Diversity of Expression Patterns of Lr34, Lr67, and Candidate Genes towards Lr46 with Analysis of Associated miRNAs in Common Wheat Hybrids in Response to Puccinia triticina Fungus.

Leaf rust caused by Puccinia triticina (Pt) is one of the most dangerous diseases causing significant losses in common wheat crops. In adult plants resistant to rust, a horizontal adult plant resistance (APR) type is observed, which protects the plant against multiple pathogen races and is distinguished by greater persistence under production conditions. Crucial pleiotropic slow-rust genes such as Lr34, Lr46, Lr67, and Lr68, in combination with other genes of lesser influence, continue to increase durable resistance to rust diseases. Based on our previous results, we selected four candidate genes for Lr46 out of ten candidates and analysed them for expression before and after inoculation by P. triticina. As part of our study, we also investigated the expression patterns of miRNA molecules complementary to Lr34 and the candidate genes. The aim of the study was to analyse the expression profiles of candidate genes for the Lr46 gene and the Lr34 and Lr67 genes responsible for the differential leaf-rust resistance of hybrid forms of the F1 generation resulting from crosses between the Glenlea cultivar and cultivars from Polish breeding companies. In addition, the expression of five miRNAs (tae-miR9653b, tae-miR5384-3p, tae-miR9780, tae-miR9775 and tae-miR164), complementary to Lr34, and selected candidate genes were analysed using stem-loop RT-PCR and ddPCR. Biotic stress was induced in adult plants by inoculation with Pt fungal spores, under controlled conditions. Plant material was collected before and 6, 12, 24, and 48 h after inoculation (hpi). Differences in expression patterns of Lr34, Lr67, and candidate genes (for Lr46) were analysed by qRT-PCR and showed that gene expression changed at the analysed time points. Identification of molecular markers coupled to the Lr genes studied was also carried out to confirm the presence of these genes in wheat hybrids. qRT-PCR was used to examine the expression levels of the resistance genes. The highest expression of Lr46/Yr29 genes (Lr46-Glu2, Lr46-RLK1, Lr46-RLK2, and Lr46-RLK3) occurred at 12 and 24 hpi, and such expression profiles were obtained for only one candidate gene among the four genes analysed (Lr46-Glu2), indicating that it may be involved in resistance mechanisms of response to Pt infection.

Characteristic of the gene candidate SecARS encoding alkylresorcinol synthase in Secale.

BACKGROUND: Alkylresorcinols (ARs) are compounds belonging to the class of phenolic lipids. A rich source of ARs are cereal grains such as rye, wheat, triticale or barley. ARs found in plants are characterized by a variety of biological properties such as antimicrobial, antifungal and cytotoxic activity. Moreover, they are proven to have a positive influence on human health. Here, we aimed to find and characterize the gene with ARs synthase activity in the species Secale cereale. METHODS AND RESULTS: Using BAC library screening, two BAC clones containing the gene candidate were isolated and sequenced. Bioinformatic analyses of the resulting contigs were used to examine the structure and other features of the gene, including promoter, intron, 3'UTR and 5'UTR. Mapping using the FISH procedure located the gene on the 4R chromosome. Comparative analysis showed that the gene is highly similar to sequences coding for type III polyketide synthase. The level of gene expression in various parts of the plant was investigated, and the biochemical function of the gene was confirmed by heterologous expression in yeast. CONCLUSIONS: The conducted analyses contributed to a better understanding of the processes related to ARs synthesis. Although the research concerned the rye model, the knowledge gained may help in understanding the genetic basis of ARs biosynthesis in other species of the Poaceae family as well.

[Infectious complications in a course of multiple myeloma]. / Powiklania infekcyjne w przebiegu szpiczaka plazmocytowego.

Infections are the leading cause of mortality among patients with multiple myeloma (MM). This review presents etiology and epidemiological data concerning infectious complications in the course of MM, risk factors for its development and current indications for prophylaxis and treatment of infections in this group of patients.

[INHIBITORY EZH2 NOWA OPCJA TERAPEUTYCZNA W LECZENIU NAWROTOWEGO LUB OPORNEGO CHLONIAKA GRUDKOWEGO EZH2 INHIBITORS AS A NEW THERAPEUTIC OPTION FOR THE TREATMENT OF RELAPSED OR RECURRENT FOLLICULAR LYMPHOMA].

Follicular lymphoma (FL) is the most common of indolent non-Hodgkin's lymphomas. Its first-line treatment, based on immuno-chemotherapy with the anti-CD20 monoclonal antibody rituximab, is characterized by a high overall response rate to therapy. However, the disease is not curable in most cases and frequent relapses and transformations to other higher-grade lymphomas are observed. The effectiveness of the treatment of relapsed or refractory FL is not satisfactory, therefore the novel drugs are under investigation. Data from clinical trials with tazemetostat (an EZH2 inhibitor) in a group of patients with confirmed presence of the EZH2 mutation showed that this treatment may be an alternative to currently used chemotherapy regimens.

Expression patterns of candidate genes for the Lr46/Yr29 "slow rust" locus in common wheat (Triticum aestivum L.) and associated miRNAs inform of the gene conferring the Puccinia triticina resistance trait.

Leaf rust caused by Puccinia triticina (Pt) is one of the most impactful diseases causing substantial losses in common wheat (Triticum aestivum L.) crops. In adult plants resistant to Pt, a horizontal adult plant resistance (APR) is observed: APR protects the plant against multiple pathogen races and is distinguished by durable persistence under production conditions. The Lr46/Yr29 locus was mapped to chromosome 1B of common wheat genome, but the identity of the underlying gene has not been demonstrated although several candidate genes have been proposed. This study aimed to analyze the expression of nine candidate genes located at the Lr46/Yr29 locus and their four complementary miRNAs (tae-miR5384-3p, tae-miR9780, tae-miR9775, and tae-miR164), in response to Pt infection. The plant materials tested included five reference cultivars in which the molecular marker csLV46G22 associated with the Lr46/Yr29-based Pt resistance was identified, as well as one susceptible control cultivar. Biotic stress was induced in adult plants by inoculation with fungal spores under controlled conditions. Plant material was sampled before and at 6, 12, 24, 48 hours post inoculation (hpi). Differences in expression of candidate genes at the Lr46/Yr29 locus were analyzed by qRT-PCR and showed that the expression of the genes varied at the analyzed time points. The highest expression of Lr46/Yr29 candidate genes (Lr46-Glu1, Lr46-Glu2, Lr46-Glu3, Lr46-RLK1, Lr46-RLK2, Lr46-RLK3, Lr46-RLK4, Lr46-Snex, and Lr46-WRKY) occurred at 12 and 24 hpi and such expression profiles were obtained only for one candidate gene among the nine genes analyzed (Lr46-Glu2), indicating that it may be a contributing factor in the resistance response to Pt infection.

Molecular, physicochemical and rheological characteristics of introgressive Triticale/Triticum monococcum ssp. monococcum lines with wheat 1D/1A chromosome substitution.

Three sets of hexaploid introgressive triticale lines, with Triticum monococcum ssp. monococcum (cultivated einkorn wheat) genes and a bread wheat chromosome 1D substituted for chromosome 1A, and one set of secondary triticale lines were evaluated for grain and flour physicochemical and dough rheological characteristics in two generations (F7 and F8). Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) confirmed the 1D/1A chromosome substitution. The presence or absence of einkorn high-molecular-weight (HMW) glutenin subunits and the wheat Glu-D1d locus encoding the 5 + 10 subunits was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary zone electrophoresis, and allele-specific molecular markers. Significant differences were found among physicochemical properties (with the exception of the Hagberg falling number) of all introgressive Triticale/T. monococcum lines and the secondary triticale lines. The wheat 1D/1A chromosome substitution also affected these properties. The results showed that in all introgressive triticale lines, the protein and gluten content, Zeleny sedimentation value, and water absorption capacity, were increased. The rheological parameters estimated using micro-farinograph, reomixer, and Kieffer dough extensibility systems also showed an appreciable increase in dough-mixing properties, maximum resistance to extension (Rmax), and dough extensibility. Introgressive Triticale/T. monococcum lines with 5 + 10 subunits have particularly favorable rheological parameters. The results obtained in this study suggest that the cultivated einkorn genome Am, in the context of hexaploid secondary triticale lines and with a wheat 1D/1A substitution, has the potential to improve gluten polymer interactions and be a valuable genetic resource for triticale quality improvement.

Multiplex PCR assay for the simultaneous identification of race specific and non-specific leaf resistance genes in wheat (Triticum aestivum L.).

Race-nonspecific resistance is a key to sustainable management of pathogens in bread wheat (Triticum aestivum L.) breeding. It is more durable compared to race-specific immunity, conferred by the major genes (R), which are often overcome by pathogens. The accumulation of the genes, which provide the resistance to a specific race of a pathogen, together with the introduction of race-non-specific resistance genes is the most effective strategy aimed at preventing the breakdown of genetically conditioned immunity. PCR markers improved the productivity and accuracy of classical plant breeding by means of marker-assisted selection (MAS). Multiplexing assays provide increased throughput, reduced reaction cost, and conservation of limited sample material, which are beneficial for breeding purposes. Here, we described the process of customizing multiplex PCR assay for the simultaneous identification of the major leaf rust resistance genes Lr19, Lr24, Lr26, and Lr38, as well as the slow rusting, race-nonspecific resistance genes: Lr34 and Lr68, in thirteen combinations. The adaptation of PCR markers for multiplex assays relied on: (1) selection of primers with an appropriate length; (2) selection of common annealing/extension temperature for given primers; and (3) PCR mixture modifications consisting of increased concentration of primers for the scanty band signals or decreased concentration of primers for the strong bands. These multiplex PCR protocols can be integrated into a marker-assisted selection of the leaf rust-resistant wheat genotypes.

Expression Profiling of the Slow Rusting Resistance Genes Lr34/Yr18 and Lr67/Yr46 in Common Wheat (Triticum aestivum L.) and Associated miRNAs Patterns.

The main efforts in common wheat (Triticum aestivum L.) breeding focus on yield, grain quality, and resistance to biotic and abiotic stresses. One of the major threats affecting global wheat cultivation and causing significant crop production losses are rust diseases, including leaf rust caused by a biotrophic fungus Puccinia triticina Eriks. Genetically determined resistance to leaf rust has been characterized in young plants (seedling resistance) as well as in plants at the adult plant stage. At the seedling stage, resistance is controlled vertically by major R genes, conferring a race-specific response that is highly effective but usually short-lived due to the rapid evolution of potentially virulent fungi. In mature plants, horizontal adult plant resistance (APR) was described, which provides long-term protection against multiple races of pathogens. A better understanding of molecular mechanisms underlying the function of APR genes would enable the development of new strategies for resistance breeding in wheat. Therefore, in the present study we focused on early transcriptomic responses of two major wheat APR genes, Lr34 and Lr67, and three complementary miRNAs, tae-miR9653b, tae-miR9773 and tae-miR9677b, to inoculation with P. triticina. Plant material consisted of five wheat reference varieties, Artigas, NP846, Glenlea, Lerma Rojo and TX89D6435, containing the Lr34/Yr18 and Lr67/Yr46 resistance genes. Biotic stress was induced by inoculation with fungal spores under controlled conditions in a phytotron. Plant material consisted of leaf tissue sampled before inoculation as well as 6, 12, 24 and 48 h postinoculation (hpi). The APR gene expression was quantified using real-time PCR with two reference genes, whereas miRNA was quantified using droplet digital PCR. This paper describes the resistance response of APR genes to inoculation with races of leaf rust-causing fungi that occur in central Europe. The study revealed high variability of expression profiles between varieties and time-points, with the prevalence of downregulation for APR genes and upregulation for miRNAs during the development of an early defense response. Nevertheless, despite the downregulation initially observed, the expression of Lr34 and Lr67 genes in studied cultivars was significantly higher than in a control line carrying wild (susceptible) alleles.

Novel Tetraploid Triticale (Einkorn Wheat × Rye)-A Source of Stem Rust Resistance.

Among cereals, triticale (×Trititcoseale Wittmack ex A. Camus) represents a number of advantages such as high grain yield even in marginal environments, tolerance to drought, cold and acid soils, as well as lower production costs. Together with high biomass of grain and straw, triticale is also considered as an industrial energy crop. As an artificial hybrid, it has not evolved naturally, which is reflected in narrow genetic diversity causing a resistance collapse in recent years. Here, we describe a novel, synthetic tetraploid triticale, which was developed by the crossing of rye (Secale cereale L.) with einkorn wheat (Triticum monococcum spp. monococcum), which possess Sr35 stem rust resistance gene. Three subsequent generations of alloploids were obtained by chromosome doubling followed by self-pollination. The cytogenetic analyses revealed that the amphiploids possess a set of 28 chromosomes (14 of Am-genome and 14 of R-genome). The values of the most important yield-shaping traits for these tetraploid triticale form, including thousand-grain weight, plant height and stem length were higher compared to parental genotypes, as well as standard hexaploid triticale cultivars. This study shows that this tetraploid triticale genetic stock can be an interesting pre-breeding germplasm for triticale improvement or can be developed as a new alternative crop.

Diagnostic accuracy of genetic markers for identification of the Lr46/Yr29 "slow rusting" locus in wheat (Triticum aestivum L.).

Wheat leaf rust, caused by fungal pathogen Puccinia triticina Erikss, annually contributes to production losses as high as 40% in susceptible varieties and remains as one of the most damaging diseases of wheat worldwide. Currently, one of the major challenges of wheat geneticists and breeders is to accumulate major genes for durability of rust resistance called "slow rusting" genes using marker-assisted selection (MAS). Until now, eight genes (Lr34/Yr18, Lr46/Yr29, Lr67/Yr46, Lr68, Lr74, Lr75, Lr77, and Lr78) conferring resistance against multiple fungal pathogens have been identified in wheat gene pool and the molecular markers were developed for them. In MAS practice, it is a common problem that cultivars exhibiting desirable marker genotypes may not necessarily have the targeted genes or alleles and vice versa, which is known as "false positives." The aim of this study was to compare the available four markers: Xwmc44, Xgwm259, Xbarc80, and csLV46G22 markers (not published yet), for the identification of the Lr46/Yr29 loci in 73 genotypes of wheat, which were reported as sources of various "slow rusting" genes, including 60 with confirmed Lr46/Yr29 gene, reported in the literature. This research revealed that csLV46G22 together with Xwmc44 is most suitable for the identification of resistance allele of the Lr46/Yr29 gene; however, there is a need to clone the Lr46/Yr29 loci to identify and verify the allelic variation of the gene and the function.

Cytomolecular analysis of mutants, breeding lines, and varieties of camelina (Camelina sativa L. Crantz).

Camelina sativa L. Crantz (Brassicaceae family), known as camelina, has gained new attention as a re-emerging oil seed crop. With a unique seed oil profile, with the majority of the fatty acids consisting of linolenic (C18:3), oleic (C18:1), linoleic (C18:2), and eicosenoic (C20:1), camelina oil is reported to be useful as a food oil and biofuel. However, there are still many unknown factors about the structure and genetic variability of this crop. Chromosomal localization of ribosomal DNA was performed using fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as molecular probes on mitotic chromosomes of enzymatically digested root-tip meristematic cells. Here, we present for the first time a comparative analysis of selected genotypes (cultivars, breeding lines and mutants) of C. sativa with the use of cytogenetic techniques. The main aim of the study was to determine the intraspecific and interspecific polymorphisms in the structure of chromosomes of selected accessions using conserved 5S and 25S rDNA repetitive sequences as molecular probes. The results were compared with C. microcarpa (closely related to C. sativa) rDNA gene loci distribution. The presence of minor rDNA sites was discussed and compared with other Brassicaceae species. In addition, demonstration karyograms of C. sativa and C. microcarpa mapped with rDNA probes were prepared based on the cv. "Przybrodzka" and GE2011-02 genotype, respectively. The use of 5S and 25S rDNA probes provided an insight on the genome structure of C. sativa at the cytogenetic level and can help to understand the genome organization of this crop. The putative role of cytogenetic markers in phylogenetic analyses of camelina was discussed, as well.

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.

Identification of leaf rust resistance genes Lr34 and Lr46 in common wheat (Triticum aestivum L. ssp. aestivum) lines of different origin using multiplex PCR.

Leaf rust caused by the fungus Puccinia recondita f. sp. tritici is one of the most dangerous diseases of common wheat. Infections caused by fungal pathogens reduce the quantity and quality of yields of many cereal species. The most effective method to limit plant infection is to use cultivars that show rust resistance. Genetically conditioned horizontal-type resistance (racial-nonspecific) is a desirable trait because it is characterized by more stable expression compared to major (R) genes that induce racially specific resistance, often overcome by pathogens. Horizontal resistance is conditioned by the presence of slow rust genes, which include genes Lr34 and Lr46. This study aimed to identify markers linked to both genes in 64 common wheat lines and to develop multiplex PCR reaction conditions that were applied to identify both genes simultaneously. The degree of infestation of the analyzed lines was also assessed in field conditions during the growing season of 2017 and 2018. Simple sequence repeat anchored-polymerase chain reaction (SSR-PCR) marker csLV was identified during analysis in line PHR 4947. The presence of a specific sequence has also been confirmed in multiplex PCR analyses. In addition to gene Lr34, gene Lr46 was identified in this genotype. Lines PHR 4947 and PHR 4819 were characterized by the highest leaf rust resistance in field conditions. During STS-PCR analyses, the marker wmc44 of gene Lr46 was identified in most of the analyzed lines. This marker was not present in the following genotypes: PHR 4670, PHR 4800, PHR 4859, PHR 4907, PHR 4922, PHR 4949, PHR 4957, PHR 4995, and PHR 4997. The presence of a specific sequence has also been confirmed in multiplex PCR analyses. Genotypes carrying the markers of the analyzed gene showed good resistance to leaf rust in field conditions in both 2017 and 2018. Research has demonstrated that marker assisted selection (MAS) and multiplex PCR techniques are excellent tools for selecting genotypes resistant to leaf rust.

Dependence of the Heterosis Effect on Genetic Distance, Determined using Various Molecular Markers.

A number of studies have shown that the greater the genetic diversity of parental lines, the greater the heterosis effect. Genetic or phenotypic variation can be estimated by genotype testing on the basis of the observations obtained through prediction (a priori) or the observations and studies (a posteriori). The first method uses data such as the genealogy of a given subject and the information about its geographical origin. The second method is based on the phenotypic observation and studies, as well as on the molecular research. The development of molecular genetics and genotype testing methods at the DNA level has made it possible to rapidly assess the genetic variability regardless of the modifying effect of the environment. The aim of this study was to determine the relationship between the degree of relatedness and the DNA polymorphism (determined using AFLP, RAPD, and SSR markers) of inbred maize lines and the effect of hybrid-form heterosis. Our analysis demonstrated that the parental components for heterosis crosses can be selected on the basis of the genetic similarity determined using the molecular SSR markers and the Jaccard, Kluczynski, Nei, and Rogers coefficients. Molecular AFLP markers proved less useful for selecting the parental components, but may be used to group lines with incomplete origin data. In the case of the RAPD markers, no clear relationship between genetic distance and the heterosis effect was found in this study.

Molecular identification of slow rusting resistance Lr46/Yr29 gene locus in selected triticale (× Triticosecale Wittmack) cultivars.

Recently, leaf rust and yellow rust caused by the fungi Puccinia triticina Erikss. and P. striiformis Westend f. sp. tritici Eriks and Henn are diseases of increasing threat in triticale (× Triticosecale Wittmack, AABBRR, 2n = 6x = 42) growing areas. The use of genetic resistance is considered the most economical, effective and environmentally friendly method to control the disease and minimize the use of fungicides. Currently, breeding programs mainly relied on race-specific Lr and Yr genes (R), but new races of the rust fungi frequently defeat resistance. There is a small group of genes that causes partial type of resistance (PR) that are characterized by a slow epidemic build up despite a high infection type. In wheat slow rusting resistance genes displayed longer latent periods, low infection frequencies, smaller pustule size and less spore production. Slow rusting Lr46/Yr29 gene, located on chromosome 1B, is being exploited in many wheat breeding programs. So far, there is no information about slow rusting genes in triticale. This paper showed significant differences between the results of identification of wheat molecular markers Xwmc44 and csLV46G22 associated with Lr46/Yr29 in twenty triticale cultivars, which were characterized by high levels of field resistance to leaf and yellow rust. The csLV46G22res marker has been identified in the following cultivars: Kasyno, Mamut and Puzon. Belcanto and Kasyno showed the highest resistance levels in three-year (2016-2018), leaf and yellow rust severity tests under post-registration variety testing program (PDO). Leaf tip necrosis, a phenotypic trait associated with Lr34/Yr18 and Lr46/Yr29 was observed, among others, to Belcanto and Kasyno, which showed the highest resistance for leaf rust and yellow rust. Kasyno could be considered to have Lr46/Yr29 and can be used as a source of slow rust resistance in breeding and importantly as a component of gene pyramiding in triticale.

Development of Triticale × Wheat Prebreeding Germplasm With Loci for Slow-Rusting Resistance.

There is a growing interest in breeding and production of hexaploid triticale (× Triticosecale Wittmack ex A. Camus) in European Union and in the world. It is reported that triticale can be an alternative to wheat (Triticum aestivum L.) for livestock feed production and has a potential to become preferred industrial energy crop. Fungal diseases, mainly leaf and stripe rusts, are the limiting factors of triticale growth and yield. Geneticists and breeders are now focusing on accumulation of the major genes for durability of rust resistance. Slow-rusting genes Lr34/Yr18 and Lr46/Yr19 are being exploited in many wheat breeding programs. This type of horizontal resistance is reported to be effective over space and time. Classical breeding techniques supported by marker-assisted selection (MAS) are the main tools in breeding programs. The aim of this study was to assess the possibility of transfer of slow-rusting genes from resistant genotypes of wheat into hexaploid triticale through cross-hybridizations. A total of 5,094 manual pollinations were conducted between two triticale cultivars Fredro and Twingo and 33 accessions of common wheat, which were reported as sources of slow-rusting resistance genes. The investigation of the slow-rusting gene transmission was performed using both molecular markers analyses and genomic in situ hybridization (GISH). In total, 34 F1 hybrid plants were obtained, and 29 of them carried both slow-rusting loci. Therefore, these hybrids may be used for triticale prebreeding program.

Development and Cytomolecular Identification of Monosomic Alien Addition and Substitution Lines of Triticale (×Triticosecale Wittmack) With 2Sk Chromosome Conferring Leaf Rust Resistance Derived From Aegilops kotschyi Boiss.

Alien chromosome introgression has become a valuable tool to broaden the genetic variability of crop plants via chromosome engineering. This study details the procedure to obtain monosomic addition and monosomic substitution lines of the triticale carrying 2Sk chromosome from Aegilops kotchyi Boiss., which harbors Lr54 + Yr37 leaf and stripe rust-resistant gene loci, respectively. Initially, A. kotschyi × Secale cereale artificial amphiploids (2n = 6x = 42 chromosomes, UUSSRR) were crossed with triticale cv. "Sekundo" (2n = 6x = 42, AABBRR) in order to obtain fertile offspring. Cyto-molecular analyses of five subsequent backcrossing generations revealed that 2Sk chromosome was preferentially transmitted. This allowed for the selection of monosomic 2Sk addition (MA2Sk) lines of triticale. Finally, the 2Sk(2R) substitution plants were obtained by crossing MA2Sk with the nullisomic (N2R) plants of triticale. The presence of 2Sk chromosome in subsequent generations of plants was evaluated using SSR markers linked to Lr54 + Yr37 loci. Disease evaluation of the monosomic 2Sk(2R) substitution plants for the reaction to leaf and stripe rust infection were carried out under controlled conditions in a growth chamber. The results showed significant improvement of leaf rust resistance severity of monosomic substitution plants compared with control ("Sekundo"). In contrast, the introgression of the Lr54 + Yr37 loci did not lead to improvement of stripe rust resistance. In summary, the creation of monosomic addition and monosomic substitution lines of triticale is the starting point for the precise and guided transfer of Lr54 + Yr37 loci. The results showed that the developed materials could be exploited for the development of triticale varieties with resistance to leaf rust.

Analysis of miRNA expression associated with the Lr46 gene responsible for APR resistance in wheat (Triticum aestivum L.).

Lr46/Yr29/Pm39 (Lr46) is a gene for slow rusting resistance in wheat. The aim of the study was to analyze the miRNA expression in selected common wheat cultivars carrying resistance genes, Lr46 among others (HN Rod, Pavon'S', Myna'S', Frontana'S', and Sparrow'S') in response to leaf rust infection caused by Puccinia triticina Erikss. In the Pavon 'S', Myna 'S', Frontana'S', and Sparow'S' varieties a product with a length of 242 bp has been identified, which is specific to the Xwmc44 marker linked to the brown rust resistance gene Lr46. In the next step, the differences in the expression of microRNA (miR5085 and miR164) associated with the Lr46 gene, which is responsible for different resistance of selected wheat cultivars to leaf rust, were examined using emulsion PCR (ddPCR). In the experiment, biotic stress was induced in mature plants by infecting them with fungal spores under controlled conditions in a growth chamber. For analysis the plant material was collected before inoculation and 6, 12, 24, and 48 h after inoculation. The experiments also showed that plant infection with Puccinia triticina resulted in an increase in miR164 expression in cultivars carrying the Lr46 gene. The expression of miR164 remained stable in a control cultivar (HN ROD) lacking this gene. This has proved that miR164 can be involved in leaf rust resistance mechanisms.

Development of multiplex PCR to detect slow rust resistance genes Lr34 and Lr46 in wheat.

Leaf rust caused by Puccinia triticina belongs to one of the most dangerous fungal diseases of wheat (Triticum aestivum L.) and is the cause of large yield losses every year. Here we report a multiplex polymerase chain reaction (PCR) assay, which was developed for detection of two important wheat slow rust resistance genes Lr34 and Lr46, using two molecular markers: csLV34 and Xwmc44, respectively. The presence of genes was analyzed in one winter wheat variety TX89D6435 and five spring wheat varieties: Pavon F76, Parula 'S', Rayon 89, Kern, Mochis 88. Both Lr34 and Lr46 genes were identified in variety TX89D6435, gene Lr34 was also identified in Parula 'S' and Kern varieties, and gene L46 occurs in Pavon F76 and Mochis 88 variety. None of the resistance genes tested was detected in the Rayon 89 variety. The use of the multiplex PCR method allowed to shorten the analysis time, reduce costs of analyses, and reduce the workload.

Comparison of the Androgenic Response of Spring and Winter Wheat (Triticum aestivum L.).

Androgenesis is potentially the most effective technique for doubled haploid production of wheat. It is not however widely used in breeding programmes due to its main limitation: the genotype dependence. Due to genetic differences between spring and winter wheat, it was assumed that both phenotypes are different in their capacity to conduct androgenesis. And so, the aim of this investigation was to verify the effectiveness of androgenesis induction and plant regeneration of spring and winter wheat genotypes while considering varying amounts of growth hormones in the induction medium. Fifteen genotypes of spring wheat and fifteen of winter wheat were used in the experiment. Six hundred anthers of each of the 30 genotypes were plated and analysed. Previous studies have allowed selection of the best medium for wheat androgenesis and a combination of growth hormones that are the most effective in stimulating microspore proliferation. Therefore, C17 induction media with two combinations of growth hormones were used: I-supplemented only by auxins (2,4-D and dicamba), and II-supplemented by auxin and cytokinin (2,4-D and kinetin). Data was recorded according to the efficiency of androgenic structure formation (ASF), green plant regeneration (GPR), and albino plant regeneration (APR). The results showed that the induction and regeneration of androgenesis in the spring wheat were more efficient than in the winter ones. The spring genotypes formed more androgenic structures and green plants on anthers plated on the medium supplemented only by auxins, in contrast to the winter genotypes which were better induced and regenerated on the medium supplemented by auxin and cytokinin. The study showed that to increase the efficiency of androgenesis, it is necessary to select appropriate factors such as concentration and type of hormones in medium composition, affecting the course of the culturing procedure according to the winter or spring phenotype of donor plants.