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Diagnostic accuracy of genetic markers for identification of the Lr46/Yr29 “slow rusting” locus in wheat (Triticum aestivum L.)
Abstract 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.
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.
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.
Analysis of miRNA expression associated with gene Lr34 responsible for resistance mechanisms to wheat leaf rust
Structural Polymorphisms of Chromosome 3Am Containing Lr63 Leaf Rust Resistance Loci Reflect the Geographical Distribution of Triticum monococcum L. and Related Diploid Wheats
Wheat is one of the world’s crucial staple food crops. In turn, einkorn wheat (Triticum monococcum L.) is considered a wild relative of wheat (Triticum aestivum L.) and can be used as a source of agronomically important genes for breeding purposes. Cultivated T. monococcum subsp. monococcum originated from T. monococcum subsp. aegilopoides (syn. T. boeticum). For the better utilization of valuable genes from these species, it is crucial to discern the genetic diversity at their cytological and molecular levels. Here, we used a fluorescence in situ hybridization toolbox and molecular markers linked to the leaf rust resistance gene Lr63 (located on the short arm of the 3Am chromosome—3AmS) to track the polymorphisms between T. monococcum subsp. monococcum, T. boeticum and T. urartu (A-genome donor for hexaploid wheat) accessions, which were collected in different regions of Europe, Asia, and Africa. We distinguished three groups of accessions based on polymorphisms of cytomolecular and leaf rust resistance gene Lr63 markers. We observed that the cultivated forms of T. monococcum revealed additional marker signals, which are characteristic for genomic alternations induced by the domestication process. Based on the structural analysis of the 3AmS chromosome arm, we concluded that the polymorphisms were induced by geographical dispersion and could be related to adaptation to local environmental conditions.
Analiza molekularna genów warunkujących odporność poziomą u pszenicy (Triticum aestivum L.) na porażenie przez grzyby patogeniczne z rodzaju Puccinia sp.
The use of high-throughput DArTseq-based silicoDArT and SNP markers to identify yellow rust resistance genes in common wheat
Unraveling Effects of miRNAs Associated with APR Leaf Rust Resistance Genes in Hybrid Forms of Common Wheat (Triticum aestivum L.)
The fungus Puccinia triticina Eriks (Pt) is the cause of leaf rust, one of the most damaging diseases, which significantly reduces common wheat yields. In Pt-resistant adult plants, an APR-type resistance is observed, which protects the plant against multiple pathogen races and is distinguished by its persistence under production conditions. With a more complete understanding of the molecular mechanisms underlying the function of APR genes, it will be possible to develop new strategies for resistance breeding in wheat. Currently, mainly APR genes, such as Lr34, Lr46, and Lr67, are principally involved in resistance breeding as they confer durable resistance to multiple fungal races occurring under different climatic and environmental conditions. However, the mechanisms underlying the defence against pathogens mediated by APR genes remain largely unknown. Our research aimed to shed light on the molecular mechanisms related to resistance genes and miRNAs expression, underlying APR resistance to leaf rust caused by Pt. Furthermore, the present study aimed to identify and functionally characterize the investigated miRNAs and their target genes in wheat in response to leaf rust inoculation. The plant material included hybrid forms of wheat from the F2 and BC1F1 generations, obtained by crossing the resistance cultivar Glenlea (CItr 17272) with agriculturally important Polish wheat cultivars. Biotic stress was induced in adult plants via inoculation with Pt fungal spores under controlled conditions. The RT-qPCR method was used to analyze the expression profiles of selected APR genes at five time points (0, 6, 12, 24, and 48 hpi). The results presented here demonstrate the differential expression of APR genes and miRNAs at stages of leaf rust development at selected timepoints after inoculation. We analyzed the expression of three leaf rust resistance genes, using different genetic backgrounds in F2 and BC1F1 segregation materials, in leaf tissues after Pt infection. Our goal was to investigate potential differences resulting from the genetic background found in different generations of hybrid forms of the same parental forms. Gene ontology analysis predicted 190 target genes for tae-miR5384-3p and 167 target genes for tae-miR9653b. Our findings revealed distinct expression profiles for genes, with the highest expression levels observed mainly at 6, 24, and 48 hpi. The candidate gene Lr46-Glu2 displayed an upregulation, suggesting its potential involvement in the immune response against Pt infection.
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.
Application of Artificial Neural Network Sensitivity Analysis to Identify Key Determinants of Harvesting Date and Yield of Soybean (Glycine max [L.] Merrill) Cultivar Augusta
Genotype and weather conditions play crucial roles in determining the volume and stability of a soybean yield. The aim of this study was to identify the key meteorological factors affecting the harvest date (model M_HARV) and yield of the soybean variety Augusta (model M_YIELD) using a neural network sensitivity analysis. The dates of the start of flowering and maturity, the yield data, the average daily temperatures and precipitation were collected, and the Selyaninov hydrothermal coefficients were calculated during a fifteen-year study (2005–2020 growing seasons). During the experiment, highly variable weather conditions occurred, strongly modifying the course of phenological phases in soybean and the achieved seed yield of Augusta cultivar. The harvesting of mature soybean seeds took place between 131 and 156 days after sowing, while the harvested yield ranged from 0.6 t·ha−1 to 2.6 t·ha−1. The sensitivity analysis of the MLP neural network made it possible to identify the factors which had the greatest impact on the tested dependent variables among all the analyzed factors. It was revealed that the variables assigned ranks 1 and 2 in the sensitivity analysis of the neural network forming the M_HARV model were total rainfall in the first decade of June and the first decade of August. The variables with the highest impact on the Augusta soybean seed yield (model M_YIELD) were the mean daily air temperature in the second decade of May and the Seljaninov coefficient values calculated for the sowing–flowering date period.
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.
Application Marker-Assisted Selection (MAS) and Multiplex PCR Reactions in Resistance Breeding of Maize (Zea mays L.)
Cultivated maize (Zea mays L.) is the oldest and one of the most important crop species in the world. Changing climatic conditions in recent years, warm weather, expansion of acreage and intensification of maize cultivation have resulted in an increase in the threat posed by diseases caused by, among others, Fusarium fungi. Breeding success in all plant species is determined by access to starting materials with possible high genetic diversity also in terms of disease resistance. Identification of parental combinations that produce offspring that are high-yielding and resistant to Fusarium, among other diseases, is one of the costliest steps in breeding programs. We used maize lines which, as a result of five-year field observations, were divided into resistant and susceptible to F. verticillioides. It is known that resistance to fusarium is a trait strongly dependent on environmental conditions. Due to the fact that the years of observation of the degree of infestation were hot and dry, the resistance of some lines could result from favorable environmental conditions. In view of the above, the aim of this study was to analyze the genetic basis of the resistance of these lines and to correlate molecular analyses with field observations. Comprehensive field and molecular analyses will allow the selection of reference lines that will be resistant to fusarium in the field and, at the same time, will have pyramidized resistance genes. Such lines can be used for crossbreeding to obtain fusarium-resistant varieties. In addition, an attempt was made to develop Multiplex PCR conditions for faster identification of the analyzed markers. As a result of the analyses, it was found that the resistance of the studied maize lines was correlated with the number of molecular markers identified in them. Both field and laboratory analyses have shown that the best line that can be used for crossbreeding as a source of fusarium resistance genes is the line number 25. It has a resistance level of 8–9 on the nine-point COBORU scale. In this line, as a result of molecular analyses, 10 out of 12 markers were identified (SSR 85, Bngl 1063, Bngl 1740, Umc 2082, Bngl 1621, Umc 2059, Umc 2013, SSR 93, SSR 105, STS 03) related to fusarium resistance genes, which may be the reason for such a high resistance to this pathogen. Similarly, 9 markers were identified for line number 35 (SSR 85, Bngl 1063, Bngl 1740, Umc 2082, Bngl 1621, Umc 2059, Umc 2013, SSR 93, STS 03). This line, however, was characterized by a slightly lower resistance at the level of 7–8. Line 254 turned out to be the least resistant, as the resistance was at the level of 4–5, and the number of identified molecular markers was 5. Lines numbered 25 and 35 can be successfully used as a source of fusarium resistance genes.