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Using NGS technology and association mapping to identify candidate genes associated with fusarium stalk rot resistance
Stalk rot caused by Fusarium fungi is one of the most widespread and devastating diseases of maize, and the introduction of resistant genotypes is one of the most effective strategies for controlling the disease. Breeding genotypes with genetically determined resistance will also allow less use of crop protection products. The aim of the research was to identify molecular markers and associated candidate genes determining maize plant resistance to Fusarium stalk rot. The plant material for this study consisted of 122 maize hybrids. The experiment was conducted in two localities: Smolice and Kobierzyce. The Fusarium stalk rot values ranged from 1.65% (for genotype G01.10) to 31.18% (for genotype G03.07) in Kobierzyce and from 0.00% (for 58 genotypes) to 6.36% (G05.03) in Smolice. The analyzed genotypes were simultaneously subjected to next-generation sequencing using the Illumina platform. Illumina sequencing identified 60,436 SilicoDArT markers and 32,178 SNP markers (92,614 in total). For association mapping, 32,900 markers (26,234 SilicoDArT and 6666 SNP) meeting the criteria (MAF > 0.25 and the number of missing observations <10%) were used. The results of the observation of the degree of infection and sequencing were used for association mapping, which ultimately resulted in the selection of ten molecular markers important at both places. Among the identified markers, two SNP markers that are located inside candidate genes play an important role. Marker 4772836 is located inside the serine/threonine-protein kinase bsk3 gene, while marker 4765764 is located inside the histidine kinase 1 gene. Both genes can be associated with plant resistance to Fusarium stalk rot, and these genes can also be used in breeding programs to select resistant varieties.
Genetic Parameters for Selected Traits of Inbred Lines of Maize (Zea mays L.)
This paper presents an estimation of the parameters connected with the additive (a) effect, additive by additive (aa) epistatic effect, and additive by additive by additive (aaa) interaction gene effect for nine quantitative traits of maize (Zea mays L.) inbred lines. To our knowledge, this is the first report about aaa interaction of maize inbred lines. An analysis was performed on 252 lines derived from Plant Breeding Smolice Ltd. (Smolice, Poland)—Plant Breeding and Acclimatization Institute-National Research Institute Group (151 lines) and Małopolska Plant Breeding Ltd. (Kobierzyce, Poland) (101 lines). The total additive effects were significant for all studied cases. Two-way and three-way significant interactions were found in most analyzed cases with a considerable impact on phenotype. Omitting the inclusion of higher-order interactions effect in quantitative genetics may result in a substantial underestimation of additive QTL effects. Expanding models with that information may also be helpful in future homozygous line crossing projects.
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 silicoDArT—2,435,784, 4,772,587, 4,776,334, 2,507,310, 25,981,291, and one SNP—2,386,217) 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.
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.
Analysis of Linkage on Interaction of Main Aspects (Genotype by Environment Interaction, Stability and Genetic Parameters) of 1000 Kernels in Maize (Zea mays L.)
The assessment of 1000-kernel weight holds significant importance in determining maize grain yield, and elucidating its underlying genetic mechanisms is imperative for enhancing its overall performance. The material for the study consisted of 26 doubled-haploid (DH) maize lines obtained from crossing two cultivars with flint kernels. Lines were planted in the northern part of the Lower Silesia voivodship in Poland over ten years (2013–2022). The 1000-kernel weight was assessed. The purposes of the research were as follows: (1) to assess genotype by environment interaction (GEI by the additive main effects and multiplicative interaction (AMMI) model; (2) the selection of stable DH lines and environment-specific lines; and (3) the estimation of parameters related to additive and additive–additive gene interaction (epistasis). The results indicate the significant effects of genotype and environment, as well as the GEI, on the 1000-kernel weight. Estimates of additive gene action effects were statistically significant in every year of the study, except 2022. Estimates of epistasis (total additive-by-additive interaction) effects for 1000-kernel weight were statistically significant in 2013, 2015, and 2017 (positive effects), as well as in 2018 and 2020 (negative effects). The lines KN07 and KN10 are recommended for further inclusion in the breeding program due to their stability and highest average of 1000-kernel weight.
Observation of changes in Fusarium mycotoxin profiles in maize grain over the last decade in Poland
Transcriptomic Characterization of Genes Harboring Markers Linked to Maize Yield
Background: It is currently believed that breeding priorities, including maize breeding, should focus on introducing varieties with greater utility value, specifically higher yields, into production. Global modern maize breeding relies on various molecular genetics techniques. Using the above mentioned technologies, we can identify regions of the genome that are associated with various phenotypic traits, including yield, which is of fundamental importance for understanding and manipulating these regions. Objectives: The aim of the study was to analyze the expression of candidate genes associated with maize yield. To better understand the function of the analyzed genes in increasing maize yield, their expression in different organs and tissues was also assessed using publicly available transcriptome data. Methods: RT-qPCR analyses were performed using iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each of the performed RT-qPCR experiments consisted of three biological replicates and three technical replicates, the results of which were averaged. Results: The research results allowed us to select three out of six candidate genes (cinnamoyl-CoA reductase 1—CCR1, aspartate aminotransferase—AAT and sucrose transporter 1—SUT1), which can significantly affect grain yield in maize. Not only our studies but also literature reports clearly indicate the participation of CCR1, AAT and SUT1 in the formation of yield. Identified molecular markers located within these genes can be used in breeding programs to select high yielding maize genotypes.
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 Małopolska 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.
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.
Studies of Oat-Maize Hybrids Tolerance to Soil Drought Stress
The ontogenesis and yield formation in crop plants are modified by environmental conditions. Due to climatic change detected over two decades, the harmful influence of abiotic factors is increasing. One of the most threatening issues reducing plant productivity is drought stress. The strength of plant response to water shortages could differ depending on the strength of the drought stress, type of crop, genetic background, presence of additional stresses, and stage of plant development. There are examples of sexual hybridization between crop plants like oat (Avena sativa L.) and maize (Zea mays L.) with which stable fertile hybrids were generated. Additional maize chromosomes in oat plants (oat × maize addition, OMA) often infer morphological and physiological (e.g., PS II photosystem activity and chlorophyll production) changes modulated by the interaction of certain maize chromosomes added to the oat genome. The aim of the research was to evaluate the chosen physiological, biochemical, and agronomic parameters of OMA plants subjected to soil drought. Analysis of variance indicated that the main effects of genotype as well as treatment × genotype interaction were significant for all the traits studied (photosynthetic pigment content, selected PSII indices, mass of stem, number of grains/plant, mass of grains/plant). Most of the examined lines severely reduced PSII photosystem parameters, pigment content, and yield-related traits under drought stress. The results indicated that two lines (9 and 78b) retained high yielding potential under drought stress compared to commercial cv. Bingo.
Wykorzystanie technologii NGS, mapowania asocjacyjnego i fizycznego w celu identyfikacji genów kandydujących związanych z plonem kukurydzy
Meta-Analysis of Influence of Diversity of Parental Forms on Heterosis and Specific Combining Ability of Their Hybrids
An important stage in any breeding activity is selection of suitable individuals for further breeding. Thus, the main goal of breeders becomes such a selection of parental forms that leads to the consolidation and maximization of the value of traits of significant utility and economic importance. Heterosis and specific combining ability are very important parameters in plant and animal breeding. The ability to predict their value and relevance could significantly shorten the breeding process. One way to predict the effects of heterosis and specific combining ability is to select parental forms for crosses. This selection can be made on the basis of variation in parental forms. An analysis was made of publicly available data that contain information about the effects of heterosis, the effects of specific combining ability, and phenotypic and genetic diversity of parental forms. Preliminary studies show that the best approach for obtaining favorable hybrids would be selection of parental forms that are very genetically diverse while being phenotypically equal.
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.
Effect of Soil Drought Stress on Selected Biochemical Parameters and Yield of Oat × Maize Addition (OMA) Lines
Plant growth and the process of yield formation in crops are moderated by surrounding conditions, as well as the interaction of the genetic background of plants and the environment. In the last two decades, significant climatic changes have been observed, generating unfavorable and harmful impacts on plant development. Drought stress can be considered one of the most dangerous environmental factors affecting the life cycle of plants, reducing biomass production and, finally, the yield. Plants can respond to water deficit in a wide range, which depends on the species, genetic variability within the species, the plant’s ontogenesis stage, the intensity of the stress, and other potential stress factors. In plants, it is possible to observe hybrids between different taxa that certain traits adopted to tolerate stress conditions better than the parent plants. Oat × maize addition (OMA) plants are good examples of hybrids generated via wide crossing. They can exhibit morphological, physiological, and biochemical variations implemented by the occurrence of extra chromosomes of maize, as well as the interaction of maize and oat chromatin. The initial goal of the study was to identify OMA lines among plants produced by wide crossing with maize. The main goal was to investigate differences in OMA lines according to the Excised Leaf Water Loss (ELWL) test and to identify specific biochemical changes and agronomic traits under optimal water conditions and soil drought. Additionally, detection of any potential alterations that are stable in F2 and F3 generations. The aforementioned outcomes were the basis for the selection of OMA lines that tolerate growth in an environment with limited water availability. The molecular analysis indicated 12.5% OMA lines among all tested descendants of wide oat-maize crossing. The OMA lines significantly differ according to ELWL test results, which implies some anatomical and physiological adaptation to water loss from tissues. On the first day of drought, plants possessed 34% more soluble sugars compared to control plants. On the fourteen day of drought, the amount of soluble sugars was reduced by 41.2%. A significant increase of phenolic compounds was observed in the fourteen day of drought, an average of 6%, even up to 57% in line 9. Soil drought substantially reduced stem biomass, grains number, and mass per plant. Lower water loss revealed by results of the ELWL test correlated with the high yield of OMA lines. Phenolic compound content might be used as a biochemical indicator of plant drought tolerance since there was a significant correlation with the high yield of plants subjected to soil drought.
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.