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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.
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.
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 Identification of Novel Molecular Markers for the Detection of Fusarium Resistance in Maize
Modern maize breeding worldwide relies on a broad range of molecular genetics research techniques. These technologies allow us to identify genomic regions associated with various phenotypic traits, including resistance to fungi of the genus Fusarium. Therefore, the aim of this publication was to identify new molecular markers linked to candidate genes that confer maize resistance to Fusarium fungi, using next-generation sequencing, association mapping, and physical mapping. In the study, a total of 5714 significant molecular markers related to maize plant resistance to Fusarium fungi were identified. Of these, 10 markers were selected that were significantly associated (with the highest LOD values) with the disease. These markers were identified on chromosomes 5, 6, 7, 8, and 9. The authors were particularly interested in two markers: SNP 4583014 and SilicoDArT 4579116. The SNP marker is located on chromosome 5, in exon 8 of the gene encoding alpha-mannosidase I MNS5. The SilicoDArT marker is located 240 bp from the gene for peroxisomal carrier protein on chromosome 8. Our own research and the presented literature review indicate that both these genes may be involved in biochemical reactions triggered by the stress caused by plant infection with Fusarium fungal spores. Molecular analyses indicated their role in resistance processes, as resistant varieties responded with an increase in the expression level of these genes at various time points after plant inoculation with Fusarium fungal spores. In the negative control, which was susceptible to Fusarium, no significant fluctuations in the expression levels of either gene were observed. Analyses concerning the identification of Fusarium fungi showed that the most abundant fungi on the infected maize kernels were Fusarium poae and Fusarium culmorum. Individual samples were very sparsely colonized by Fusarium or not at all. By using various molecular technologies, we identified genomic regions associated with maize resistance to Fusarium fungi, which is of fundamental importance for understanding these regions and potentially manipulating them.