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.

Identification and Analysis of Candidate Genes Associated with Maize Fusarium Cob Resistance Using Next-Generation Sequencing Technology.

The pressure to reduce mineral fertilization and the amount of pesticides used has become a factor limiting production growth, as has the elimination of many crop protection chemicals from the market. A key condition for this to be an effective form of protection is the use of varieties with higher levels of resistance. The most effective and fastest way to assist in the selection and control of pathogens is the conducting of genome-wide association studies. These are useful tools for identifying candidate genes, especially when combined with QTL mapping to map and validate loci for quantitative traits. The aim of this study was to identify new markers coupled to genes that determine maize plant resistance to fusarium head blight through the use of next-generation sequencing, association and physical mapping, and to optimize diagnostic procedures to identify selected molecular markers coupled to plant resistance to this fungal disease. As a result of field experiments and molecular analyses, molecular markers coupled to potential genes for resistance to maize ear fusariosis were selected. The newly selected markers were tested against reference genotypes. As a result of the analyses, it was found that two markers (11801 and 20607) out of the ten that were tested differentiated between susceptible and resistant genotypes. Marker number 11801 proved to be the most effective, with a specious product of 237 bp appearing for genotypes 1, 3, 5, 9 and 10. These genotypes were characterized by a field resistance of 4-6 on the 9° scale (1 being susceptible, 9 being resistant) and for all genotypes except 16 and 20, which were characterized by a field resistance of 9. In the next step, this marker will be tested on a wider population of extreme genotypes in order to use it for the preliminary selection of fusarium-resistant genotypes, and the phosphoenolpyruvate carboxylase kinase 1 gene coupled to it will be subjected to expression analysis.

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.

The Use of DArTseq Technology to Identify Markers Related to the Heterosis Effects in Selected Traits in Maize.

Spectacular scientific advances in the area of molecular biology and the development of modern biotechnological tools have had a significant impact on the development of maize heterosis breeding. One technology based on next-generation sequencing is DArTseq. The plant material used for the research consisted of 13 hybrids resulting from the crossing of inbred maize lines. A two-year field experiment was established at two Polish breeding stations: Smolice and Lagiewniki. Nine quantitative traits were observed: cob length, cob diameter, core length, core diameter, number of rows of grain, number of grains in a row, mass of grain from the cob, weight of one thousand grains, and yield. The isolated DNA was subjected to DArTseq genotyping. Association mapping was performed using a method based on the mixed linear model. A total of 81602 molecular markers (28571 SNPs and 53031 SilicoDArTs) were obtained as a result of next-generation sequencing. Out of 81602, 15409 (13850 SNPs and 1559 SilicoDArTs) were selected for association analysis. The 105 molecular markers (8 SNPs and 97 SilicoDArTs) were associated with the heterosis effect of at least one trait in at least one environment. A total of 186 effects were observed. The number of statistically significant relationships between the molecular marker and heterosis effect varied from 8 (for cob length) and 9 (for yield) to 42 (for the number of rows of grain). Of particular note were three markers (2490222, 2548691 and 7058267), which were significant in 17, 8 and 6 cases, respectively. Two of them (2490222 and 7058267) were associated with the heterosis effects of yield in three of the four environments.

Identification and Analysis of Candidate Genes Associated with Yield Structure Traits and Maize Yield Using Next-Generation Sequencing Technology.

The main challenge of agriculture in the 21st century is the continuous increase in food production. In addition to ensuring food security, the goal of modern agriculture is the continued development and production of plant-derived biomaterials. Conventional plant breeding methods do not allow breeders to achieve satisfactory results in obtaining new varieties in a short time. Currently, advanced molecular biology tools play a significant role worldwide, markedly contributing to biological progress. The aim of this study was to identify new markers linked to candidate genes determining grain yield. Next-generation sequencing, gene association, and physical mapping were used to identify markers. An additional goal was to also optimize diagnostic procedures to identify molecular markers on reference materials. As a result of the conducted research, 19 SNP markers significantly associated with yield structure traits in maize were identified. Five of these markers (28629, 28625, 28640, 28649, and 29294) are located within genes that can be considered candidate genes associated with yield traits. For two markers (28639 and 29294), different amplification products were obtained on the electrophorograms. For marker 28629, a specific product of 189 bp was observed for genotypes 1, 4, and 10. For marker 29294, a specific product of 189 bp was observed for genotypes 1 and 10. Both markers can be used for the preliminary selection of well-yielding genotypes.

The Use of DArTseq Technology to Identify Markers Linked to Genes Responsible for Seed Germination and Seed Vigor in Maize.

Seed vigor and seed germination are very important traits, determined by several factors including genetic and physical purity, mechanical damage, and physiological condition, characterized by maintaining a high seed vigor and stable content after storage. The search for molecular markers related to improvement in seed vigor under adverse condition is an important issue in maize breeding currently. Higher sowing quality of seeds is necessary for the development of the agriculture production and better ability to resist all kinds of adversity in the seeds' storage. Condition is a very important factor affecting the yield of plants, thanks to the construction of their vitality. Identification of molecular markers associated with seed germination and seed vigor may prove to be very important in the selection of high-yielding maize varieties. The aim of this study was to identify and select new markers for maize (SNP and SilicoDArT) linked to genes influencing the seed germination and seed vigor in inbred lines of maize (Zea mays L.). The plant material used for the research was 152 inbred maize lines. The seed germination and seed vigor were analyzed. For identification of SNP and SilicoDArT markers related to the seed germination and seed vigor, the SilicoDarT technique developed by Diversity Arrays Technology was used. The analysis of variance indicated a statistically significant differentiation between genotypes for both observed traits. Positive (r = 0.41) correlation (p < 0.001) between seed germination and seed vigor was observed. As a result of next-generation sequencing, the molecular markers SilicoDArT (53,031) and SNP (28,571) were obtained. Out of 81,602 identified SilicoDArT and SNP markers, 15,409 (1559 SilicoDArT and 13,850 SNP) were selected as a result of association mapping, which showed them to be significantly related to the analyzed traits. The 890 molecular markers were associated with seed vigor, and 1323 with seed germination. Fifty-six markers (47 SilicoDArT and nine SNP) were significant for both traits. Of these 56 markers, the 20 most significant were selected (five of these markers were significant at the level of 0.001 for seed vigor and at the level of 0.05 for seed germination, another five markers were significant at the level of 0.001 for seed germination and at the level of 0.05 for seed vigor, five markers significant at the level of 0.001 only for seed vigor and five significant at the level of 0.001 only for seed germination also selected). These markers were used for physical mapping to determine their location on the genetic map. Finally, it was found that six of these markers (five silicoDArT­2,435,784, 4,772,587, 4,776,334, 2,507,310, 25,981,291, and one SNP­2,386,217) are located inside genes, the action of which may affect both seed germination and seed vigor. These markers can be used to select genotypes with high vigor and good seed germination.

Associative and Physical Mapping of Markers Related to Fusarium in Maize Resistance, Obtained by Next-Generation Sequencing (NGS).

On the basis of studies carried out in the last few years, it is estimated that maize diseases cause yield losses of up to 30% each year. The most dangerous diseases are currently considered to be caused by fungi of the genus Fusarium, which are the main culprits of root rot, ear rots, and stalk rot. Early plant infection causes grain diminution, as well as a significant deterioration in nutritional value and fodder quality due to the presence of harmful mycotoxins. Therefore, the aim of the research was to identify new markers of the SilicoDArT and SNP type, which could be used for the mass selection of varieties resistant to fusarium. The plant material consisted of 186 inbred maize lines. The lines came from experimental plots belonging to two Polish breeding companies: Plant Breeding Smolice Ltd., (Co., Kobylin, Poland). Plant Breeding and Acclimatization Institute-National Research Institute Group (51°41'23.16″ N, 17°4'18.241″ E), and Malopolska Plant Breeding Kobierzyce, Poland Ltd., (Co., Kobierzyce, Poland) (50°58'19.411″ N, 16°55'47.323″ E). As a result of next-generation sequencing, a total of 81,602 molecular markers were obtained, of which, as a result of the associative mapping, 2962 (321 SilicoDArT and 2641 SNP) significantly related to plant resistance to fusarium were selected. Out of 2962 markers significantly related to plant resistance in the fusarium, seven markers (SilicoDArT, SNP) were selected, which were significant at the level of 0.001. They were used for physical mapping. As a result of the analysis, it was found that two out of seven selected markers (15,097-SilicoDArT and 58,771-SNP) are located inside genes, on chromosomes 2 and 3, respectively. Marker 15,097 is anchored to the gene encoding putrescine N-hydroxycinnamoyltransferase while marker 58,771 is anchored to the gene encoding the peroxidase precursor 72. Based on the literature data, both of these genes may be associated with plant resistance to fusarium. Therefore, the markers 15,097 (SilicoDArT) and 58,771 (SNP) can be used in breeding programs to select lines resistant to fusarium.

The Use of DArTseq Technology to Identify New SNP and SilicoDArT Markers Related to the Yield-Related Traits Components in Maize.

In the last decade, many scientists have used molecular biology methods in their research to locate the grain-yield-determining loci and yield structure characteristics in maize. Large-scale molecular analyses in maize do not only focus on the identification of new markers and quantitative trait locus (QTL) regions. DNA analysis in the selection of parental components for heterotic crosses is a very important tool for breeders. The aim of this research was to identify and select new markers for maize (SNP and SilicoDArT) linked to genes influencing the size of the yield components in maize. The plant material used for the research was 186 inbred maize lines. The field experiment was established in twolocations. The yield and six yield components were analyzed. For identification of SNP and SilicoDArT markers related to the yield and yield components, next-generation sequencing was used. As a result of the biometric measurements analysis, differentiation in the average elevation of the analyzed traits for the lines in both locations was found. The above-mentioned results indicate the existence of genotype-environment interactions. The analysis of variance for the observed quality between genotypes indicated a statistically significant differentiation between genotypes and a statistically significant differentiation for all the observed properties betweenlocations. A canonical variable analysis was applied to present a multi-trait assessment of the similarity of the tested maize genotypes in a lower number of dimensions with the lowest possible loss of information. No grouping of lines due to the analyzed was observed. As a result of next-generation sequencing, the molecular markers SilicoDArT (53,031) and SNP (28,571) were obtained. The genetic distance between the analyzed lines was estimated on the basis of these markers. Out of 81,602 identified SilicoDArT and SNP markers, 15,409 (1559 SilicoDArT and 13,850 SNPs) significantly related to the analyzed yield components were selected as a result of association mapping. The greatest numbers of molecular markers were associated with cob length (1203), cob diameter (1759), core length (1201) and core diameter (2326). From 15,409 markers significantly related to the analyzed traits of the yield components, 18 DArT markers were selected, which were significant for the same four traits (cob length, cob diameter, core length, core diameter) in both Kobierzyce and Smolice. These markers were used for physical mapping. As a result of the analyses, it was found that 6 out of 18 (1818; 14,506; 2317; 3233; 11,657; 12,812) identified markers are located inside genes. These markers are located on chromosomes 8, 9, 7, 3, 5, and 1, respectively.

DArTseq-Based High-Throughput SilicoDArT and SNP Markers Applied for Association Mapping of Genes Related to Maize Morphology.

Today, agricultural productivity is essential to meet the needs of a growing population, and is also a key tool in coping with climate change. Innovative plant breeding technologies such as molecular markers, phenotyping, genotyping, the CRISPR/Cas method and next-generation sequencing can help agriculture meet the challenges of the 21st century more effectively. Therefore, the aim of the research was to identify single-nucleotide polymorphisms (SNPs) and SilicoDArT markers related to select morphological features determining the yield in maize. The plant material consisted of ninety-four inbred lines of maize of various origins. These lines were phenotyped under field conditions. A total of 14 morphological features was analyzed. The DArTseq method was chosen for genotyping because this technique reduces the complexity of the genome by restriction enzyme digestion. Subsequently, short fragment sequencing was used. The choice of a combination of restrictases allowed the isolation of highly informative low copy fragments of the genome. Thanks to this method, 90% of the obtained DArTseq markers are complementary to the unique sequences of the genome. All the observed features were normally distributed. Analysis of variance indicated that the main effect of lines was statistically significant (p < 0.001) for all 14 traits of study. Thanks to the DArTseq analysis with the use of next-generation sequencing (NGS) in the studied plant material, it was possible to identify 49,911 polymorphisms, of which 33,452 are SilicoDArT markers and the remaining 16,459 are SNP markers. Among those mentioned, two markers associated with four analyzed traits deserved special attention: SNP (4578734) and SilicoDArT (4778900). SNP marker 4578734 was associated with the following features: anthocyanin coloration of cob glumes, number of days from sowing to anthesis, number of days from sowing to silk emergence and anthocyanin coloration of internodes. SilicoDArT marker 4778900 was associated with the following features: number of days from sowing to anthesis, number of days from sowing to silk emergence, tassel: angle between the axis and lateral branches and plant height. Sequences with a length of 71 bp were used for physical mapping. The BLAST and EnsemblPlants databases were searched against the maize genome to identify the positions of both markers. Marker 4578734 was localized on chromosome 7, the closest gene was Zm00001d022467, approximately 55 Kb apart, encoding anthocyanidin 3-O-glucosyltransferase. Marker 4778900 was located on chromosome 7, at a distance of 45 Kb from the gene Zm00001d045261 encoding starch synthase I. The latter observation indicated that these flanking SilicoDArT and SNP markers were not in a state of linkage disequilibrium.