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Wykorzystanie technologii NGS, mapowania asocjacyjnego i fizycznego w celu identyfikacji genów kandydujących związanych z plonem kukurydzy
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.
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.
Transcriptomic Characterization of Candidate Genes for Fusarium Resistance in Maize (Zea mays L.)
Fusarium diseases are among the most dangerous fungal diseases of plants. To date, there are no plant protectants that completely prevent fusariosis. Current breeding trends are therefore focused on increasing genetic resistance. While global modern maize breeding relies on various molecular genetics techniques, they are useless without a precise characterization of genomic regions that determine plant physiological responses to fungi. The aim of this study was thus to characterize the expression of candidate genes that were previously reported by our team as harboring markers linked to fusarium resistance in maize. The plant material included one susceptible and four resistant varieties. Biotic stress was induced in adult plants by inoculation with fungal spores under controlled conditions. qRT-PCR was performed. The analysis focused on four genes that encode for GDSL esterase/lipase (LOC100273960), putrescine hydroxycinnamyltransferase (LOC103649226), peroxidase 72 (LOC100282124), and uncharacterized protein (LOC100501166). Their expression showed differences between analyzed time points and varieties, peaking at 6 hpi. The resistant varieties consistently showed higher levels of expression compared to the susceptible variety, indicating their stronger defense responses. Moreover, to better understand the function of these genes, their expression in various organs and tissues was also evaluated using publicly available transcriptomic data. Our results are consistent with literature reports that clearly indicate the involvement of these genes in the resistance response to fusarium. Thus, they further emphasize the high usefulness of the previously selected markers in breeding programs to select fusarium-resistant maize genotypes.
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.
Using Genome-Wide Association Studies to Reveal DArTseq and SNP Loci Associated with Agronomic Traits and Yield in Maize
Next-generation sequencing (NGS) has revolutionized genetic research, enabling the massive, rapid, and relatively inexpensive analysis of the genomes, transcriptomes, and epigenomes of various organisms, including maize. Therefore, this paper uses NGS, association mapping, and physical mapping to identify candidate genes associated with yield structure traits and yield in maize (Zea mays L.). Furthermore, expression analysis of selected candidate genes was performed to confirm their contribution to yield formation. The plant material used for the study was 186 F1 hybrids and 20 reference genotypes (high-yielding and low-yielding). Field experiments were conducted simultaneously in two locations (in Smolice and Kobierzyce). NGS yielded a total of 45,876 molecular markers (24,437 SilicoDArT markers and 21,439 SNP markers) relevant to yield and crop structure. The largest number of markers in both localities (Smolice and Kobierzyce) was related to: the number of grain rows (6960), dry matter content after harvest (6616), the number of grains in a row (6721), mass of grain from the cob (6616), and cob length (6564). The smallest number of markers in both localities was related to yield (t ha−1) (1114) and yield from the plot (1237). To narrow down the number of markers for physical mapping, ten were selected from all the significant ones associated with the same traits in both localities (Kobierzyce and Smolice). Significant markers included eight silicoDArT markers (459199, 2447305, 4768759, 4579916, 4764335, 2448946, 2492509, 4774802) and two SNP markers (9692004, 5587791). These markers were used for physical mapping. These markers are located on chromosomes 7, 8, and 10. Some of these markers are located at a considerable distance from characterized genes or within uncharacterized genes. Two markers caught our attention: SNP 5587791 and silicoDArT 4774802. The first one is located on chromosome 8 inside exon 5 of the LOC100383455 U-box domain-containing protein 7 gene, the second marker is also located on chromosome 8 near (300 bp) the LOC103635953 putative WUSCHEL-related homeobox 2 protein gene. Our own research and literature reports indicate the usefulness of next-generation sequencing, association mapping, and physical mapping for identifying candidate genes associated with economically important traits in maize. Furthermore, two genes characterized in detail in the publication, LOC100383455 U-box domain-containing protein 7 gene and LOC103635953 putative WUSCHEL-related homeobox 2 protein gene, may be involved in processes related to maize yield.
Molecular selection of soybean towards adaptation to Central European agroclimatic conditions
AbstractEurope is highly dependent on soybean meal imports and anticipates an increase of domestic plant protein production. Ongoing climate change resulted in northward shift of plant hardiness zones, enabling spring-sowing of freezing-sensitive crops, including soybean. However, it requires efficient reselection of germplasm adapted to relatively short growing season and long-day photoperiod. In the present study, a PCR array has been implemented, targeting early maturity (E1–E4, E7, E9, and E10), pod shattering (qPHD1), and growth determination (Dt1) genes. This array was optimized for routine screening of soybean diversity panel (204 accessions), subjected to the 2018–2020 survey of phenology, morphology, and yield-related traits in a potential cultivation region in Poland. High broad-sense heritability (0.84–0.88) was observed for plant height, thousand grain weight, maturity date, and the first pod height. Significant positive correlations were identified between the number of seeds and pods per plant, between these two traits and seed yield per plant as well as between flowering, maturity, plant height, and first pod height. PCR array genotyping revealed high genetic diversity, yielding 98 allelic combinations. The most remarkable correlations were identified between flowering and E7 or E1, between maturity and E4 or E7 and between plant height and Dt1 or E4. The study demonstrated high applicability of this PCR array for molecular selection of soybean towards adaptation to Central Europe, designating recessive qPHD1 and dominant Dt1, E3, and E4 alleles as major targets to align soybean growth season requirements with the length of the frost-free period, improve plant performance, and increase yield.
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.
Analiza molekularnych mechanizmów odporności wybranych odmian pszenicy zwyczajnej w odpowiedzi na porażenie przez rdzę brunatną.
Identification of SNP and SilicoDArT Markers and Characterization of Their Linked Candidate Genes Associated with Maize Smut Resistance
The implementation of biological advancements in agricultural production is the response to the needs of the agricultural sector in the 21st century, enabling increased production and improved food quality. Biological progress in the maize breeding and seed industries is unique in terms of their social and ecological innovation aspects. It affects agricultural productivity and the adaptation of cultivated maize varieties to market demands and changing climate conditions without compromising the environment. Modern maize resistance breeding relies on a wide range of molecular genetic research techniques. These technologies enable the identification of genomic regions associated with maize smut resistance, which is crucial for characterizing and manipulating these regions. Therefore, the aim of this study was to identify molecular markers (SilicoDArT and SNP) linked to candidate genes responsible for maize smut resistance, utilizing next-generation sequencing, as well as association and physical mapping. By using next-generation sequencing (NGS) and statistical tools, the analyzed maize genotypes were divided into heterotic groups, which enabled the prediction of the hybrid formula in heterosis crosses. In addition, 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. Among the selected markers, 61 were highly statistically significant (LOD > 2.3). Among the selected 61 highly statistically significant markers (LOD > 2.3), 10 were significantly associated with plant resistance to maize smut in two locations (Smolice and Kobierzyce). Of the 10 selected markers, 3 SilicoDArT (24016548, 2504588, 4578578) and 3 SNP (4779579, 2467511, 4584208) markers were located within genes. According to literature reports, of these six genes, three (ATAD3, EDM2, and CYP97A3) are characterized proteins that may play a role in the immune response that develops in response to corn smut infection. In the case of genotypes belonging to the same origin groups, markers linked to these genes can be used to select varieties resistant to corn smut. These markers will also be tested on genotypes belonging to other maize origin groups to demonstrate their universality.
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.
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.
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 high-throughput DArTseq-based silicoDArT and SNP markers to identify yellow rust resistance genes in common wheat
Analiza genetycznych uwarunkowań związanych z efektem heterozji oraz odpornością na fuzarium u kukurydzy (Zea mays L.).
Pasta with Kiwiberry (Actinidia arguta): Effect on Structure, Quality, Consumer Acceptance, and Changes in Bioactivity during Thermal Treatment
In this study, kiwiberry lyophilizate (KBL) was incorporated into pasta at different levels (5%, 10%, and 15% w/w). Kiwiberry fruits’ characteristics (ascorbic acid, carotenoids, phenolic compounds, and antioxidant activity determination) as well as physical (cooking properties, color, microscopic structure determination, texture, and water molecular dynamics analysis by low-field NMR) and chemical analyses (proximate composition phenolic compounds composition and antioxidant activity) of KBL-enriched pasta were investigated. The replacement of semolina with KBL in the production of pasta significantly changed its culinary properties. Results showed that the addition of KBL leads to a reduction in optimal cooking time and cooking weight (47.6% and 37.3%, respectively). Additionally, a significant effect of the KBL incorporation on the color of both fresh and cooked pasta was observed. A significant reduction in the L* value for fresh (27.8%) and cooked (20.2%) pasta was found. The KBL-enriched pasta had a different surface microstructure than the control pasta and reduced firmness (on average 44.7%). Low-field NMR results have confirmed that the ingredients in kiwiberry fruit can bind the water available in fresh pasta. The heat treatment resulted in increasing the availability of phenolic compounds and the antioxidant activity (64.7%) of cooked pasta. Sensory evaluation scores showed that the use of 5–10% of the KBL additive could be successfully accepted by consumers.
Development and application of duplex and triplex assays for simultaneous detection of resistance genes to leaf rust, Fusarium head blight, powdery mildew, Septoria tritici blotch, eyspot, stem rust and yellow rust in wheat
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 Łagiewniki. 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.
