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Currently, production of wheat cultivars (Triticum aestivum L.) that show durable field resistance against fungal pathogens is a priority of many breeding programs. This type of resistance involves race-nonspecific mechanisms and can be identified at adult-plant stages. Until now, seven genes (Lr34/Yr18, Lr46/Yr29, Lr67/Yr46, Lr68, Lr75, Lr77 and Lr78) conferring durable types of resistance against multiple fungal pathogens have been identified in the wheat gene pool. In this study we showed a multiplex Polymerase Chain Reaction (multiplex PCR) assay, which was developed for detection of slow rusting resistance genes Lr34, Lr46, Lr68, using molecular markers: csLV34, Xwmc44 and csGS, respectively. Identification of molecular markers was performed on 40 selected wheat genotypes which are the sources of slow rusting genes according to literature reports. Multiplex PCR is an important tool to reduce the time and cost of analysis. This multiplex PCR protocol can be applicable for genotyping processes and marker assisted resistance breeding of wheat.

Background: Wheat is one of the most important crops cultivated all over the world. New high-yielding cultivars that are more resistant to fungal diseases have been permanently developed. The present study aimed at the possibility of accelerating the process of breeding new cultivars, resistant to eyespot, by using doubled haploids (DH) system supported by marker-assisted selection. Results: Two highly resistant breeding lines (KBP 0916 and KBH 4942/05) carrying Pch1 gene were crossed with the elite wheat genotypes. Hybrid plants of early generations were analyzed using endopeptidase EpD1 and two SSR markers linked to the Pch1 locus. Selected homozygous and heterozygous genotypes for the Pch1-linked EpD1b allele were used to produce haploid plants. Molecular analyses were performed on haploids to identify plants possessing Pch1 gene. Chromosome doubling was performed only on haploid plants with Pch1 gene. Finally, 65 DH lines carrying eyespot resistance gene Pch1 and 30 lines without this gene were chosen for the eyespot resistance phenotyping in a field experiment. Conclusions: Results of the experiment confirmed higher resistance to eyespot of the genotypes with Pch1 in comparison to those without this gene. This indicates the efficiency of selection at the haploid level.How to cite: Wiśniewska H, Majka M, Kwiatek M, et al. Production of wheat doubled haploids resistant to eyespot supported by marker-assisted selection. Electron J Biotechnol 2019;37. https://doi.org/10.1016/j.ejbt.2018.10.003 Keywords: Breeding, Crops, Doubled haploids, Eyespot, Gene expression, Marker-assisted selection, Molecular breeding, Triticum aestivum, Wheat cultivars, Wheat genotypes, Wheat

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.

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.

Eyespot is one of the most important fungal diseases of the stem base of wheat (Triticum aestivum L.). The presented study clearly demonstrated that the Pch1 gene was the main effective source for reducing the eyespot disease score in the analyzed winter wheat lines. Nevertheless, Pch1 was present only in 8−9% of the investigated lines. Using an isoenzymatic marker and molecular markers, the presence of the Pch1 gene and lack of the Pch2 gene was identified in six lines. Two lines, SMH 9409 and DL 358/13/4, were polymorphic in an isoenzymatic marker study. In the remaining three lines, C 3373/11-1, KBH 15.15 and KBP 1416, the Pch1 gene was identified only with the use of an isoenzymatic marker. Both genes Pch1 and Pch2, as well as the resistant variety Rendezvous, were found in three lines: DD 248/12, KBP 15.2 and STH 4431. In line DD 708/13, the presence of the Pch1 and Pch2 genes was identified, where the association between the Pch1 and the locus of the Xorw5 marker was broken. It was shown that the presence or absence of Pch1 and Pch2 genes did not significantly affect the grain yield (from the plot), although the yield was highest in the presence of both genes. A significant effect of the presence of the Pch1 gene on thousand kernel weight (TKW) was observed. Lines with the Pch1 gene showed significantly higher TKW values than lines without both genes or with the Pch2 gene only.

Fluorescent in situ hybridization (FISH) relies on fluorescent-labeled probes to detect specific DNA sequences in the genome, and it is widely used in cytogenetic analyses. The aim of this study was to determine the karyotype of T. aestivum and T. spelta hybrids and their parental components (three common wheat cultivars and five spelt breeding lines), to identify chromosomal aberrations in the evaluated wheat lines, and to analyze the distribution of polymorphisms of repetitive sequences in the examined hybrids. The FISH procedure was carried out with four DNA clones, pTa-86, pTa-535, pTa-713 and 35S rDNA used as probes. The observed polymorphisms between the investigated lines of common wheat, spelt and their hybrids was relatively low. However, differences were observed in the distribution of repetitive sequences on chromosomes 4A, 6A, 1B and 6B in selected hybrid genomes. The polymorphisms observed in common wheat and spelt hybrids carry valuable information for wheat breeders. The results of our study are also a valuable source of knowledge about genome organization and diversification in common wheat, spelt and their hybrids. The relevant information is essential for common wheat breeders, and it can contribute to breeding programs aimed at biodiversity preservation.

[This corrects the article DOI: 10.1371/journal.pone.0192862.].

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.

Triticale was used here as a model to recognize new components of molecular mechanism of resistance to Fusarium head blight (FHB) in cereals. Fusarium-damaged kernels of two lines distinct in levels of resistance to FHB were applied into a proteome profiling using two-dimensional gel electrophoresis (2-DE) to create protein maps and mass spectrometry to identify the proteins differentially accumulated between the analyzed lines. This proteomic research was supported by a measurement of alpha- and beta-amylase activities, mycotoxin content and fungal biomass in the analyzed kernels. The 2-DE analysis indicated a total of 23 spots with clear differences in a protein content between the more resistant and more susceptible triticale lines after infection with F. culmorum. A majority of the proteins were involved in a cell carbohydrate metabolism, stressing the importance of this protein group in a plant response to Fusarium infection. The increased accumulation levels of different isoforms of plant beta-amylase were observed for a more susceptible triticale line after inoculation but these were not supported by a total level of beta-amylase activity, showing the highest value in the control conditions. The more resistant line was characterized by a higher abundance of alpha-amylase inhibitor CM2 subunit and simultaneously a lower activity of alpha-amylase after inoculation. We suggest that the level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to Fusarium head blight.

Triticum genus encloses several tetraploid species that are used as genetic stocks for expanding the genetic variability of wheat (Triticum aestivum L.). Although the T. aestivum (2n = 6x = 42, AABBDD) and T. durum (2n = 4x = 28, AABB) karyotypes were well examined by chromosome staining, Giemsa C-banding and FISH markers, other tetraploids are still poorly characterized. Here, we established and compared the fluorescence in situ hybridization (FISH) patterns on chromosomes of 20 accessions of T. polonicum species using different repetitive sequences from BAC library of wheat 'Chinese Spring'. The chromosome patterns of Polish wheat were compared to tetraploid (2n = 4x = 28, AABB) Triticum species: T. durum, T. diccocon and T. turanicum, as well. A combination of pTa-86, pTa-535 and pTa-713 probes was the most informative among 6 DNA probes tested. Probe pTa-k374, which is similar to 28S rDNA sequence enabled to distinguish signal size and location differences, as well as rDNA loci elimination. Furthermore, pTa-465 and pTa-k566 probes are helpful for the detection of similar organized chromosomes. The polymorphisms of signals distribution were observed in 2A, 2B, 3B, 5B, 6A and 7B chromosomes. Telomeric region of the short arm of 6B chromosome was the most polymorphic. Our work is novel and contributes to the understanding of T. polonicum genome organization which is essential to develop successful advanced breeding strategies for wheat. Collection and characterization of this germplasm can contribute to the wheat biodiversity safeguard.

Numerous potential components involved in the resistance to Fusarium head blight (FHB) in cereals have been indicated, however, our knowledge regarding this process is still limited and further work is required. Two winter wheat (Triticum aestivum L.) lines differing in their levels of resistance to FHB were analyzed to identify the most crucial proteins associated with resistance in this species. The presented work involved analysis of protein abundance in the kernel bulks of more resistant and more susceptible wheat lines using two-dimensional gel electrophoresis and mass spectrometry identification of proteins, which were differentially accumulated between the analyzed lines, after inoculation with F. culmorum under field conditions. All the obtained two-dimensional patterns were demonstrated to be well-resolved protein maps of kernel proteomes. Although, 11 proteins were shown to have significantly different abundance between these two groups of plants, only two are likely to be crucial and have a potential role in resistance to FHB. Monomeric alpha-amylase and dimeric alpha-amylase inhibitors, both highly accumulated in the more resistant line, after inoculation and in the control conditions. Fusarium pathogens can use hydrolytic enzymes, including amylases to colonize kernels and acquire nitrogen and carbon from the endosperm and we suggest that the inhibition of pathogen amylase activity could be one of the most crucial mechanisms to prevent infection progress in the analyzed wheat line with a higher resistance. Alpha-amylase activity assays confirmed this suggestion as it revealed the highest level of enzyme activity, after F. culmorum infection, in the line more susceptible to FHB.