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.

Robertsonian translocations (RobTs) in the progeny of triticale (×Triticosecale Wittmack) plants with monosomic substitution of Aegilops kotschyi chromosome 2S k (2R) were investigated by fluorescence in-situ hybridization. Chromosome 2S k of Ae. kotschyi is reported to possess many valuable loci, such as Lr54 + Yr37 leaf and stripe (yellow) rust resistance genes. We used a standard procedure to produce RobTs, which consisted of self-pollination of monosomic triticale plants, carrying 2R and 2S k chromosomes in monosomic condition. This approach did not result in RobTs. Simultaneously, we succeeded in producing 11 plants carrying 2R.2S k compensatory RobTs using an alternative approach that utilized ditelosomic lines of triticale carrying 2RS (short arm) and 2RL (long arm) telosomic chromosomes. Identification of molecular markers linked to Lr54 + Yr37 genes in the translocation plants confirmed that these resources can be exploited in current triticale breeding programmes.Agronomy 2019, 9, 646 2 of 12 from wild relatives into the wheat genetic background [10]. Recently, several attempts were made to transfer rust resistance genes from Aegilops, Agropyron and Triticum species into triticale [11][12][13][14][15].Aegilops species are closely related to wheat (and triticale, per se) and carry a number of valuable traits, which have been effectively incorporated into wheat by developing wheat-Aegilops hybrids and deriving addition, substitution and translocation lines [16]. Aegilops kotschyi Boiss. (2n = 4x = 28 chromosomes, U-and S-genomes) is a wild tetraploid goatgrass native to Northern Africa, the Mid-East, and Western Asia. Ae. kotschyi germplasm is exploited in wheat breeding [17] as a source of high grain protein, iron and zinc [18]. Moreover, Antonov and Marais [19] observed leaf rust resistance that was effective against the infection of Puccinia triticina in Ae. kotschyi. Marais et al. [20] identified the Lr54 and Yr37 leaf rust and stripe rust resistance genes, and developed aT2DS.2S k L wheat-Ae. kotschyi translocation line. The first Lr54 + Yr37 marker was developed by Heyns et al. [21]. Moreover, translocation gene sequences were cloned and specific SSR markers were developed [22].Homoeologous recombination based engineering is the most common way for efficiently utilizing the wild relative gene pool for crop improvement [23]. The generation of translocation lines is the most promising pathway for the exploitation of alien germplasm in crop breeding [23]. In distant hybrids, unpaired chromosomes are present as univalents during meiosis. Monosomic chromosomes are prone to centric breaks at anaphase I of meiosis, which misdivide and the broken ends fuse during the interkinesis of meiosis II [24][25][26]. Fusion of the misdivided products may result in the formation of a Robertsonian translocation (RobT) [27].Several steps are required to generate RobTs (Figure 1a), with self-pollination of double-monosomic plants being the most common method used in the induction of RobTs [26]. Wheat breeders can use a l...

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 F 1 hybrid plants were obtained, and 29 of them carried both slow-rusting loci. Therefore, these hybrids may be used for triticale prebreeding program.

Ten leading wheat cultivars originating from the Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute (Poland) and the Department of Gene Bank (Czech Republic) were used to establish a field experiment in 2017 and 2018 at the Dłoń Experimental Farm. The analyzed wheat genotypes were characterized by diversified field resistance to leaf rust. Jubilatka, Thatcher and Sparta were the most resistant cultivars in field conditions in both 2017 and 2018. The aim of the work was to identify the Lr11, L13, Lr16 and Lr26 genes encoding resistance to leaf rust using molecular SSR markers (wmc24, wmc261, Xgwm630, Xwmc764 and P6M12) and to develop multiplex PCR conditions to accelerate identification of these genes. Markers of three leaf rust resistance genes have been identified simultaneously in these cultivars. Jubilatka, Thatcher and Sparta cultivars may serve as a good source of the analyzed leaf rust resistance genes. In addition, multiplex PCR conditions have been developed for the simultaneous identification of the Lr11 and Lr16 and Lr11 and Lr26 gene pairs.

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.

The aim of the study was to identify the Pm2, Pm3a, Pm4b and Pm6 genes and to develop multiplex PCR reaction conditions to reduce time and limit analysis costs. The following molecular markers were used for gene identification: Xcfd81, Whs350 and Xgwm205 (for Pm2), Pm3a (for Pm3a), STS_241 and Xgwm382 (for Pm4b), NAU/BCDSTS 135-2 (for Pm6). Plant material consisted of 7 popular European wheat varieties from the wheat collection at the Department of Genetics and Plant Breeding of the Poznań University of Life Sciences. The field experiment was established in 2017 and 2018 on 10 m2 plots in a randomized complete block design in three replicates in the Dłoń Agricultural Experimental Farm of the Poznań University of Life Sciences (51°41’23.835”N 017°4’1.414”E). The analyses demonstrated that the accumulation of all identified Pm genes was found in the Assosan variety. The accumulation of the Pm2, Pm4b and Pm6 genes was found in Atomic, Bussard, Lear, Sparta, Tonacja and Ulka varieties. The work also involved developing multiplex PCR conditions for Xcfd81 and STS_241 and Xcfd81 and Xgwm382 primer pairs, allowing the simultaneous identification of the Pm2 and Pm4b genes.

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.

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.