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.

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.

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.

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.

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.

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.

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.

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.

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.