We identified 15 main genomic regions located in ten wheat chromosomes (1B, 2B, 2D, 3A, 3D, 5A, 5B, 6A, 6B, and 7B) that explained from 7.9 to 20.3% of the variation in grain yield and 13.3% of the yield stability. Loci identified in the reduced genepool are important for wheat improvement using marker-assisted selection. We found marker-trait associations between three genes involved in starch biosynthesis and grain yield. Two starch synthase genes (TraesCS2B03G1238800 and TraesCS2D03G1048800) and a sucrose synthase gene (TraesCS3D03G0024300) were found in regions of QGy.rut-2B.2, QGy.rut-2D.1, and QGy.rut-3D, respectively. These loci and other significantly associated SNP markers found in this study can be used for pyramiding favorable alleles in high-yielding varieties or to improve the accuracy of prediction in genomic selection., (© 2023. The Author(s).)

Investigating genetic structure and diversity is crucial for the rye hybrid breeding strategy, leading to improved plant productivity and adaptation. The present study elucidated the population structure and genetic diversity of 188 rye accessions, comprising 94 pollen fertility restoration lines (RF) and 94 cytoplasmic male-sterile (CMS) lines with Pampa sterilizing cytoplasm using SNP and silicoDArT markers from the diversity array technology (DArT)-based sequencing platform (DArTseq). Expected heterozygosity (H e ) and Shanon's diversity (I) indexes varied slightly between marker systems and groups of germplasms (H e  = 0.34, I = 0.51 for RF and CMS lines genotyped using SNPs; H e  = 0.31, I = 0.48, and H e  = 0.35, I = 0.53 for RF and CMS using silicoDArTs, respectively). ANOVA indicated moderate variation (7%) between RF and CMS breeding materials. The same parameter varied when chromosome-assigned markers were used and ranged from 5.8% for 5R to 7.4% for 4R. However, when silicoDArT markers were applied, the respective values varied from 6.4% (1R) to 8.2% (3R and 4R). The model-based (Bayesian) population structure analysis based on the total marker pool identified two major subpopulations for the studied rye germplasm. The first one (P1) encompasses 93 RF accessions, and the second one (P2) encompasses 94 CMS and one RF accession. However, a similar analysis related to markers assigned to selected chromosomes failed to put plant materials into any of the populations in the same way as the total marker pool. Furthermore, the differences in grouping depended on marker types used for analysis., (© 2023. The Author(s).)

Babia Góra massif is the only site of occurrence of the Cerastium alpinum L. in Poland, an arctic-alpine perennial plant with a wide distribution in North America, northwestern Asia, and Europe. To determine whether the isolated Polish populations are genetically distinct, we have performed an evaluation of C. alpinum from Babia Góra with the use of iPBS markers. A total number of 133 individuals of C. alpinum from seven populations representing four localizations of the species were analyzed, i.e., from Babia Góra (Poland), Alps (Switzerland), Nuolja massif (Sweden), and Kaffiøyra (Svalbard, Norway). Genetic analysis of all C. alpinum samples using eight PBS primers identified 262 bands, 79.4% of which were polymorphic. iPBS markers revealed low genetic diversity (average H e  = 0.085) and high population differentiation (F ST  = 0.617). AMOVA results confirmed that the majority of the genetic variation (62%) was recorded among populations. The grouping revealed by PCoA showed that C. alpinum from Svalbard is the most diverged population, C. alpinum from Switzerland and Sweden form a pair of similar populations, whereas C. alpinum from the Babia Góra form a heterogeneous group of four populations. Results of isolation by distance analysis suggested that the spatial distance is the most probable cause of the observed differentiation among populations. Although significant traces of a bottleneck effect were noted for all populations of C. alpinum from Babia Góra, the populations still maintain a low but significant level of genetic polymorphism. These results are of great importance for developing conservation strategies for this species in Poland., (© 2022. The Author(s).)

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.

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.

Plant tissue culture techniques are handy tools for obtaining unique plant materials that are difficult to propagate or important for agriculture. Homozygous materials derived through in vitro cultures are invaluable and significantly accelerate the evaluation of new varieties, e.g., cereals. The induction of somatic embryogenesis/androgenesis and the regeneration and its efficiency can be influenced by the external conditions of tissue culture, such as the ingredients present in the induction or regeneration media. We have developed an approach based on biological system, molecular markers, Fourier Transform Infrared spectroscopy, and structural equation modeling technique to establish links between changes in sequence and DNA methylation at specific symmetric (CG, CHG) and asymmetric (CHH) sequences, glutathione, and green plant regeneration efficiency in the presence of variable supplementation of induction medium with copper ions. The methylation-sensitive Amplified Fragment Length Polymorphism was used to assess tissue culture-induced variation, Fourier Transform Infrared spectroscopy to describe the glutathione spectrum, and a structural equation model to develop the relationship between sequence variation , de novo DNA methylation within asymmetric sequence contexts, and copper ions in the induction medium, as well as, glutathione, and green plant efficiency. An essential aspect of the study is demonstrating the contribution of glutathione to green plant regeneration efficiency and indicating the critical role of copper ions in influencing tissue culture-induced variation, glutathione, and obtaining green regenerants. The model presented here also has practical implications, showing that manipulating the concentration of copper ions in the induction medium may influence cell function and increases green plant regeneration efficiency., Competing Interests: The authors declare that the research was conducted without any commercial or financial relationships construed as a potential conflict of interest., (Copyright © 2022 Bednarek, Orłowska, Mańkowski, Zimny, Kowalczyk, Nowak and Zebrowski.)

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.

Pampa cytoplasmic male sterility phenomenon is used extensively in the rye hybrid breeding programs. It relies on sterilizing action of the cytoplasm resulting in non-viable pollen of female lines. The sterilizing effect is problematic for reversion, and efficient restores are needed. The most promising QTL is located on chromosome 4R, but other chromosomes may also code the trait. Advanced recombinant inbred lines formed bi-parental mapping population genotyped with DArTseq markers. Genetic mapping allowed the seven linkage groups to construct with numerous markers and represent all rye chromosomes. Single marker analysis and composite interval mapping were conducted to identify markers linked to the pollen fertility. Association mapping was used to detect additional markers associated with the trait. A highly significant QTL (QRfp-4R) that explained 42.3% of the phenotypic variation was mapped to the distal part of the long arm of the 4R chromosome. The markers localized in the QRfp-4R region achieve R 2 association values up to 0.59. The homology of the 43 marker sequences to the loci responsible for fertility restoration in other species and transcription termination factor (mTERF) linked to Rf genes was established. Ten markers were successfully converted into PCR-specific conditions, and their segregation pattern was identical to that of unconverted DArTs., (© 2021. The Author(s).)

The process of anther culture involves numerous abiotic stresses required for cellular reprogramming, microspore developmental switch, and plant regeneration. These stresses affect DNA methylation patterns, sequence variation, and the number of green plants regenerated. Recently, in barley ( Hordeum vulgare L.), mediation analysis linked DNA methylation changes, copper (Cu 2+ ) and silver (Ag + ) ion concentrations, sequence variation, β-glucans, green plants, and duration of anther culture (Time). Although several models were used to explain particular aspects of the relationships between these factors, a generalized complex model employing all these types of data was not established. In this study, we combined the previously described partial models into a single complex model using the structural equation modeling approach. Based on the evaluated model, we demonstrated that stress conditions (such as starvation and darkness) influence β-glucans employed by cells for glycolysis and the tricarboxylic acid cycle. Additionally, Cu 2+ and Ag + ions affect DNA methylation and induce sequence variation. Moreover, these ions link DNA methylation with green plants. The structural equation model also showed the role of time in relationships between parameters included in the model and influencing plant regeneration via anther culture. Utilization of structural equation modeling may have both scientific and practical implications, as it demonstrates links between biological phenomena (e.g., culture-induced variation, green plant regeneration and biochemical pathways), and provides opportunities for regulating these phenomena for particular biotechnological purposes.

In vitro tissue culture plant regeneration is a complicated process that requires stressful conditions affecting the cell functioning at multiple levels, including signaling pathways, transcriptome functioning, the interaction between cellular organelles (retro-, anterograde), compounds methylation, biochemical cycles, and DNA mutations. Unfortunately, the network linking all these aspects is not well understood, and the available knowledge is not systemized. Moreover, some aspects of the phenomenon are poorly studied. The present review attempts to present a broad range of aspects involved in the tissue culture-induced variation and hopefully would stimulate further investigations allowing a better understanding of the phenomenon and the cell functioning.

A plant genome usually encompasses different families of transposable elements (TEs) that may constitute up to 85% of nuclear DNA. Under stressful conditions, some of them may activate, leading to sequence variation. In vitro plant regeneration may induce either phenotypic or genetic and epigenetic changes. While DNA methylation alternations might be related, i.e., to the Yang cycle problems, DNA pattern changes, especially DNA demethylation, may activate TEs that could result in point mutations in DNA sequence changes. Thus, TEs have the highest input into sequence variation (SV). A set of barley regenerants were derived via in vitro anther culture. High Performance Liquid Chromatography (RP-HPLC), used to study the global DNA methylation of donor plants and their regenerants, showed that the level of DNA methylation increased in regenerants by 1.45% compared to the donors. The Methyl-Sensitive Transposon Display (MSTD) based on methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach demonstrated that, depending on the selected elements belonging to the TEs family analyzed, varying levels of sequence variation were evaluated. DNA sequence contexts may have a different impact on SV generated by distinct mobile elements belonged to various TE families. Based on the presented study, some of the selected mobile elements contribute differently to TE-related SV. The surrounding context of the TEs DNA sequence is possibly important here, and the study explained some part of SV related to those contexts.

Cytoplasmic male sterility (CMS) is a widely applied plant breeding tool for hybrid seed production. The phenomenon is often caused by chimeric genes with altered open reading frames (ORFs) located in the mitochondrial genomes and expressed as novel genotoxic products that induce pollen abortion. The fertility of CMS plants can be restored by nuclear-encoded genes that inhibit the action of ORFs responsible for pollen sterility. A recombinant inbred line (RIL) mapping population S64/04/01, encompassing 175 individuals, was used for genetic map construction and identification of quantitative trait loci (QTLs) responsible for fertility restoration in rye (Secale cereale L.) with CMS Pampa. The genetic map of all seven rye chromosomes included 15,516 SNP and silicoDArT markers and covered 1070.5 cm. Individual QTLs explaining 60% and 5.5% of the fertility trait's phenotypic variance were mapped to chromosomes 4R (QRft-4R) and 5R (QRft-5R), respectively. Association mapping identified markers with the highest R 2 value of 0.58 (p value = 2.21E-28). Markers showing the highest associations with the trait were also mapped to the 4R chromosome within the QRft-4R region. Based on marker sequence homology, putative genes involved in pollen fertility restoration were suggested. Five silicoDArTs were converted into PCR-based markers for further breeding purposes.

In vitro tissue culture could be exploited to study cellular mechanisms that induce sequence variation. Altering the metal ion composition of tissue culture medium affects biochemical pathways involved in tissue culture-induced variation. Copper ions are involved in the mitochondrial respiratory chain and Yang cycle. Copper ions may participate in oxidative mutations, which may contribute to DNA sequence variation. Silver ions compete with copper ions to bind to the complex IV subunit of the respiratory chain, thus affecting the Yang cycle and DNA methylation. The mechanisms underlying somaclonal variation are unknown. In this study, we evaluated embryo-derived barley regenerants obtained from a single double-haploid plant via embryo culture under varying copper and silver ion concentrations and different durations of in vitro culture. Morphological variation among regenerants and the donor plant was not evaluated. Methylation-sensitive Amplified Fragment Length Polymorphism analysis of DNA samples showed DNA methylation pattern variation in CG and CHG (H = A, C, or T) sequence contexts. Furthermore, modification of in vitro culture conditions explained DNA sequence variation, demethylation, and de novo methylation in the CHG context, as indicated by analysis of variance. Linear regression indicated that DNA sequence variation was related to de novo DNA methylation in the CHG context. Mediation analysis showed the role of copper ions as a mediator of sequence variation in the CHG context. No other contexts showed a significant sequence variation in mediation analysis. Silver ions did not act as a mediator between any methylation contexts and sequence variation. Thus, incorporating copper ions in the induction medium should be treated with caution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Orłowska, Zimny and Bednarek.)

Cytoplasmic male sterility (CMS) phenomenon is widely exploited in commercial hybrid seed production in economically important crop species, including rye, wheat, maize, rice, sorghum, cotton, sugar beets, and many vegetables. Although some commercial successes, little is known about QTLs responsible for the trait in case of triticale with sterilizing Triticum timopheevii (Tt) cytoplasm. Recombinant inbred line (RIL) F6 mapping population encompassing 182 individuals derived from the cross of individual plants representing the HT352 line and cv Borwo was employed for genetic map construction using SNP markers and identification of QTLs conferring pollen sterility in triticale with CMS Tt. The phenotypes of the F1 lines resulting from crossing of the HT352 (Tt) with HT352 (maintainer) × Borwo were determined by assessing the number of the F2 seeds per spike. A genetic map with 21 linkage groups encompasses 29,737 markers and spanned over the distance of 2549 cM. Composite (CIM) and multiple (MIM) interval mappings delivered comparable results. Single QTLs mapped to the 1A, 1B, 2A, 2R, 3B, 3R, 4B, and 5B chromosomes, whereas the 5R and 6B chromosomes shared 3 and 2 QTLs, respectively. The QTLs with the highest LOD score mapped to the 5R, 3R, 1B, and 4B chromosomes; however, the QRft-5R.3 has the highest explained variance of the trait.

Plant anther culture allows for the regeneration of uniform and homozygous double haploids. However, off-type regenerants may appear as a result of so-called tissue culture-induced variation (TCIV). In addition, the presence of Cu 2+ and Ag + ions in the culture medium might influence the number of green plants. The regenerants were obtained via anther cultures of barley under varying Cu 2+ and Ag + ion concentrations in the induction medium during distinct time conditions. DArTseqMet markers were evaluated based on regenerants and donor plants and delivering data on DNA demethylation (DM) and de novo methylation (DNM) and changes in methylation (Delta). The number of green regenerated plants per 100 anthers (GPs) was evaluated. The Cu 2+ and Ag + ion concentrations moderated relationships between Delta and the number of green plants conditional on time of tissue cultures. Depending on the ions, moderated moderation is valid within the different time of anther culture. When the highest concentration of copper is analyzed, plant regeneration is possible under short 'Time' (21 days) of anther culture wherein Delta is negative or under elongated Time when Delta is positive. Under 21 days of culture, the highest concentration of silver ions and when Delta is negative, some regenerants could be evaluated. However, under high Ag + concentration when Time of culture is long and Delta positive, the highest number of green plants could be obtained.

Tissue culture is an essential tool for the regeneration of uniform plant material. However, tissue culture conditions can be a source of abiotic stress for plants, leading to changes in the DNA sequence and methylation patterns. Despite the growing evidence on biochemical processes affected by abiotic stresses, how these altered biochemical processes affect DNA sequence and methylation patterns remains largely unknown. In this study, the methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach was used to investigate de novo methylation, demethylation, and sequence variation in barley regenerants derived by anther culture. Additionally, we used Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy to identify the spectral features of regenerants, which were then analyzed by mediation analysis. The infrared spectrum ranges (710-690 and 1010-940 cm -1 ) identified as significant in the mediation analysis were most likely related to β-glucans, cellulose, and S-adenosyl-L-methionine (SAM). Additionally, the identified compounds participated as predictors in moderated mediation analysis, explaining the role of demethylation of CHG sites (CHG_DMV) in in vitro tissue culture-induced sequence variation, depending on the duration of tissue culture. The data demonstrate that ATR-FTIR spectroscopy is a useful tool for studying the biochemical compounds that may affect DNA methylation patterns and sequence variation, if combined with quantitative characteristics determined using metAFLP molecular markers and mediation analysis. The role of β-glucans, cellulose, and SAM in DNA methylation, and in cell wall, mitochondria, and signaling, are discussed to highlight the putative cellular mechanisms involved in sequence variation.

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO 4 and AgNO 3 ) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu 2+ and Ag + ion concentrations ('Cu*Ag') acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of 'Cu*Ag', and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis.

Key Message: The Taguchi method and metAFLP analysis were used to optimise barley regenerants towards maximum and minimum levels of tissue culture-induced variation. The subtle effects of symmetric and asymmetric methylation changes in regenerants were identified. Plant tissue cultures (PTCs) provide researchers with unique materials that accelerate the development of new breeding cultivars and facilitate studies on off-type regenerants. The emerging variability of regenerants derived from PTCs may have both genetic and epigenetic origins, and may be desirable or degrade the value of regenerated plants. Thus, it is crucial to determine how the PTC variation level can be controlled. The easiest way to manipulate total tissue culture-induced variation (TTCIV) is to utilise appropriate stress factors and suitable medium components. This study describes the optimisation of in vitro tissue culture-induced variation in plant regenerants derived from barley anther culture, and maximizes and minimizes regenerant variation compared with the source explants. The approach relied on methylation amplified fragment length polymorphism (metAFLP)-derived TTCIV characteristics, which were evaluated in regenerants derived under distinct tissue culture conditions and analysed via Taguchi statistics. The factors that may trigger TTCIV included CuSO 4 , AgNO 3 and the total time spent on the induction medium. The donor plants prepared for regeneration purposes had 5.75% and 2.01% polymorphic metAFLP loci with methylation and sequence changes, respectively. The level of TTCIV (as the sum of all metAFLP characteristics analyzed) identified in optimisation and verification experiments reached 7.51 and 10.46%, respectively. In the trial designed to produce a minimum number of differences between donor and regenerant plants, CuSO 4 and AgNO 3 were more crucial than time, which was not a significant factor. In the trial designed to produce a maximum number of differences between donor and regenerant plants, all factors had comparable impact on variation. The Taguchi method reduced the time required for experimental trials compared with a grid method and suggested that medium modifications were required to control regenerant variation. Finally, the effects of symmetric and asymmetric methylation changes on regenerants were identified using novel aspects of the metAFLP method developed for this analysis.

We studied an invasion of Poa annua on King George Island (Maritime Antarctic). The remoteness of this location, its geographic isolation, and its limited human traffic provided an opportunity to trace the history of an invasion of the species. Poa annua was recorded for the first time at H. Arctowski Polish Antarctic Station in the austral summer of 1985/6. In 2008/9, the species was observed in a new locality at the Ecology Glacier Forefield (1.5 km from "Arctowski"). We used AFLP to analyze the genetic differences among three populations of P. annua : the two mentioned above ( Station and Forefield ) and the putative origin of the introduction, Warsaw (Poland). There was 38% genetic variance among the populations. Pairwise Ф PT was 0.498 between the Forefield and Warsaw populations and 0.283 between Warsaw and Station . There were 15 unique bands in the Warsaw population (frequency from 6% to 100%) and one in the Station/Forefield populations (which appears in all analyzed individuals from both populations). The Δ( K ) parameter indicated two groups of samples: Warsaw/Station and Forefield . As indicated by Fu's F s statistics and an analysis of mismatch distribution, the Forefield population underwent a bottleneck and/or founder effect. The Forefield population was likely introduced by secondary dispersal from the Station population.

Background: We present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms., Methods: Five Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward's Agglomeration method whereas MSAP characteristics for ANOVA., Results: Relative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines., Conclusions: Our relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII-MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control and stressed Al-tolerant and non-tolerant triticale inbred lines. The method could also be used to analyze methylation events affecting CG and CHG contexts, which were differentially methylated under Al stress. We cannot exclude that the methylation changes revealed among lines as well as between Al-tolerant and non-tolerant groups of lines were due to some experimental errors or that the number of lines was too small for ANOVA to prove the influence of Al stress. Nevertheless, we suspect that Al tolerance in triticale could be partly regulated by epigenetic factors acting at the level of DNA methylation. This method provides a valuable tool for studies of abiotic stresses in plants.

Background: In vitro plant regeneration via androgenesis or somatic embryogenesis is capable of inducing (epi)mutations that may affect sexual progenies. While epimutations are associated with DNA methylation, mutations could be due to the movement of transposons. The common notion is that both processes are linked. It is being assumed that demethylation activates transposable elements (TEs). Analysis of methylation changes and their relation with TEs activation in tissue cultures requires uniquely derived donor plants (Ds), their regenerants (Rs) and respective progeny (Ps) that would allow discrimination of processes not related to changes introduced via in vitro cultures. Moreover, a set of methods (RP-HPLC, SSAP, and MSTD) is needed to study whether different TEs families are being activated during in vitro tissue culture plant regeneration and whether their activity could be linked to DNA methylation changes or alternative explanations should be considered., Results: The in vitro tissue culture plant regeneration in barley was responsible for the induction of DNA methylation in regenerants and conservation of the methylation level in the progeny as shown by the RP-HPLC approach. No difference between andro- and embryo-derived Rs and Ps was observed. The SSAP and MSTD approach revealed that Ds and Rs were more polymorphic than Ps. Moreover, Rs individuals exhibited more polymorphisms with the MSTD than SSAP approach. The differences between Ds, Rs and Ps were also evaluated via ANOVA and AMOVA., Conclusions: Stressful conditions during plant regeneration via in vitro tissue cultures affect regenerants and their sexual progeny leading to an increase in global DNA methylation of Rs and Ps compared to Ds in barley. The increased methylation level noted among regenerants remains unchanged in the Ps as indicated via RP-HPLC data. Marker-based experiments suggest that TEs are activated via in vitro tissue cultures and that, independently of the increased methylation, their activity in Rs is greater than in Ps. Thus, the increased methylation level may not correspond to the stabilization of TEs movement at least at the level of regenerants. The presence of TEs variation among Ds that were genetically and epigenetically uniform may suggest that at least some mobile elements may be active, and they may mask variation related to tissue cultures. Thus, tissue cultures may activate some TEs whereas the others remain intact, or their level of movement is changed. Finally, we suggest that sexual reproduction may be responsible for the stabilization of TEs.

Genetic diversity analysis of triticale populations is useful for breeding programs, as it helps to select appropriate genetic material for classifying the parental lines, heterotic groups and predicting hybrid performance. In our study 232 breeding forms were analyzed using diversity arrays technology markers. Principal coordinate analysis followed by model-based Bayesian analysis of population structure revealed the presence of weak data structuring with three groups of data. In the first group, 17 spring and 17 winter forms were clustered. The second and the third groups were represented by 101 and 26 winter forms, respectively. Polymorphic information content values, as well as Shannon's Information Index, were higher for the first (0.319) and second (0.309) than for third (0.234) group. AMOVA analysis demonstrated a higher level of within variation (86 %) than among populations (14 %). This study provides the basic information on the presence of structure within a genetic pool of triticale breeding forms.

Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

The tolerance of triticale ( x Triticosecale Wittmack) cultivars to aluminum (Al) stress observed in acid soils is an important agronomic trait affecting seed yield. Traditionally, breeding of Al-tolerant cultivars was selection based; for example, using a physiological test. However, such selection methods are relatively slow and require numerous plants for phenotype evaluation. Alternatively, DNA-based molecular marker systems could be applied to identify markers useful for selection purposes. Among many marker platforms available, Diversity Arrays Technology (DArT) is one of the most promising. DArT markers preselected for conversion to specific PCR assays were chosen based on association mapping studies using diverse materials. Forty-nine DArT markers were selected and tested for redundancy based on their segregation patterns and sequences, and 40 were successfully converted into specific PCR assays. However, only 24 of these proved to be polymorphic. Where possible, the chromosomal locations of the converted markers were verified. The markers assigned to chromosome 7R that were the most highly correlated with Al-tolerant and non-tolerant plants were chosen for marker assisted selection using genetically diverse triticale materials.

Triticale (x Triticosecale Wittmack) is a relatively new cereal crop. In Poland, triticale is grown on 12 % of arable land ( http://www.stat.gov.pl ). There is an increasing interest in its cultivation due to lowered production costs and increased adaptation to adverse environmental conditions. However, it has an insufficient tolerance to the presence of aluminum ions (Al(3+)) in the soil. The number of genes controlling aluminum tolerance in triticale and their chromosomal location is not known. Two F2 mapping biparental populations (MP1 and MP15) segregating for aluminum (Al) tolerance were tested with AFLP, SSR, DArT, and specific PCR markers. Genetic mapping enabled the construction of linkage groups representing chromosomes 7R, 5R and 2B. Obtained linkage groups were common for both mapping populations and mostly included the same markers. Composite interval mapping (CIM) allowed identification of a single QTL that mapped to the 7R chromosome and explained 25 % (MP1) and 36 % (MP15) of phenotypic variation. The B1, B26 and Xscm150 markers were 0.04 cM and 0.02 cM from the maximum of the LOD function in the MP1 and MP15, respectively and were highly associated with aluminum tolerance as indicated by Kruskal-Wallis nonparametric test. Moreover, the molecular markers B1, B26, Xrems1162 and Xscm92, previously associated with the Alt4 locus that encoded an aluminum-activated malate transporter (ScALMT1) that was involved in Al tolerance in rye (Secale cereale) also mapped within QTL. Biochemical analysis of plants represented MP1 and MP15 mapping populations confirmed that the QTL located on 7R chromosome in both mapping populations is responsible for Al tolerance.

We present the development of the theoretical background of the metAFLP approach which allows for partition of complex variation into sequence changes, de novo methylation and demethylation of the regenerants derived via in vitro tissue culture methods in the case of triticale. It was demonstrated that, independent of whether andro- or embryogenesis was used for plant regeneration, the level of sequence changes identified between regenerants is about 10 %. Moreover, DNA demethylation prevails over de novo methylation of the regenerants compared to the donor plant. The metAFLP approach allows for the evaluation of numerous quantitative characteristics. For instance, one may quantify the number of sites unaffected by tissue culture approaches, global site DNA methylation etc. It is suggested that the approach could be useful for breeders in order to control plant material uniformity or for the evaluation of modified in vitro tissue culture approaches allowing for control of the (epi)mutation level. The extended metAFLP approach presented here delivers sufficient background for the evaluation of software that could facilitate analyses of the tissue culture induced variation.

Patterns of morphological, genetic and epigenetic variation (DNA methylation pattern) were investigated in metallicolous (M) and non-metallicolous (NM) populations of Armeria maritima. A morphological study was carried out using plants from six natural populations grown in a greenhouse. Morphological variation was assessed using seven traits. On the basis of this study, three representative populations were selected for molecular analyses using metAFLP to study sequence- and methylation-based DNA variation. Only one morphological trait (length of outer involucral bracts) was common to both metallicolous populations studied; however, the level of variation was sufficient to differentiate between M and NM populations. Molecular analyses showed the existence of naturally occurring epigenetic variation in A. maritima populations, as well as structuring into distinct between and within population components. We show that patterns of population genetic structure differed depending on the information used in the study. Analysis of sequence-based information data demonstrates the presence of three well-defined and genetically differentiated populations. Methylation-based data show that two major groups of individuals are present, corresponding to the division into M and NM populations. These results were confirmed using different analytical approaches, which suggest that the DNA methylation pattern is similar in both M populations. We hypothesise that epigenetic processes may be involved in microevolution leading to development of M populations in A. maritima., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Background: Crop production practices and industrialization processes result in increasing acidification of arable soils. At lower pH levels (below 5.0), aluminum (Al) remains in a cationic form that is toxic to plants, reducing growth and yield. The effect of aluminum on agronomic performance is particularly important in cereals like wheat, which has promoted the development of programs directed towards selection of tolerant forms. Even in intermediately tolerant cereals (i.e., triticale), the decrease in yield may be significant. In triticale, Al tolerance seems to be influenced by both wheat and rye genomes. However, little is known about the precise chromosomal location of tolerance-related genes, and whether wheat or rye genomes are crucial for the expression of that trait in the hybrid., Results: A mapping population consisting of 232 advanced breeding triticale forms was developed and phenotyped for Al tolerance using physiological tests. AFLP, SSR and DArT marker platforms were applied to obtain a sufficiently large set of molecular markers (over 3000). Associations between the markers and the trait were tested using General (GLM) and Multiple (MLM) Linear Models, as well as the Statistical Machine Learning (SML) approach. The chromosomal locations of candidate markers were verified based on known assignments of SSRs and DArTs or by using genetic maps of rye and triticale.Two candidate markers on chromosome 3R and 9, 15 and 11 on chromosomes 4R, 6R and 7R, respectively, were identified. The r2 values were between 0.066 and 0.220 in most cases, indicating a good fit of the data, with better results obtained with the GML than the MLM approach. Several QTLs on rye chromosomes appeared to be involved in the phenotypic expression of the trait, suggesting that rye genome factors are predominantly responsible for Al tolerance in triticale., Conclusions: The Diversity Arrays Technology was applied successfully to association mapping studies performed on triticale breeding forms. Statistical approaches allowed the identification of numerous markers associated with Al tolerance. Available rye and triticale genetic maps suggested the putative location of the markers and demonstrated that they formed several linked groups assigned to distinct chromosomes (3R, 4R, 6R and 7R). Markers associated with genomic regions under positive selection were identified and indirectly mapped in the vicinity of the Al-tolerant markers. The present findings were in agreement with prior reports.

A set of 90 doubled haploid (DH) lines derived from F(1) plants that originated from a cross between × Triticosecale Wittm. 'Saka3006' and ×Triticosecale Wittm. 'Modus', via wide crossing with maize, were used to create a genetic linkage map of triticale. The map has 21 linkage groups assigned to the A, B, and R genomes including 155 simple sequence repeat (SSR), 1385 diversity array technology (DArT), and 28 amplified fragment length polymorphism (AFLP) markers covering 2397 cM with a mean distance between two markers of 4.1 cM. Comparative analysis with wheat consensus maps revealed that triticale chromosomes of the A and B genomes were represented by 15 chromosomes, including combinations of 2AS.2AL#, 2AL#2BL, 6AS.6AL#, and 2BS.6AL# instead of 2A, 2B, and 6A. In respect to published maps of rye, substantial rearrangements were found also for chromosomes 1R, 2R, and 3R of the rye genome. Chromosomes 1R and 2R were truncated and the latter was linked with 3R. A nonhomogeneous distribution of markers across the triticale genome was observed with evident bias (48%) towards the rye genome. This genetic map may serve as a reference linkage map of triticale for efficient studies of structural rearrangements, gene mapping, and marker-assisted selection.

Background: When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level., Results: A variant of methylation sensitive AFLP, based on the isoschizomeric combinations Acc65I/MseI and KpnI/MseI was applied to analyze, at both the sequence and methylation levels, the outcomes of regeneration from tissue culture in barley. Both sequence mutation and alteration in methylation pattern were detected. Two sets of regenerants from each of five DH donor lines were compared. One set was derived via androgenesis, and the other via somatic embryogenesis, developed from immature embryos. These comparisons delivered a quantitative assessment of the various types of somaclonal variation induced. The average level of variation was 6%, of which almost 1.7% could be accounted for by nucleotide mutation, and the remainder by changes in methylation state. The nucleotide mutation rates and the rate of epimutations were substantially similar between the andro- and embryo-derived sets of regenerants across all the donors., Conclusion: We have developed an AFLP based approach that is capable of describing the qualitative and quantitative characteristics of the tissue culture-induced variation. We believe that this approach will find particular value in the study of patterns of inheritance of somaclonal variation, since non-heritable variation is of little interest for the improvement of plant species which are sexually propagated. Of significant biological interest is the conclusion that the mode of regeneration has no significant effect on the balance between sequence and methylation state change induced by the tissue culture process.

The aim of this study was to identify genetic changes in rye seeds induced by natural ageing during long-term storage and consecutive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples varying in their initial viability after one and three cycles of reproduction was analyzed by AFLP (amplified fragment length polymorphism) fingerprinting. Seven EcoRI/MseI primer combinations defined 663 fragments, and seven PstI/MseI primer combinations defined 551 fragments. The variation in the frequency of the seventy-four EcoRI/MseI bands was statistically significant between samples. These changes could be attributed to genetic changes occurring during storage and regeneration. However, the PstI/MseI fragments appeared to be uninfluenced by seed ageing, regeneration and propagation. A combined Principle Coordinate Analysis revealed differences between samples with different initial viability. We showed that materials with low initial viability differ in their response from highly viable ones, and that the changes exhibited in the former case are preserved through regeneration cycles.

A linkage map of rye, previously developed using DS2 x RXL10 F2 mapping population, was enriched with 179 AFLP and 19 RAPD marker loci. The current map covers 1386 cM and contains 480 markers including 200 RFLPs, 179 AFLPs, 88 RAPDs, 12 protein loci and one dwarfing gene. AFLPs generated by EcoRI/MseI primer combinations were distributed over the entire genome as distinct loci or clusters of 2-14 tightly linked DNA fragments. New marker loci mapped distally to the existing framework, significantly increased coverage of chromosomes 1R, 2R and 5R. The average marker distance is now 2.9 cM, but in seven regions the closest markers are still more than 20 cM apart. A detailed description of the newly mapped AFLP and RAPD loci is presented. The relationship with other published rye maps is discussed.

Application of AFLPs linked to pollen fertility restoration and non-performing genes evaluated in the C394-F2 hybrid was studied using a set of male sterile lines in the sterilising Pampa cytoplasm, several restorers and maintainer lines and, finally, two inbred lines backcrossed into cms-P, cms-R, cms-S and cms-C cytoplasms each. The set of male sterile lines based on the Pampa cytoplasm exhibited gradual variation in their ability to restore pollen fertility (starting from low and closing with high) in crosses with three unrelated restorers. Variations in the AFLPs between the analysed materials were observed, however, no clustering of the lines according to their sterile and fertile phenotypes was observed. The same markers, when applied to the population restorer (cv. Walet) that formed the C394-F2 cross permitted identification of plants with genotypes that could be recognized as restorers.

23 AFLP bands were assigned to different rye chromosomes by means of two different sets of wheat-rye addition lines. Only one AFLP band could be assigned to 4R, and no specific AFLPs were found on the 5R chromosome. Only one AFLP band was explicitly assigned to 4R, and no specific AFLPs were found on the 5R chromosome. At least seven co-migrating AFLPs showed the same chromosomal location in both sets of addition lines. A total of 22 AFLPs were assigned to chromosome 1R using wheat-rye substitution lines. Six of them have counterparts in one of the addition lines analyzed, but only four have the same chromosomal location. Six and four of the total AFLPs located using addition (23) and substitution (22) lines segregated in the mapping population DS2 x RXL10, but only six were simultaneously assigned to the same chromosome by both approaches. Although co-migrating AFLPs could be located on different rye chromosomes using addition and substitution lines, we believe that AFLPs can be useful as rye chromosome markers.

Combined morphological and molecular techniques were used to characterize variation in Elymus repens. We studied the morphological variability of E. repens in relation to the degree of its genetic differentiation, in order to unravel the causes of conspicuous intraspecific morphological variation. Four populations of E. repens from different habitats were analyzed for 35 morphological characters, and their genetic differentiation was assessed by Amplified Fragment Length Polymorphism (AFLP). Four pairs of selective primers were used to detect a total of 279 AFLP bands, of which 104 (37.28%) were polymorphic between populations. Cluster analysis based on AFLP fingerprint data showed that individuals were arranged in population-specific groups. The analyses of variance (ANOVA and AMOVA) indicated significant morphological and genetic differentiation among populations (P<0.01). This study revealed low levels of AFLP variation, which suggests that conspicuous morphological variation of E. repens is caused by plasticity. E. repens is an evolutionarily young species, of hybrid origin, in which microevolutionary processes continue. This study showed that common analysis of genetic diversity and morphology is a powerful tool in low-level taxonomy.

Twenty-five AFLPs, previously linked to fertility restoration genes for the male-sterilizing PAMPA cytoplasm (cms-P) in a restricted rye population, were studied in an enlarged population of 120 plants. A strong association with the trait was verified for 19 of the markers. The recombination of these markers was tested and two linkage groups were identified: one consisting of six and the other of eight AFLPs. The remaining markers were segregated as independent loci. Using wheat-rye addition and substitution lines, the AFLPs were assigned to individual rye chromosomes. AFLP profiles of such lines were generated to identify the DNA fragments co-migrating with individual markers. This identified 1R and 3R as the two chromosomes corresponding to the linkage groups of eight and six markers, respectively. Mapping in a DS2 x RXL10 population linked four additional AFLPs to chromosome arms 1RS, 3RL, 4RL and 6RL. RAPD and SSR markers mapped in various populations and known to be located on the appropriate chromosomes did not disrupt the C394-F2 population into sterile and fertile phenotypes. It was concluded that the identified markers would reduce by one half the number of primer pair combinations needed for molecular breeding programs or for the selection of parental forms for rye hybrid crosses.

The aim of this study was to identify the genetic changes in rye seeds induced by natural aging during long-term storage and successive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples (cv. Dańkowskie Złote), varying in their initial viability and having gone through one or three reproduction cycles, were analysed using specific PCR targeting of a secalin locus, and various repetitive fragments defined by the R173 sequence. A statistical analysis of the band frequencies for both secalin and R173.3 primer pairs revealed no changes in their frequencies. Similar data on R173.1 demonstrated significant changes between samples of different initial viability showing a lack of a band of the expected length (987 bp) in progeny originating from low viability seeds lots. These changes were inherited even after three regeneration cycles. Our results may indicate that long-term storage that leads to loss of viability also generates heritable changes in the preserved germplasm. However, it remains to be discovered where these changes occur and whether they are connected with coding or with non-coding DNA regions.

The aim of these studies was to analyse the genetic changes induced by natural aging during long-term seed storage of rye. For this purpose, the AFLP (Amplified Fragment Length Polymorphism) technique was applied. In the experiment, DNA variation was demonstrated in seven-day-old seedlings of four seed samples of cv. Dańkowskie Złote, showing different levels of viability following long-term storage. Among the 362 AFLP fragments analysed, 73 had significantly different frequencies in at least one of the series. Principle Coordinate Analysis (PCA) based on molecular data revealed differences between the progenies of naturally aged seed samples with variable initial viability. It was clearly shown that materials with low viability differed in structure from highly viable ones, and that the population changes exhibited in the first case are preserved through regenerations. Although changes that were observed for initially viable samples were not so significant, they still occurred - probably as a result of genetic shift

Cytoplasmic male sterility (cms) in rye (Secale cereale L.), especially cytoplasma PAMPA, is used commercially in hybrid breeding programmes. The development of molecular markers that are tightly linked to the numerous genes coding for pollen fertility is expected to have great impact in the field. Morphological and cytological analyses of plants from a three-way cross C394: [(S67P/94 x S38/94) x CHD296] indicated the presence of at least several genes acting at different stages of pollen grain development, and proved the concurrence of both approaches in plant classification. The AFLP technique combined with the Bulk Segregant Analysis (BSA) were applied to identify DNA fragments linked to the genes of interest. All the 256 possible primer pair combinations based on the MseI and EcoRI restriction sites generated distinct band patterns allowing the identification of 31143 DNA fragments, visualised using the isotopic method. On average, any given primer combination generated 122 fragments. Among 1111 and 431 potential genetic markers respectively identified in the restorer form and the maternal lines, 775 and 295 were present in the F2 population. These numbers were then reduced to 109 and 51. The identified DNA fragments were tested on a limited segregating population, C394-F2, in order to eliminate false signals and to select markers for a future marker-assisted selection programme. Twenty-five markers were selected. Four of these markers were not identified via the BSA approach, indicating that if a highly polymorphic component is used for a cross, or a polygenic trait is studied, then the use of a limited population may be required.

Amplified fragment length polymorphism of DNA has been used to analyse the equality of plants obtained from isolated microspores. Although the control parental material was regarded as being highly homozygous, the analysis of the banding patterns of single plants showed a certain level of polymorphism. The analysis of regenerants with a doubled chromosome number did not show any diversity within the progeny of a single line. The differences in banding patterns coming from single plants were only observed in microspore donor lines. These results have proven the high purity of homozygous lines obtained via androgenesis from isolated microspores.