Landessaatzuchtanstalt

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Improving host resistance to Fusarium head blight in wheat (Triticum aestivum L.) and Gibberella ear rot in maize (Zea mays L.)
(2023) Akohoue, Félicien; Miedaner, Thomas
Fusarium head blight (FHB) in wheat and Fusarium (FER) and Gibberella ear rot (GER) in maize are major cereal diseases which reduce yield and contaminate kernels with several mycotoxins. In Europe, these diseases contribute to significant yield gaps and high mycotoxin risks across countries. However, existing management strategies related to agronomic practices are not fully effective, with some of them being cost-prohibitive. Enhancing host plant resistance is additionally required for managing the diseases more effectively and sustainably. Unfortunately, breeding for FHB resistance is challenged by complex interactions with morphological traits and the quantitative nature of the trait. In maize, available genetic resources have not been fully exploited to improve GER resistance in elite materials. In this work, we elucidated the complex interactions between FHB resistance and morphological traits, like plant height (PH) and anther retention (AR) in wheat. The effect of reduced height (Rht) gene Rht24 on AR and the contribution of genomic background (GB) to FHB resistance in semi-dwarf genotypes were also assessed. GB refers to all genomic loci, except major Rht genes, that affect the traits. To achieve this, 401 winter wheat cultivars were evaluated across five environments (location × year combination). All cultivars were genotyped using Illumina 25 K Infinium single-nucleotide polymorphism array. We performed correlation and path coefficient analysis, and combined single and multi-trait genome-wide association studies (GWAS). Our findings revealed significant genotypic correlations and path effects between FHB severity with PH and AR, which were controlled by several pleiotropic loci. FHB severity and PH shared both negatively and positively acting pleiotropic loci, while only positively acting pleiotropic loci were detected between FHB severity and AR. Rht-D1 is a major pleiotropic gene which exerted a negative effect on FHB resistance. These pleiotropic loci contribute to our understanding of the complex genetic basis of FHB resistance, and their exploitation can help to simultaneously select for FHB resistance with PH and AR. Contrary to Rht-D1b, Rht24b had no negative effect on FHB resistance and AR. This exhibits Rht24 as an important FHB-neutral Rht gene which can be integrated into breeding programs. Genomic estimated breeding values (GEBV) were calculated for each cultivar to assess GB. We observed highly negative GEBV for FHB severity within resistant wheat cultivars. Susceptible cultivars exhibited positive GEBV. Genomic prediction has a great potential and can be exploited by selecting for semi-dwarf winter wheat genotypes with higher FHB resistance due to their genomic background resistance. To tackle maize ear rot diseases, refined and stable quantitative trait loci (QTL) harboring candidate genes conferring resistances to FER and GER were identified. The effectiveness of introgression of two European flint landraces, namely “Kemater Gelb Landmais” (KE) and “Petkuser Ferdinand Rot” (PE) was evaluated. The prediction accuracy of using line performance as a predictor of hybrid performance for GER resistance was also evaluated within the two landraces. We applied a meta-QTL (MQTL) analysis based on 15 diverse SNP-based QTL mapping studies and performed gene expression analysis using published RNA-seq data on GER resistance. In total, 40 MQTL were identified, of which 14 most refined MQTL harbored promising candidate genes for use in breeding programs for improving FER and GER resistances. 28 MQTL were common to both FER and GER, with most of them being shared between silk (channel) and kernel resistances. This highlights the co-inheritance of FER and GER resistances as well as types of active resistance. Resistance genes can be transferred into elite cultivars by integrating refined MQTL into genomics-assisted breeding strategies. Afterwards, four GER resistant doubled haploid (DH) lines from both KE and PE landraces were crossed with two susceptible elite lines to generate six bi-parental populations with a total of 534 DH lines which were evaluated for GER resistance. GER severity within the six landrace-derived populations were reduced by 39−61% compared to the susceptible elite lines. Moderate to high genetic advance was observed within each population, and the use of KE landrace as a donor was generally more effective than PE landrace. This shows promise in enhancing resistance to GER in elite materials using the European flint landraces as donors. Furthermore, per se performance of 76 DH lines from both landraces was used to predict GER resistance of their corresponding testcrosses (TC). Moderate phenotypic and genomic prediction accuracy between TC and line per se performance was found for GER resistance. This implies that pre-selecting lines for GER resistance is feasible; however, TC should be additionally tested on a later selection stage to aim for GER-resistant hybrid cultivars.
Deciphering the potential of large-scale proteomics to improve product quality and nutritional value in different wheat species
(2022) Afzal, Muhammad; Longin, Friedrich
Wheat (Triticum aestivum) is one of the most important staple crops globally, which provides on average ~20% of the dietary intake of protein, starch and further important ingredients like fiber, minerals, vitamins, and essential amino acids for humans. Besides common wheat, there exist further wheat species with global to only local importance, i.e., durum, spelt, emmer and einkorn. Common wheat and durum are relatively widely cultivated whereas the other three species are cultivated only in specific regions. Apart from other functions, wheat proteins largely influence the end-use quality of products such as bread and pasta quality. Furthermore, wheat proteins can induce inflammatory reactions in humans such as celiac disease, wheat allergy and non-celiac wheat sensitivity. Thus, proteome profiles of different wheat species and cultivars within these species are of high relevance for stakeholders along the wheat supply chain. Proteomic technology has made breakthrough advancements in the recent times capable of quantifying thousands of proteins in 1.5–2 hours. Also, the wheat reference genome has been published and extended recently. These developments are extremely helpful in studying the wheat proteome at a high resolution. However, the modern large-scale proteomics has yet neither been applied to perform comparative investigation of the proteomes of different wheat species nor to study the proteomes of different types of breads and flours nor to study its application in the context of plant breeding. Therefore, we utilized modern large-scale proteomics to fill these gaps within the framework of this PhD work. First of all, an optimized data analysis pipeline was designed to deal with big proteomics data. This was necessary to estimate a multitude of quantitative genetics parameters for each protein and perform a comparative investigation of the proteomes. Optimization included implementation of data filtering based on the quantification of a protein in a given proportion of the samples, cultivars and environments. Different tests such as test for normal distribution of each protein in the context of statistical modelling and test to check the equality of variance between groups to apply the appropriate t-test were incorporated into a semi-automated workflow. In parallel, we adjusted and improved the lab methodology to deal with hundreds of samples within a short time period. We introduced a novel hybrid liquid chromatography-mass spectrometry (LC-MS) approach that combines quantification concatamer (QconCAT) technology with short microflow LC gradients and data-independent acquisition (DIA). The proposed approach measures the proteome by label-free quantification (LFQ) while concurrently providing accurate QconCAT-based absolute quantification of the key amylase/trypsin inhibitors (ATIs). These methods were then applied to compare different wheat species based on dozens of cultivars grown at multiple locations. First, we compared common wheat and spelt and identified 3,050 proteins overall. Of total proteins, 1,555 proteins in spelt and 1,166 in common wheat were only detected in a subset of the field locations. There were 1,495 and 1,604 proteins in spelt and common wheat, respectively, which were consistently expressed across all test locations in at least one cultivar. Finally, there were 84 and 193 unique proteins for spelt and common wheat, respectively, as well as 396 joint proteins, which were significantly differentially expressed between the two species. Using potentially allergenic proteins – annotated as amylase/trypsin inhibitors, serpins, and wheat germ agglutinin – we calculated an equally weighted “allergen index” that largely varied across cultivars ranging from –13.32 to 10.88 indicating the potential to select for cultivars with favorable proteome profiles. Next, we examined the proteomes of six different flours (wholegrain and superfine flours) and 14 different bread types (yeast and sourdough fermented breads and common wheat breads plus/minus bread improver) from common wheat, spelt and rye. Proteins that could cause allergies were functionally classified and comparatively measured by LFQ in flours and breads. Our findings showed that allergenic proteins were more prevalent in common wheat and spelt than rye and were not specifically degraded during bread manufacturing. In terms of abundance of the allergenic proteins, there was almost no difference between spelt and common wheat and the type of grain is likely more important for allergenicity than milling or traditional fermentation techniques. In a further study, we generated the flour reference proteomes for five wheat species, identifying at least 2,540 proteins in each species. More than 50% of the proteins significantly differed between species. Particularly, einkorn expressed 5.4 and 7.2 times less allergens and amylase/trypsin inhibitors than common wheat, respectively, emerging as a potential alternative cereal crop for people with sensitivities to cereal allergens. Lastly, we studied the application of large-scale proteomics for plant breeding. We found a significant impact of the environmental factors on protein expression. Only a fraction of proteins was stably expressed in all environments in at least one cultivar. Environmental influence was observed not only in the form of absolute expression or suppression of a certain protein at one or more environments but also in the form of low heritability (H2). High coefficients of variation across wheat cultivars indicate that the protein profiles of different cultivars vary considerably. Although, heritability was low for many proteins, we were able to identify hundreds of proteins with H²>0.5 – including key proteins for baking quality and human health. It should be possible to specifically manipulate the expression of functionally important proteins with high heritability by selecting and breeding for superior wheat cultivars along the wheat supply chain. Nevertheless, a successful implementation in plant breeding programs needs an improvement in the speed of protein quantification methods and in the validation of protein functions and annotations. In a nutshell, high number of proteins can be quantified in cereal grains utilizing cutting-edge proteomics techniques, opening new avenues for their use in the wheat supply chain. We generated lists of intriguing candidate proteins for further investigations on wheat sensitivity, and proteins with high heritability and important biological functions. Current research work has significant implications for the scientific and business communities across multiple disciplines including breeding, agriculture, cereal technology, nutritional science, health, and medicine. Political decision-makers and stakeholders in the food supply chain can benefit from the findings of this PhD project.
Mapping stem rust and leaf rust resistances in winter rye (Secale cereale L.)
(2023) Gruner, Paul; Miedaner, Thomas
Rye (Seale cereale L.) is one of the few cross-pollinating small-grain cereals and is mainly used for bread baking, biogas production and as animal feed. In its largest cultivation area (Northern, Central and Eastern Europe, including the Russian Federation) two major rust diseases, stem rust (SR) caused by Puccinia graminis f. sp. secalis and leaf rust (LR) caused by Puccinia recondita, can cause severe yield losses. Whereas LR can be found in most rye growing areas every year, SR is occurring less regularly, but can become epidemic in some years. The general occurrence of stem rust in Germany is becoming more regular, especially when hot summers provide optimum conditions for the growth and the spread of this fungus. Resistant cultivars can be a successful way to control both diseases, but SR is not assessed in the (German) variety registration and still several cultivars can be found that are susceptible or medium resistant for LR. Before the studies of this thesis were conducted, no marker-associated SR resistance gene locus was known and only six LR resistance loci had been reported. Rust resistances can be classified into all-stage resistances (ASR), that are usually caused by single R-genes and adult-plant resistances (APR), that are characterized by smaller (quantitative) effects and can only be observed in the adult-plant stage and thus make field tests mandatory. This thesis aimed on identifying resistant genotypes and respective resistance loci for SR and LR resistances in the rye genome. Two different material groups were used: biparental populations composed of inbred lines and populations composed of self-incompatible single plants. In total ten biparental populations and two additional testcross populations were studied, each constituting 68-90 genotypes. Self-incompatible populations were genetic resources from the Russian Federation, Austria and the United States of America and had 68-74 single plants each. Inbred lines were assessed in multi-environmental field trials (4-6 environments per population) and to guarantee high disease pressure, SR was artificially inoculated in contrast to naturally occurring LR in all environments. In addition, two different kind of seedling tests, one based on inoculations of entire seedling plants and one based on inoculation of detached leaves, were used to assess SR resistance. Mixed linear models were used to analyze the phenotypic data from field experiments and (mixed) cumulative logit models were used to analyze ordinal data resulting from seedling tests. Due to small sample size of a single detached leaf per genotype and isolate in self-incompatible populations, the results based on cumulative modes were cross checked with a non-parametric test. Both, progenies from biparental populations and single plants from self-incompatible populations were genotyped with single nucleotide polymorphism (SNP) based markers (Illumina iSelect 10K SNP chip or DArTseqTM) and appropriate statistical tests for phenotype-marker association were applied. This was achieved by extending phenotypic models with additive and dominant marker effects and their respective interaction with the environment or the isolates. Two marker-associated SR ASR loci (Pgs1, Pgs3.1) could be identified in biparental populations that were responsible for (large) qualitative differences between resistant and susceptible plants in the field and/or seedling stage. Additionally, 14 quantitative trait loci (QTLs) were shown to be responsible for SR APR. For LR, except one QTL found at similar position compared to a previous study, two new genes (Pr7, Pr8) and three QTLs were identified. Self-incompatible rye populations were used for the first time for association mapping and three SR resistance loci (Pgs1 - Pgs3) could be identified. Two thereof were also found within biparental mapping populations by means of QTL mapping and this was considered as prove of this new method. Throughout all studies, the natural cross-pollinating character of rye had to be considered in choosing appropriate methods and for developing rust resistant rye hybrids. This thesis includes breeding material from the largest European rye breeding companies and experiments were conducted in close cooperation with them. The characterization of breeding material for SR and LR infection, development of (new) mapping approaches, detection of resistance loci and marker candidates in the rye genome and finally the discussion of selection strategies provides a solid basis for breeders to develop the most durable SR and LR resistant rye cultivars. For scientists, new research topics could be, for example, the cloning of rye genes or a more thorough understanding of pathogen dynamics to finally achieve durable resistance in future.
Phenotypic and genomics-assisted breeding of soybean for Central Europe : from environmental adaptation to tofu traits
(2022) Kurasch, Alena; Würschum, Tobias
Soybean (Glycine max Merr.) is one of the major crops in the world providing an important source of protein and oil for food and feed; however it is still a minor crop in Central Europe. Soybean cultivation can play an important role in a more sustainable agricultural system by increasing local and regional protein production in Europe. The demand for locally produced soybean products is still growing in Europe. The key for a successful establishment of soybean cultivation in Europe is adaptation of soybean varieties to the Central European growing conditions. For the latitudinal adaptation to long-day conditions in Central to Northern Europe, an adapted early flowering and maturity time is of crucial importance for a profitable cultivation. The key traits flowering and maturity are quantitatively inherited and mainly affected by photoperiod responsiveness and temperature sensitivity. The most important loci for an early flowering and maturity are E1-E4 and the various allelic combinations condition soybean flowering and maturity time and therefore strongly contribute to the wide adaptability (Jiang et al., 2014; Tsubokura et al., 2014; M. Xu et al., 2013). Besides the main usage as protein source for animal feeding, soybean is also a very valuable source for human consumption. Tofu is enjoying ever greater popularity in Europe, as it is one of the best sources of plant protein with additional health benefits, rich in essential amino acids, beneficial lipids, vitamins, and minerals, as well as other bioactive compounds, such as isoflavones, soyasaponin, and others, (Lima et al., 2017; Zhang et al., 2018). Thus, plant breeding has to provide not only well-adapted varieties with good agronomic and quality properties, but also provide varieties well-suited to the further processing into soymilk and tofu. Therefore, a good knowledge about the breeding target, how to assess it and how it is inherited is crucial. The conducted studies covered a broad range of aspects relevant to improve a soybean breeding program. By combining environmental analysis, E-gene analysis, genomic approaches (QTL mapping and genomic prediction), and tofu phenotyping, breeder decisions become more accurate and targeted in the way of selection thereby increasing the genetic gain. In addition, combining the results of the different aspects helps to optimize the resources of a breeding program. Increasing the knowledge about the different aspects from environment to tofu QTL enables a breeder to be more precise and focused. But the more targeted and specific, the more complex a breeding program gets, which requires adequate tools to handle all the different information in a meaningful and efficient way to enable a quick and precise breeding decision.
Molecular and phenotypic diversity in populations of Fusarium culmorum on cereal hosts
(2022) Castiblanco Vargas, Eveline Valheria; Miedaner, Thomas
Fusarium head blight is one of the most devastating diseases of cereals globally and responsible for large harvest losses, not only due to the reduction in productivity but also due to the contamination of the grain with mycotoxins. The major causal agent worldwide is Fusarium graminearum; in Europe also other Fusarium species, among them Fusarium culmorum (FC) play an important role. The interaction between Fusarium species and cereals has been categorized as quantitative according to previous phenotypic and genetic observations. We studied the molecular and phenotypic diversity of natural populations of FC and how they interact with four cereals (bread wheat, durum wheat, triticale, rye) as host. Specifically, we sought (i) to understand the interaction between host and isolate, and between isolate and environment using the variance partition approach offered by mixed models applied to analyze multi-environmental studies; (ii) to identify or validate the association of Fusarium genes previously assigned as candidates using field aggressiveness and deoxynivalenol (DON) production; and (iii) to compare the application and results of the candidate gene association mapping approach applied to the same population of FC isolates but with different phenotypic data obtained from inoculation in different hosts-bread wheat and rye. Phenotyping was based on multi-environmental field experiments where each plot of the host plant was artificially inoculated with spores of the respective isolate in accordance with the experimental design. Aggressiveness was visually quantified as the percentage of spikelets with symptoms per plot and was repeatedly evaluated over time. The content of the mycotoxin deoxynivalenol (DON) in the harvested grain was evaluated by double enzyme linked immunosorbent assays (ELISA). Genes previously reported in the literature as related to aggressiveness were selected for sequencing. Using the available F. graminearum genome sequence, specific primers were constructed to amplify and sequence the most variable regions of the respective genes. The partitioning of the phenotypic variance using mixed models, for a subpopulation of 38 FC isolates in four cereal hosts, allowed to disaggregate the magnitude of the genotypic and environmental variance, and the environmental variance in turn into its different components. The genotypic variance was significant, but was exceeded by the magnitude of the environmental variance and its interactions with genotype, showing that the role of plasticity in the pathosystem of Fusarium culmorum and its cereal hosts is highly important. In contrast, the variance associated with the host factor and the interactions with host were not significant, confirmed by high values of genetic correlation amogn host. This result supports the categorization of the cereal/Fusarium culmorum interaction as unspecific and quantitatively inherited also from the view of the pathogen. For the present study, plasticity was understood as the changes in the phenotype of the pathogen that could be attributed to changes induced by the environment. Our data revealed the year as factor with the highest influence on plasticity, meaning that the isolates with high performance values under humid conditions did not exhibit the same high values under dry conditions. Because the environmental conditions are erratic between the years, the lack of a constant selection pressure in the same direction reduces the probability of achieving a speciation event per environment. The phenotypic data of the DON content in harvested grain showed a high correlation with the aggressiveness data. An association mapping study with 17 candidate genes for aggressiveness using a population of 100 isolates of FC inoculated on bread wheat revealed the significant association of the HOG1 gene, explaining 10.29% of the genetic variance of aggressiveness and 6.05% of the genetic variance corresponding to the accumulation of DON in mature grain. HOG1 is a kinase-like protein involved in the communication within the oxidative metabolism of the fungus. In a similar study using the same population of FC isolates and the same candidate genes but rye as host, the gene CUT showed a significant association with aggressiveness, explaining 16.05% of the genetic variance. The CUT gene encodes a cutinase protein, belonging to the secretome and involved in the process of unleashing the membranes and cuticles of the host plant. Taken together, our results suggest that i) field trials of breeding for resistance to FC in cereals should be carried out in several years to properly account for the genotype-by-year interaction; ii) despite the fact that molecular communication may present some type of host specificity the high plasticity guarantees that the effects on the phenotype are very similar among the cereal hosts; and iii) the high genetic correlation of aggressiveness for different cereals invites to involve non-cereal crops in the rotation plans focused on Fusarium disease management.
Analyzing resistance to ergot caused by Claviceps purpurea [Fr.] Tul. and alkaloid contamination in winter rye (Secale cereale L.)
(2022) Kodisch, Anna; Miedaner, Thomas
Ergot caused by Claviceps purpurea [Fr.] Tul. is one of the oldest well-known plant diseases leading already in medieval times to severe epidemic outbreaks. After the occurrence of honeydew, the characteristic ergot bodies called sclerotia are formed on the ear. These are containing toxic ergot alkaloids (EAs). Strict limits are set within the European Union. Rye (Secale cereale L.) as cross-pollinating crop is particularly vulnerable to ergot since the competitive situation of fungal spores and pollen during flowering. Nevertheless, even today the threat is real as agricultural practice is changing and screening studies revealed EAs in samples of the whole cereal value chain frequently. The aims were to establish a harmonized method to test ergot resistance and EA contamination in winter rye, to clarify major significant factors and their relevance and to reveal the suitability of one commercial ELISA test. Further, effort was paid to examine the covariation of ergot amount and EA content considering different factors because of prospective legislative changes. Genotypes showed significant variation for ergot severity and pollen-fertility restoration after natural infection as well as artificial inoculation whereas a high positive correlation could be found between both treatments. Additionally, variances of environment, general combining ability (GCA), specific combining ability (SCA), and interactions were significant. Although male pollen-fertility restoration was of utmost importance, the female component was also significant. This illustrates that apart from promising selection of high restoration ability the maternal restorability could be exploited in future breeding programs especially when a high pollen amount is already reached. A large-scale calibration study was performed to clarify the covariation of ergot severity, EA content (HPLC, ELISA) considering genotypes, locations, countries, years, and isolates. EA profile was rather stable across country-specific isolates although large differences regarding the EA content were detected. The moderate covariation between ergot severity and EA content (HPLC) indicates that a reliable prediction of the EA content based on ergot severity is not possible what can also not be explained by grouping effects of the factors. Further, EAs seem not to act as virulence factor in the infection process since EA content showed no relationship to disease severity. Additionally, the missing correlation of ELISA and HPLC leads to the conclusion that the ELISA is not an appropriate tool what can be used safely to screen samples regarding ergot in the daily life. The genetic variation of male-sterile CMS-single crosses was analysed in a special design without pollen in field and greenhouse to identify resistance mechanisms and to clarify whether ergot can be reduced in the female flower. At this, comparison of needle and spray inoculation revealed medium to high correlations illustrating that both methods were suitable for this research. Significant environment and genotype by environment interaction variances were detected. So, testing across several environments is necessary also without pollen. Further, small but significant genotypic variation and identification of one more ergot-resilient candidate revealed that selection of female lines could be promising to further reduce ergot. The EA content was lower for less susceptible genotypes. Thus, EA content can be considerably reduced by breeding. A strong positive correlation could be found for ergot severity and EA content when analysing 15 factorial single crosses. The male pollen-fertility restoration was also here the most relevant component but the female component contributed an obviously higher proportion for the EA content than for ergot severity. In conclusion, this thesis demonstrate that implementing of a high and environmental stable male fertility restoration ability via exotic Rf genes can effectively reduce ergot although also the female restorability enables great opportunities. The unpredictable covariation between ergot amount and EA content illustrates that both traits have to be assessed, in particular the EA content by a valid HPLC approach to guarantee food and feed safety.
Advancing soybean adaptation to Central European growth conditions with novel breeding tools
(2020) Jähne, Felix; Würschum, Tobias
According to the European Soy Monitor 2018 (European Soy Monitor, 2018), there is a wide discrepancy in the EU between market demands and general sustainability aims regarding soybean products. Europe needs to take action, if it wants to maintain its protein demands and at the same time requests a reduction in the destruction of globally important tropical and subtropical ecosystems. One step towards more sustainable soybean products lies in the increase of domestic production which has the potential to decrease soybean imports from areas of unsustainable cultivation. An augmented EU production of soybeans can be achieved for example by increasing the yield potential of soybeans in areas where successful cultivation already takes place or by expanding the cultivation area to more northern parts of Central Europe. Breeding for new, improved and adapted soybean cultivars that meet those terms, is a key activity towards that aim. This dissertation elucidates three different ways how the adaptation of soybeans to the climatic and photoperiodic conditions of Central Europe can be assisted and even accelerated: 1) By using off-season climate-controlled LED chambers to enable a speed breeding single seed descent approach. A 10 h light regime, rich in blue and deprived of far-red light emission is capable to significantly reduce and synchronise the generation time of soybeans. It was possible to shorten the life cycle for a panel of 8 soybean cultivars from different maturity groups to 77 days. This allows several generations of soybeans to be grown within one year. For the short day crops rice and amaranth on the other hand, different light quality parameters were favoured. In those crops mean flowering time was accelerated when far-red light was included in the light protocol. This underlines the importance of a crop-specific light regime in order to realise the full potential of LED-based speed breeding single seed descent. 2) By including experiments in climate-control chambers in combination with molecular tools (i.e. genomic prediction) to advance cold tolerance in soybeans. This quantitatively inherited key trait is necessary to adapt soybeans to colder regions and consequently extend growing areas of this crop to higher latitudes in Europe. In the biparental soybean population Merlin × Sigalia (103 recombinant inbred lines) three QTL for cold tolerance during pod onset were found on chromosomes 7, 11 and 13. The relatively small proportion of genotypic variance for this trait explained by these QTL underlines the quantitative nature of cold tolerance. Genomic prediction was shown to be a promising approach to select for cold stress tolerance. Scenarios with different test set sizes and prediction models were evaluated. In scenarios with smaller test set sizes prediction accuracies increased if known and confirmed QTL were included in the prediction model. 3) By incorporating citizen science into the breeding process. The citizen science project ‘1000 Gärten’ from 2016 approached this topic. Phenotypic data from soybean cultivars and breeding lines were collected by citizen scientists in 2492 gardens throughout Germany which generated a unique dataset. Among many other results this study was able to show that in 2016 and within the early maturity segment of soybeans the factor temperature influenced flowering and maturity to a higher degree than photoperiod although day length differed by over an hour between the north and the south of Germany during the time of flowering. It was shown that this admittedly challenging tool can realise a significant impact not only regarding the possibility of a highly multi-environmental screening of breeding material but also by connecting plant breeding, agriculture and potential future costumers in order to raise awareness and acceptance of a crop in larger parts of the society - a factor that may not be highlighted enough when a new crop is introduced to our agriculture. These approaches should not be seen as an alternative to classical plant breeding, but rather considered as valuable additional tools that can contribute to conventional breeding of soybeans, as well as other crops. If applied, the presented tools may assist plant breeding to pave Europe’s way towards a greener and more sustainable future that is urgently needed.
Integration of hyperspectral, genomic, and agronomic data for early prediction of biomass yield in hybrid rye (Secale cereale L.)
(2021) Galán, Rodrigo José; Miedaner, Thomas
Currently, the combination of a growing bioenergy demand and the need to diversify the dominant cultivation of energy maize opens a highly attractive scenario for alternative biomass crops. Rye (Secale cereale L.) stands out for its vigorous growth and increased tolerance to abiotic and biotic stressors. In Germany, less than a quarter of the total harvest is used for food production. Consequently, rye arises as a source of renewables with a reduced bioenergy-food tradeoff, emerging biomass as a new breeding objective. However, rye breeding is mainly driven by grain yield while biomass is destructively evaluated in later selection stages by expensive and time-consuming methods. The overall motivation of this research was to investigate the prospects of combining hyperspectral, genomic, and agronomic data for unlocking the potential of hybrid rye as a dual-purpose crop to meet the increasing demand for renewable sources of energy affordably. A specific aim was to predict the biomass yield as precisely as possible at an early selection stage. For this, a panel of 404 elite rye lines was genotyped and evaluated as testcrosses for grain yield and a subset of 274 genotypes additionally for biomass. Field trials were conducted at four locations in Germany in two years (eight environments). Hyperspectral fingerprints consisted of 400 discrete narrow bands (from 410 to 993 nm) and were collected in two points of time after heading for all hybrids in each site by an uncrewed aerial vehicle. In a first study, population parameters were estimated for different agronomic traits and a total of 23 vegetation indices. Dry matter yield showed significant genetic variation and was stronger correlated with plant height (r_g=0.86) than with grain yield (r_g=0.64) and individual vegetation indices (r_g: =<|0.35|). A multiple linear regression model based on plant height, grain yield, and a subset of vegetation indices surpassed the prediction ability for dry matter yield of models based only on agronomic traits by about 6 %. In a second study, whole-spectrum data was used to indirectly estimate dry matter yield. For this, single-kernel models based on hyperspectral reflectance-derived (HBLUP) and genomic (GBLUP) relationship matrices, a multi-kernel model combining both matrices, and a bivariate model fitted also with plant height as a secondary trait, were considered. HBLUP yielded superior predictive power than the models based on vegetation indices previously tested. The phenotypic correlations between individual wavelengths and dry matter yield were generally significant (p < 0.05) but low (r_p: =< |0.29|). Across environments and training set sizes, the bivariate model yielded the highest prediction abilities (0.56 – 0.75). All models profited from larger training populations. However, if larger training sets cannot be afforded, HBLUP emerged as a promising approach given its higher prediction power on reduced calibration populations compared to the well-established GBLUP. Before its incorporation into prediction models, filtering the hyperspectral data available by the least absolute shrinkage and selection operator (Lasso) was worthwhile to deal with data dimensionally. In a third study, the effects of trait heritability, as well as genetic and environmental relatedness on the prediction ability of GBLUP and HBLUP for biomass-related traits were compared. While the prediction ability of GBLUP (0.14 - 0.28) was largely affected by genetic relatedness and trait heritability, HBLUP was significantly more accurate (0.41 - 0.61) across weakly connected datasets. In this context, dry matter yield could be better predicted (up to 20 %) by a bivariate model. Nevertheless, due to environmental variances, genomic and reflectance-enabled predictions were strongly dependant on a sufficient environmental relationship between data used for model training and validation. In summary, to affordably breed rye as a double-purpose crop to meet the increasing bioenergy demands, the early prediction of biomass across selection cycles is crucial. Hyperspectral imaging has proven to be a suitable tool to select high-yielding biomass genotypes across weakly linked populations. Due to the synergetic effect of combining hyperspectral, genomic, and agronomic traits, higher prediction abilities can be obtained by integrating these data sources into bivariate models.
Genetic architecture of quality traits in wheat
(2021) Rapp, Matthias; Longin, Friedrich
Quality traits in wheat are of great importance, as they are required for the production of a wide range of food products. In Europe, bread wheat (Triticum aestivum ssp. aestivum) for human consumption is primarily used in pastries. For durum wheat (Triticum turgidum ssp. durum) that is used almost exclusively for pasta production, quality traits are at least as important as in bread wheat. In Central Europe, the bread wheat subspecies spelt (Triticum aestivum ssp. spelta) is characterized by a different quality compared to bread wheat. In addition, it is produced for a niche market with a particular focus on the final product quality. The high number of demanded quality traits of a wheat variety represents a great challenge for wheat breeders. Thus, knowledge about the genetic architecture and interrelation of quality traits is of high value for wheat breeding. Due to the long list of quality traits in wheat, we focused on currently important quality traits in each of the three wheat species. In durum wheat, I was interested in traits with a high importance for durum millers and pasta producers. The protein content and the sedimentation volume are of high importance for pasta producers as they influence the firmness of cooked pasta, better known as “al dente”. A low falling number may lead to brown instead of light yellow pasta, which goes back to an increased maillard reaction during pasta production and drying. The vitreousity, representing the glassy appearance of durum grains, and the thousand kernel mass influence the semolina yield and are therefore of great interest for durum millers. In the genome-wide association mapping, I identified several putative QTL for these quality traits. For the sedimentation volume, a genomic region on chromosome 1B appeared to be important. A BLAST search against the reference genomes of emmer and bread wheat revealed the Glu-B3 gene as a likely candidate. For vitreousity, genomic regions on chromosome 7A explained a larger proportion of the genotypic variance. One of these QTL, possibly related to the Pinb-2 locus, also slightly influenced the protein content. Thus, this genomic region might be a genomic reason for the positive correlation between vitreousity and protein content. For TKM we detected a putative QTL, which explained a large proportion of the genetic variance, but could not be attributed to a known gene. Besides a good performance for quality traits, a modern durum wheat variety should be complemented by a good agronomic performance, in particular a high grain yield. This poses a great challenge for plant breeders, since grain yield and protein content are negatively correlated. With regard to simultaneously improving grain yield and protein content, the protein yield or the grain protein deviation (GPD) were proposed. We evaluated those and further selection indices for their potential to be utilized for the simultaneous improvement of grain yield and protein content. Our results indicated that a simultaneous improvement of the two traits grain yield and protein content by means of an index seems possible. However, its efficiency largely depends on the weighting of the single traits. The selection for a high GPD would mainly increase the protein content whereas a selection based on protein yield would mainly improve the grain yield. Nevertheless, a combination of different indices allows balancing this selection. Compared to the primary traits grain yield and protein content, the selection indices did not essentially differ in the complexity of their genetic architecture. In bread wheat, we focused on the acrylamide precursor asparagine. Acrylamide is formed in potentially harmful concentrations when cereals are treated with high temperatures over a long period during the processing to food products. A promising strategy to reduce the acrylamide formation would be to decrease the precursors in the raw material. The wide range of variation for asparagine content showed that variety selection might have a large influence on the occurrence of acrylamide in the final product. In addition, the moderately high heritability suggested that successful breeding for lower asparagine content is possible. This conclusion is supported by the observation of no strong negative correlations between asparagine content and a number of other important traits. The genome-wide association mapping resulted in the detection of eight putative QTL, which jointly explained 78.5% of the genetic variance. A putative QTL on chromosome 7B explained with, 18.4%, the highest proportion of the genetic variance for a single marker. For spelt wheat, we assessed a high number of quality traits but placed a special emphasis on the flavor and odor of bread produced from 30 different varieties. Interestingly, we observed a significant genetic variation for bread flavor and a heritability estimate of moderate magnitude. This suggests that even for bread flavor a successful selection appears possible. Taken together, for most traits the genome-wide association mapping resulted in the detection of a high number of putative QTL. This indicates a complex genetic architecture, typical for predominantly quantitatively inherited traits. However, few of the putative QTL explained a large proportion of the genetic variance, so that they might have the potential to be used in marker-assisted selection. In order to examine the potential of genomic selection, I performed a five-fold cross validation for the different quality traits. I could confirm previous findings that the integration of QTL information as fixed effects in the genomic prediction model increased the prediction abilities considerably. The average prediction abilities for most traits suggested a high potential for genomic selection in breeding programs. In conclusion or results form a good basis for further research but more importantly already deliver valuable knowledge that can be used as guideline to advance wheat breeding programs for improved quality.
Genomics-assisted breeding strategies for quantitative resistances to Northern corn leaf blight in maize (Zea mays L.) and Fusarium diseases in maize and in triticale (× Triticosecale Wittm.)
(2021) Galiano Carneiro, Ana Luísa; Miedaner, Thomas
Fusarium head blight (FHB) in triticale (× Triticosecale Wittm.), Gibberella ear rot (GER) and Northern corn leaf blight (NCLB) in maize (Zea mays L.) are devastating crop diseases causing yield losses and/or reducing grain quality worldwide. Resistance breeding is the most efficient and sustainable approach to reduce the damages caused by these diseases. For all three pathosystems, a quantitative inheritance based on many genes with small effects has been described in previous studies. Hence, this thesis aimed to assess the potential of genomics-assisted breeding strategies to reduce FHB, GER and NCLB in applied breeding programs. In particular, the objectives were to: (i) Dissect the genetic architecture underlying quantitative variation for FHB, GER and NCLB through different quantitative trait loci (QTL) and association mapping approaches; (ii) assess the potential of genomics-assisted selection to select superior triticale genotypes harboring FHB resistance; (iii) phenotype and characterize Brazilian resistance donors conferring resistance to GER and NCLB in multi-environment trials in Brazil and in Europe; and (iv) evaluate approaches for the introgression and integration of NCLB and GER resistances from tropical to adapted germplasm. The genome-wide association study (GWAS) conducted for FHB resistance in triticale revealed six QTL that reduced damages by 5 to 8%. The most prominent QTL identified in our study was mapped on chromosome 5B and explained 30% of the genotypic variance. To evaluate the potential of genomic selection (GS), we performed a five-fold cross-validation study. Here, weighted genomic selection increased the prediction accuracy from 0.55 to 0.78 compared to the non-weighted GS model, indicating the high potential of the weighted genomic selection approach. The successful application of GS requires large training sets to develop robust models. However, large training sets based on the target trait deoxynivalenol (DON) are usually not available. Due to the rather moderate correlation between FHB and DON, we recommend a negative selection based on genomic estimated breeding values (GEBVs) for FHB severity in early breeding stages. In the long-run, however, we encourage breeders to build and test GS calibrations for DON content in triticale. The genetic architecture of GER caused by Fusarium graminearum in maize was investigated in Brazilian tropical germplasm in multi-environment trials. We observed high genotype-by-environment interactions which requires trials in many environments for the identification of stable QTL. We identified four QTL that explained between 5 to 22% of the genotypic variance. Most of the resistance alleles identified in our study originated from the Brazilian tropical parents indicating the potential of this exotic germplasm as resistance source. The QTL located on chromosome bin 1.02 was identified both in Brazilian and in European trials, and across all six biparental populations. This QTL is likely stable, an important feature for its successful employment across different genetic backgrounds and environments. This stable QTL is a great candidate for validation and fine mapping, and subsequent introgression in European germplasm but possible negative linkage drag should be tackled. NCLB is another economically important disease in maize and the most devastating leaf disease in maize grown in Europe. Virulent races have already overcome the majority of known qualitative resistances. Therefore, a constant monitoring of S. turcica races is necessary to assist breeders on the choice of effective resistances in each target environment. We investigated the genetic architecture of NCLB in Brazilian tropical germplasm and identified 17 QTL distributed along the ten chromosomes of maize explaining 4 to 31% of the trait genotypic variance each. Most of the alleles reducing the infections originated from Brazilian germplasm and reduced NCLB between 0.3 to 2.5 scores in the 1-9 severity scale, showing the potential of Brazilian germplasm to reduce not only GER but also NCLB severity in maize. These QTL were identified across a wide range of environments comprising different S. turcica race compositions indicating race non-specific resistance and most likely stability. Indeed, QTL 7.03 and 9.03/9.04 were identified both in Brazil and in Europe being promising candidates for trait introgression. These major and stable QTL identified for GER and NCLB can be introgressed into elite germplasm by marker-assisted selection. Subsequently, an integration step is necessary to account for possible negative linkage drag. A rapid genomics-assisted breeding approach for the introgression and integration of exotic into adapted germplasm has been proposed in this thesis. Jointly, our results demonstrate the high potential of genomics-assisted breeding strategies to efficiently increase the quantitative resistance levels of NCLB in maize and Fusarium diseases in maize and in triticale. We identified favorable QTL to increase resistance levels in both crops. In addition, we successfully characterized Brazilian germplasm for GER and NCLB resistances. After validation and fine mapping, the introgression and integration of the QTL identified in this study might contribute to the release of resistant cultivars, an important pillar to cope with global food security.
Breeding for resistance to Fusarium ear diseases in maize and small-grain cereals using genomic tools
(2021) Gaikpa, David Sewordor; Miedaner, Thomas
The world’s human and livestock population is increasing and there is the need to increase quality food production to achieve the global sustainable development goal 3, zero hunger by year 2030 (United Nations, 2015). However, biotic stresses such as Fusarium ear infections pose serious threat to cereal crop production. Breeding for host plant resistance against toxigenic Fusarium spp. is a sustainable way to produce more and safer cereal crops such as maize and small-grain winter cereals. Many efforts have been made to improve maize and small-grain cereals for ear rot (ER) and Fusarium head blight (FHB) resistances, using conventional and genomic techniques. Among small-grain cereals, rye had the shortest maturity period followed by the descendant, hexaploid triticale while both wheat species had the longest maturity period. In addition, rye and triticale were more robust to Fusarium infection and deoxynivalenol accumulation, making them safer grain sources for human and animal consumption. However, a few resistant cultivars have been produced by prolonged conventional breeding efforts in durum wheat and bread wheat. High genetic variation was present within each crop species and can be exploited for resistance breeding. In this thesis, the genetic architecture of FHB resistance in rye was investigated for the first time, by means of genome-wide association study (GWAS) and genomic prediction (GP). GWAS detected 15 QTLs for Fusarium culmorum head blight severity, of which two had major effects. Both weighted and unweighted GP approaches yielded higher prediction abilities than marker-assisted selection (MAS) for FHB severity, heading stage and plant height. Genomics-assisted breeding can shorten the duration of breeding rye for FHB resistance. In the past decade, genetic mapping and omics were used to identify a multitude of QTLs and candidate genes for ear rot resistances and mycotoxin accumulation in maize. The polygenic nature of resistance traits, high genotype x environment interaction, and large-scale phenotyping remain major bottlenecks to increasing genetic gains for ear rots resistance in maize. Phenotypic and molecular analyses of DH lines originating from two European flint landraces (“Kemater Landmais Gelb”, KE, and “Petkuser Ferdinand Rot”, PE) revealed high variation for Gibberella ear rot (GER) severity and three agronomic traits viz. number of days to female flowering, plant height and proportion of kernels per cob. By employing multi-SNP GWAS method, we found four medium-effect QTLs and many small-effect (10) QTLs for GER severity in combined DH libraries (when PCs used as fixed effects), none co-localized with the QTLs detected for the three agronomic traits analyzed. However, one major QTL was detected within KE DH library for GER severity. Two prioritized SNPs detected for GER resistance were associated with 25 protein-coding genes placed in various functional categories, which further enhances scientific knowledge on the molecular mechanisms of GER resistance. Genome-based approaches seems promising for tapping GER resistance alleles from European maize landraces for applied breeding. After several cycles of backcrossing and sufficient selection for agronomic adaptation traits, the resistant lines identified in this thesis can be incorporated into existing maize breeding programs to improve immunity against F. graminearum ear infection. Breeding progress can be faster using KE landrace than PE. A successful validation of QTLs identified in this thesis can pave way for MAS in rye and marker-assisted backcrossing in maize. Effective implementation of genomic selection requires proper design of the training and validation sets, which should include part of the current breeding population.
Prospects of genomic selection for disease resistances in winter wheat (Triticum aestivum L.)
(2019) Grote, Cathérine Pauline; Miedaner, Thomas
Die Ziele dieser Arbeit waren (i) die erstmalige Evaluierung des Effekts des Zwerggens Rht24 auf FHB- und STB-Resistenzen, Wuchshöhe und Ährenschieben im Vergleich zum weit genutzten Locus Rht-D1, (ii) die Untersuchung des Potenzials der nichtadaptierten QTL Fhb1 und Fhb5 für die Entwicklung von Kurzstrohweizen, (iii) die Analyse der Vorhersagegenauigkeit von GS innerhalb und zwischen Familien durch die Anwendung der beiden Modelle RR-BLUP (ridge-regression best linear unbiased prediction) und wRR-BLUP (weighted RR-BLUP) und (iv) die Berechnung des Selektionsgewinns bzw. die Bestimmung der korrekt selektierten Top-10 %-Genotypen für FHB- und STB-Resistenzen durch GS. Die Ergebnisse dieser Studie zeigten, dass das gibberellinsäuresensitive Zwerggen Rht24 auf Chromosom 6 die Wuchshöhe um durchschnittlich 8,96 cm senkte, ohne dabei die FHB- und STB-Resistenzen oder den Zeitpunkt des Ährenschiebens ungünstig zu beeinflussen. Demgegenüber senkte das weitläufig verwendete Allel Rht-D1b die FHB-Resistenz um durchschnittlich 10,05 Prozentpunkte in einer Winterweizenpopulation bestehend aus acht biparentalen Familien, die für diese Resistenzloci segregierten. Diese Arbeit hat zusätzlich aufgezeigt, dass die Resistenzallele von Fhb1 und Fhb5 die FHB-Anfälligkeit um 6,54 bzw. 11,33 Prozentpunkte reduzierten und somit bereits allein das nicht-adaptierte Allel Fhb5b in der Lage ist, den negativen Effekt von Rht-D1b auf die FHB-Resistenz im untersuchten Material auszugleichen. Das verdeutlicht, dass die Wahl der Zwerg- und Resistenzgene in Zuchtprogrammen, in denen FHB-Resistenz ein Selektionsmerkmal ist, von entscheidender Bedeutung ist. In dieser Studie wurde des Weiteren das Potenzial der GS innerhalb und zwischen Familien untersucht. Die Vorhersagegenauigkeiten innerhalb einer Familie waren für alle Zielmerkmale höher als die zwischen Familien und unterschieden sich zwischen den einzelnen Familien und Vorhersagekonstellationen. Die stärkere Gewichtung von signifikanten Markern durch das wRR-BLUP-Modell führte zu einer Verbesserung der Vorhersagegenauigkeit im Vergleich zum weit genutzten RR-BLUP-Modell, wenn einzelne Gene, wie Rht-D1, oder Major-QTL, wie Fhb5, vorhanden waren. In dieser Studie wurden die genomisch geschätzten Zuchtwerte (GEBVs) von 2.500 ungeprüften Genotypen bestimmt, basierend auf einer partiell verwandten Trainingspopulation von 1.120 Genotypen. Die 10 % FHB- und STB-resistentesten Linien und eine zufällige Stichprobe wurden unter Berücksichtigung der Wuchshöhe genomisch selektiert und phänotypisch in einem vierortigen Feldversuch evaluiert. Für die FHB-Resistenz wurde ein Selektionserfolg von 10,62 Prozentpunkten relativ zur zufällig selektierten Populationsstichprobe ermittelt. Die GS erhöhte die STB-Resistenz allerdings nur um 2,14 Prozentpunkte. Auch die Selektion von neuen Kreuzungseltern auf der Basis von GS erscheint nicht ausreichend zuverlässig, da nur 19 % der Top-10 %-Individuen korrekt selektiert wurden. Zusammenfassend stellt die GS ein wertvolles Werkzeug dar, um den Zuchtfortschritt für die komplex vererbte FHB-Resistenz über kürzere Zyklen und größere Populationen zu unterstützen. In Kombination mit der Nutzung geeigneter Zwerggene und des nicht adaptierten QTL Fhb5 kann dadurch eine Steigerung der FHB-Resistenz im Winterweizen erzielt werden.
Design and assessment of breeding strategies for hybrid wheat in Europe
(2018) Boeven, Philipp Hans Günter; Würschum, Tobias
Wheat is one of the top three global staple crops, possesses the largest global cultivation area, and plays a key role for the world’s future food security. However, its projected yield increase is insufficient to meet the future food and feed demand of an ever-growing world population. Consequently, the rate of breeding progress and productivity of wheat must be increased. Unfortunately, current wheat line breeding has a low return on investment mainly due to high levels of farm saved seeds, which makes wheat less attractive for the plant breeding industry and leads to lower investments and progress compared to other crops where the hybrid technology is established. Hybrid breeding is a worldwide success story in many crops but is not yet established in wheat. Hybrid wheat promises increased yield gain due to the exploitation of heterosis, higher yield stability and stabilized return on investments for breeding companies which warrants further investment and breeding progress in this important stable crop. The self-pollinating nature of wheat is a major bottleneck for hybrid seed production and efficient hybrid wheat breeding requires the redesign of the wheat floral architecture to enhance cross-pollination. Furthermore, the longterm success of hybrid wheat is crucially dependent on the establishment of heterotic groups, on the identification of a high yielding heterotic pattern, and finally, on the realized amount of heterosis and hybrid performance. Therefore, the main objectives of my thesis research were to: (i) analyze the genetic diversity and adaptation in a global winter wheat collection and evaluate how diversity trends could be used to support the development of heterotic groups in wheat; (ii) assess the relationship between heterosis and genetic distance under maximized diversity and evaluate the usefulness of exotic germplasm for hybrid wheat breeding; (iii) dissect the genetic architecture underlying male floral traits in wheat to enable genomics-assisted breeding approaches and investigate the trait seed set which is most crucial for an efficient hybrid seed production. The analyses of genetic diversity in a large worldwide panel of 1,110 winter wheat varieties released during the past decades showed no major population structure but revealed genetically distinct subgroups. Most of the global diversity trends could be explained by breeding history and were associated with geographical origin and long-term domestication. We found that the frequency of the copy number variants at the Photoperiod-B1 (Ppd-B1) and the Vernalization-A1 (Vrn-A1) loci reflect wheat adaptation to the environmental conditions of the different regions of origin. Thus, adaptation issues add an additional layer of complexity and hamper the direct introgression of genetic diversity to support the genetic divergence between heterotic pools. Based on all these analyses, we proposed HyBFrame, a unified framework illustrating how global wheat genetic diversity can be used to support and accelerate reciprocal recurrent selection for the development of genetically distinct heterotic groups in wheat. In a second experiment, we produced 2,046 wheat hybrids by crossing elite with elite lines as well as elite with exotic lines and performed multi-environmental field trials. Interestingly, we found an average midparent heterosis of about 10% in elite crosses as well as in exotic crosses and observed no evidence for a breakdown of heterosis under maximized genetic distance among the hybrid parents. Genetic distance based on genome-wide molecular markers revealed only a very weak association with midparent heterosis for grain yield. Here, we elaborated a functional Rogers’ distance giving weight to heterosis loci and observed a strong positive association between heterosis and this novel distance measure. Hence, considering the genetic architecture of heterosis revealed a more accurate picture of the relationship between heterosis and genetic distance. In addition, the genetic architecture of heterosis in wheat is crucially dependent on the genetic background. We found that a higher number of negative dominance and dominance-by-dominance epistatic effects can reduce the level of absolute heterosis in wide crosses between exotic lines and elite testers. Moreover, hybrid performance in wheat is mainly driven by parental per se performance. Thus, elite lines are favorable for hybrid breeding and should be employed as the starting material for heterotic grouping. Hybrid seed production is the major bottleneck for hybrid wheat breeding and explains the low market share of hybrid wheat varieties. Seed set on the female plants in crossing blocks is the most crucial trait for hybrid seed production in wheat. We tested 31 male lines and evaluated the hybrid seed set on two female tester lines in crossing blocks. Seed set showed a large genotypic variation and a high heritability suggesting that recurrent selection for increased seed set is feasible. The major problem is the synchronized flowering between male and female lines, making the evaluation of seed set in large panels very complex and difficult. Hence, indirect male floral traits with high correlation to the trait seed set would be promising to breed for improved hybrid seed production. We found a strong association between seed set and visual anther extrusion, underscoring that indirect male floral traits have a high potential for preliminary male screenings. We also dissected the genetic architecture underlying promising male floral traits and assessed the potential of genomics-assisted approaches for their improvement. We employed a panel of 209 diverse wheat lines and found a complex genetic architecture underlying all male floral traits. The Reduced height gene Rht-D1 was identified as the only major QTL, for which the commonly used height-reducing allele showed negative effects on male floral traits. This genetic architecture with many moderate- or small-effect QTL limits classical marker-assisted selection. In contrast, genomic prediction yielded moderate to high prediction abilities for anther extrusion. Finally, we proposed a breeding scheme to increase cross-pollination in wheat based on a combination of phenotypic and genomics-assisted selection. Taken together, hybrid breeding in wheat is a very promising approach and the next years will show if all of the current issues can be solved. This thesis research contributed to breeding strategies for hybrid wheat breeding and to the general understanding of heterosis in crops.
Phenotypic, genetic, and genomic assessment of triticale lines and hybrids
(2017) Losert, Dominik; Würschum, Tobias
Triticale (×Triticosecale Wittmack) is a small grain cereal used for livestock feeding and as renewable energy source. These diverse types of usage lead to different breeding strategies, ideally resulting in continued increase of both, grain and biomass yield. Briefly, the objectives of this thesis were to explore aspects with relevance for line and hybrid breeding in triticale by phenotypic, genetic and genomic assessment of important traits. More specifically, the objectives of this study were to (i) evaluate agronomic traits, assess trait correlations, and investigate the amount of heterosis in triticale hybrids, (ii) examine the potential of line and hybrid cultivars for production of biomass, (iii) assess the phenotypic and genotypic variability in triticale germplasm, (iv) investigate long-term phenotypic trends based on cultivars registered in the past three decades, and (v) identify QTL for agronomical relevant traits. In conclusion, hybrids of triticale possess an increased biomass yield potential compared with their mid-parent values as well as compared with commercial reference cultivars. The findings on triticale germplasm and its breeding history provide important information for breeding programs. Furthermore, based on the obtained results, genomic approaches like marker-assisted or genomic selection appear promising to assist triticale breeding in the future.
Molecular mapping of resistance and aggressiveness in the cereal/Fusarium head blight pathosystem
(2016) Kalih, Rasha; Miedaner, Thomas
Fusarium head blight (FHB) is one of the most destructive fungal diseases in small-grain cereals worldwide causing significant yield losses and contamination of grain with mycotoxins e.g., deoxynivalenol (DON). This renders the grain unsuitable for human consumption and animal feeding. Exploring the genetic mechanism of FHB resistance is considered the key tool for modern cereal breeding activities. Triticale, the intergeneric hybrid between wheat and rye, is an important cereal crop in Poland and Germany. Resistance breeding using genetic mapping to identify quantitative-trait loci (QTL) associated with FHB resistance represents the best strategy for controlling the disease. In parallel, understanding the mechanism of aggressiveness and DON production of F. graminearum will be a significant contribution to improve FHB management. The objectives of the present work were (1) identification of QTL related to FHB resistance in triticale, together with the analysis of the correlation of FHB severity with other related traits such as plant height and heading stage, (2) correlation between DON production and FHB severity, (3) mapping of dwarfing gene Ddw1 in triticale and studying its effect on FHB resistance, plant height and heading stage, (4) detection of SNPs in candidate genes associated with aggressiveness and DON production of a large Fusarium graminearum population in bread wheat. To study the genetic architecture of FHB resistance in triticale, five doubled-haploid (DH) triticale populations with 120 to 200 progenies were successfully tested under field conditions by inoculation with Fusarium culmorum (FC46) in multiple environments. All genotypes were evaluated for FHB resistance, plant height and heading stage. DArT markers were used to genotype triticale populations. Significant genotypic variances (P<0.001) were observed for FHB severity in all populations combined with high heritability. Twenty-two QTLs for FHB resistance in triticale were reported with two to five QTL per population, thus confirming the quantitative inheritance of FHB resistance in triticale. The most prominent (R2 ≥ 35%) QTLs were located on chromosomes 6A, 3B, 4R, and 5R. QTLs for plant height and heading stage were also detected in our work, some of them were overlapping with QTLs for FHB resistance. Correlation between FHB severity, DON content and Fusarium damaged kernels (FDK) in triticale was studied in the population Lasko x Alamo. Significant genotypic variance was detected for all traits. However, low correlation between FHB severity and DON content (r=0.31) was found. Interestingly, correlation between FHB severity and FDK rating was considerably higher (r=0.57). For FHB severity, two QTLs were detected in this population. A QTL located on chromosome 2A with minor effect for FHB severity was also a common QTL for DON content and FDK rating and explained ≥34% of genotypic variance for these two traits. A second QTL on chromosome 5R was a major QTL but it has no effect on DON content or FDK rating. For analyzing the rye dwarfing gene Ddw1 derived from the father Pigmej, 199 (DH) progenies were genotyped with DArT markers and in addition with conserved ortholog set (COS) markers linked to the Ddw1 locus in rye. QTL analyses detected three, four, and six QTLs for FHB severity, plant height and heading stage, respectively. Two specific markers tightly linked with Ddw1 on rye chromosome 5R explained 48, 77, and 71 % of genotypic variation for FHB severity, plant height, and heading stage, respectively. This is strong evidence, that we indeed detected the rye gene Ddw1 in this triticale population. Another objective was to highlight the association between quantitative variation of aggressiveness and DON production of 152 F. graminearum isolates with single nucleotide polymorphism (SNP) markers in seven candidate genes. One to three significant SNPs (P < 0.01 using cross-validation) were associated to FHB severity in four genes (i.e., Gmpk1, Mgv1, TRI6, and Erf2). For DON content, just one significant SNP was detected in the gene Mgv1 explaining 6.5% of the total genotypic variance. In conclusion, wide genetic variation in FHB resistance in triticale has been observed in five populations. QTL mapping analyses revealed twenty-two QTLs for FHB resistance derived from wheat and rye genomes. QTLs located on the rye genome were reported here for the first time and they are a new source for FHB resistance in triticale. In parallel, analysis of the diversity of four pathogenicity genes in F. graminearum is an important first step in inferring the genetic network of pathogenicity in this fungal pathogen.
Breeding winter durum wheat for Central Europe : assessment of frost tolerance and quality on a phenotypic and genotypic level
(2015) Sieber, Alisa-Naomi; Würschum, Tobias
Durum wheat (Triticum durum) is a tetraploid wheat that is used for pasta and other semolina products. Quality standards for semolina requested by the pasta industry are very high. Different characteristics should come with the cereal as raw material for an optimal end product. Vitreosity, the glassy and amber quality feature of durum wheat kernels, is an indicator for high semolina yield. The complex protein-starch matrix of glassy kernels breaks the grain into the typical semolina granulate instead of flour during milling. Humid conditions, like late summer rains in Central Europe, have a huge effect on this characteristic, changing this matrix irreversibly. Such processes in the kernel are less understood and challenge plant breeders to find genotypes with improved vitreosity. A set of F5 winter durum wheat lines (Chapter 2) was used to investigate the relationship between protein content and vitreosity as well as the impact of humidity on the stability of the trait. A method to evaluate the mealy part in kernels was improved and enabled to test for the influence of humidity on vitreosity. Furthermore, it was revealed that the vitreosity of a durum wheat kernel depends on the protein content up to a specific threshold as well as on the genotypic potential to form the complex endosperm matrix. The ability to maintain this kernel quality under humid conditions also highly depends on the genetics of a variety. In the Mediterranean region, durum wheat is grown as autumn-sown spring type. The mild winters as well as rain during spring allow the plants to develop well, and the dry summers enable an early harvest in June. Durum wheat production in Central Europe, on the other hand, is confronted with harsh winters and recurring severe frosts. The lack of a sufficient frost tolerance in combination with high quality, forces farmers to use the spring type with a spring sowing. Growing winter durum instead of spring durum wheat, would allow an autumn sowing. Using the winter type in this growing area, could have several advantages like an increased yield and stability due to a prolonged growing time. Further, the constant soil coverage would prevent soil erosion and the growth vigor of winter durum has advantages against weeds. The success of winter durum breeding depends on frost tolerance as a key factor for varieties with excellent winter survival. Discontinuous occurrence of frosts across years and protective snow coverage, however, limit the phenotypic selection for this trait under field conditions. Greenhouses or climate chambers could be used as alternative to test under the necessary conditions, but those fully-controlled tests are time consuming and labor-intensive. The ‘Weihenstephaner Auswinterungsanlage’ are wooden boxes with movable glass lids used as a semi-controlled test. Plants are exposed to all seasonal conditions, including frost stresses, in this test, but they can be protected from snow coverage. While this method is already successfully used to test for frost tolerance in bread wheat, the application in durum wheat has not been evaluated yet. The frost tolerance scorings of winter durum elite lines (F5 and F6) based on the ‘Weihenstephaner Auswinterungsanlage’ were compared to the field evaluation (Chapter 3). It was demonstrated that this semi-controlled test produces reliable and highly heritable (h2 = 0.83-0.86) frost tolerance data. The correlation of those results compared with the field data (r = 0.71) suggests this semi-controlled test as an indirect selection platform. Since it is now possible to test cost-efficient at early stages for frost tolerance, the next challenge was to determine whether the kernel quality or the grain yield suffers from an increased frost tolerance. In a survey with F5 winter durum elite lines, no negative association between frost tolerance and quality or other important agronomic traits could be found in European breeding material (Chapter 4). In order to support classical plant breeding, which relies predominantly on phenotypic data and parental information, molecular markers can be taken into account. Molecular markers can provide an in-depth look into the genetic architecture of traits, enable the determination of the relatedness of genotypes, identify the genetic variation in a population, or can assess the effect of geographic selection preferences. Furthermore, it is possible to assist knowledge-based selection. This improves plant breeding programs on a genetic level. The population structure in spring durum has already been examined with molecular methods in several studies. Winter durum, on the other hand, was only analyzed as a small group as part of spring durum studies or in groups of landraces. A highly diverse and unique panel of 170 winter durum and 14 spring durum lines was analyzed using a genotyping-by-sequencing (GBS) approach. A total of 30,611 markers, well distributed across the chromosomes, were obtained after filtering for marker quality. A principal coordinate analysis and a cluster analysis were applied. Together they revealed the absence of a major population structure (Chapter 5). The lines, however, grouped in a certain way, depending on their origin, associated with decreasing quality and increasing frost tolerance moving from South to Continental Europe. These groups allow breeders to conduct targeted crosses to further improve the frost tolerance in the Central European material. Another possibility is to build heterotic groups for hybrid breeding. The linkage disequilibrium (LD) decay was within 2-5 cM, indicating a high diversity in winter durum. The high marker density together with the extent of LD observed in this analysis allows to perform high-resolution association mapping in the present winter durum panel. The 30,611 markers and additional markers for candidate genes in frost tolerance were used to assess the genetic architecture of frost tolerance in durum wheat (Chapter 6). A major QTL was identified on chromosome 5A, likely being Frost Resistance-A2 (Fr-A2). Additional analysis of copy number variation (CNV) of CBF-A14 at Fr-A2 support this conclusion. CBF-A14 CNV explains about 90% of the proportion of genotypic variance. Two markers found in the QTL region were combined into a haploblock and enabled to capture the genetic variance of this QTL. Furthermore, the frequency of the QTL allele for frost tolerance shows a latitudinal gradient which is likely associated with winter conditions. In summary, the selection tools for vitreosity and frost tolerance provided in this study create a platform for winter durum breeding to select for high quality genotypes with excellent winter survival utilizing phenotypic as well as genotypic information.
Genome-wide prediction of testcross performance and phenotypic stability for important agronomic and quality traits in elite hybrid rye (Secale cereale L.)
(2016) Wang, Yu; Miedaner, Thomas
Genomic selection offers a greater potential for improving complex, quantitative traits in winter rye than marker-assisted selection. Prediction accuracies for grain yield for unrelated test populations have, however, to be improved. Nevertheless, they are already favorable for selecting phenotypic stability of quality traits.
Quantitative-trait loci (QTL) mapping of important agronomical traits of the grain and biomass production in winter rye (Secale cereale L.)
(2015) Haffke, Stefan; Miedaner, Thomas
Rye is an important crop in Northern and Eastern Europe and mainly used for food and feed and became most recently important for biogas production. Hybrid rye varieties dominate the cultivated area, which is mainly on light and sandy soils, because rye has a relatively high tolerance to biotic and abiotic stress factors. Climate change will also affect Central Europe, causing higher temperatures and less precipitation in spring and summer. Rye will be influenced more by these effects than other cereals because it is mainly grown on marginal environments. Rye has a high potential for being used as a biogas substrate, but detailed information on improving this trait in hybrid rye is missing. Until now, no study that analyzed phenotypic and genotypic agronomic traits for using rye for biogas production exists. Further, there is only one study, which dealt with the influence of periodic drought stress in rye cultivated areas. Beside this, we analyzed yield stability over a wide range of environments in consideration of drought stress in Central Europe. We analyzed an interpool hybrid population (Pop-D) in 2011 and 2012 at seven environments in Germany for the biomass yield and grain yield (Publication I). This study showed low correlations between grain yield and dry matter yield (r = 0.33). Higher correlations were obtained with two plant height measurements (at heading time, r = 0.64; before harvest, r = 0.52) and dry matter yield. The indirect selection via plant height was superior in contrast to the direct selection of dry matter yield by factor 1.24. Genotypic results confirmed phenotypic results as no overlapping QTL for grain yield and dry matter yield were detected (Publication II). However, we identified common gene regions for plant height and dry matter yield due to the high correlation between both. Plant height is a promising trait for indirectly selecting high biomass yielding varieties. The paradigm shift from shorter plants with high grain yield to taller hybrids as a resource for biogas substrate needs additional breeding efforts for lodging resistance. In Publication III we analyzed two intrapool populations (Pop-A and -B) and one interpool population (Pop-C) at 16 – 18 environments (location x year combinations) under irrigated and rainfed conditions in Germany and Poland. Yield stability was high over a wide range of environments, even when drought stress environments were included. This illustrates the adaption of rye to marginal and drought stress environments. The analyzed populations showed no differences within yield stability, but yield differences between inter- (Pop-C) and intra-pool (Pop-A and -B) crosses were visible. Selection for yield stability is possible due to the genetic variance for this trait within all three populations. Therefore, it is important to select genotypes with low genotype x environment interaction. All three populations showed high yield stability on a high yield level and were already well adapted to extreme weather events caused by climate change. It is recommended to use highly diverse environments with irrigated and rainfed conditions to select on yield stability and high yielding varieties under optimum and drought conditions
Phenotypic and genotypic assessment of traits with relevance for hybrid breeding in European winter wheat
(2015) Langer, Simon Martin; Würschum, Tobias
Hybrid breeding in wheat has recently received increased interest, especially in Europe, and large public and private projects investigating hybrid wheat breeding have been launched. Hybrid breeding has been a great success story for allogamous crops and is seen as a promising approach to increase the yield potential in wheat. Wheat covers more of the world’s surface than any other food crop and is the second main staple crop for human consumption. It can be produced under widely varying conditions and is grown all around the globe, yet, yield gain has declined and is lagging behind the needs of the constantly growing human population. Future challenges in wheat breeding such as the establishment of hybrid varieties and the adaptation of breeding germplasm to increasing stresses caused by climatic changes also in Europe require knowledge-based improvements of relevant traits and phenotyping approaches suited for applied high-throughput plant breeding. A major limitation for the establishment and the production of hybrid wheat is the lack of a cost-efficient hybrid seed production system. This requires the generation of parental ideotypes which maximize the cross-fertilization capability. Male parents should have an extended time of flowering, extrude anthers and widely shed large amounts of viable pollen. Females need increased receptivity for male pollen by opening the glumes and extruding stigmatic hair. Furthermore, male plants should be taller than females and a synchronized timing of flowering between the two parents is also of utmost importance. Employing a set of European elite winter wheat lines, we developed and evaluated phenotyping methods for important floral and flowering traits with relevance for improved cross-pollination (Publication I). We observed high heritabilities for important traits such as ‘pollen mass’ (h2=0.72) and ‘anther extrusion’ (h2=0.91). In addition, genotypic variances were significant which warrants further breeding success. Positive correlations were found among important flowering and floral characteristics which enables the improvement of outcrossing by indirect selection. ‘Pollen mass’ for example, was associated with ‘anther extrusion’, ‘anther length’ and ‘plant height’. Our findings suggest the utility of the developed phenotyping approaches for applied plant breeding and the potential of the traits to assist in the design of the male ideotype for increased cross-fertilization. We investigated the genetic architecture of flowering time and plant height (Publication II and III). A panel of 410 European winter wheat varieties was genotyped by a genotyping-by-sequencing approach and in addition, analyzed for the effects of specific candidate genes. The major factor affecting flowering time was the photoperiod regulator Ppd-D1 (58.2% of explained genotypic variance) followed by Ppd-B1 copy number variation (3.2%). For plant height, the two candidate loci Rht-D1 (37.0%) and Rht-B1 (14.0%) had the largest effects on the trait but contrary to reports in the literature did not contribute to flowering time control. In addition, we identified several small effect QTL and epistatic QTL responsible for fine-adjustments of these two traits. Population structure and genetic relatedness in European elite wheat lines was assessed using different types of markers (Publication IV). Results for relatedness differed for the marker types but consistently showed the absence of a major population structure. Regarding the large wheat genome our results revealed that a high number of markers is necessary as there are regions with only low coverage. Concordantly, we were not able to identify the major flowering locus Ppd-D1 without targeted candidate gene analysis. Observations on the findings on population structure could be confirmed in Publication II and III and in addition, the geographical distribution of important flowering time and plant height genes displayed the historical development of wheat breeding in Europe. This information on genetic relatedness among lines can also be employed to assist the establishment of hybrid wheat.
Differences in yield performance and yield stability between hybrids and inbred lines of wheat, barley, and triticale
(2015) Mühleisen, Jonathan; Reif, Jochen Christoph
Hybrids of wheat, barley, and triticale are expected to possess higher yield performance and yield stability compared to inbred lines. Assessment of yield performance as well as yield stability requires the evaluation of genotypes in plot-based yield trials across multiple environments. Evaluation of genotypes under stress conditions can be associated with increased field heterogeneity, which may result in imprecise estimates of genotypic values. The assessment of yield stability requires intensive testing in many environments, and it would be interesting to know how many test environments are required to reliably estimate yield stability. The key objectives of the present thesis were to (1) investigate optimal strategies to analyze field trials with high error variance due to spatially varying drought stress, (2) identify the required number of test environments to precisely estimate yield stability of individual barley genotypes, and (3) examine yield performance and yield stability of wheat, barley, and triticale hybrids and lines. Drought stress at two locations of a winter triticale trial caused increased field heterogeneity, resulting in lower heritabilities compared to the four non-stress locations. It was found that heritability could be increased by modeling incomplete block and row effects, by using visual scorings of drought stress intensity as covariates in an analysis of covariance, and by modeling a spatial covariance between adjacent plots. The most suitable model can be identified using the Akaike Information Criterion. In addition, it has to be ensured that the covariate is independent from genotypic effects and that it is linearly related with the response variable. Dynamic yield stability of genotypes was frequently found to depend strongly on the specific set of test environments. When the genotypes were evaluated in different environments, e.g. in the following year, the ranking in yield stability could be different. This would result in a low heritability. Theoretical assumptions and empirical studies showed that heritability can be increased when the number of test environments is increased. Five series of barley registration trials with a reduced number of 16 to 27 genotypes evaluated in 39 to 45 environments were used to investigate the relationship between magnitude of heritability of yield stability and number of test environments. Based on a cross-validation approach, it was found, that at least 40 test environments should be used to obtain a heritability of 0.5. Magnitude of heritability, however, varied strongly within and between series. Therefore, depending on the respective set of environments and genotypes, more or less test environments can be needed. Yield performance of wheat hybrids produced using chemical hybridizing agents (CHA) or cytoplasmic male sterility (CMS) was well investigated in other studies reporting around 10% midparent heterosis for grain yield. In the present thesis, CMS-based barley hybrids were compared with parental inbred lines and unrelated commercial inbred lines in breeding and registration trials. Midparent heterosis was around 10%. The comparison with commercial inbred lines in the registration trials revealed that hybrids could compete with and partially surpass outstanding inbred lines. Triticale hybrids, produced using CMS, were evaluated for grain yield at up to 20 environments with their parents and commercial inbred lines. Midparent heterosis amounted to 3% and no hybrid outyielded the best inbred line. The low yield performance of triticale hybrids is probably associated with CMS-system, since CHA-based triticale hybrids showed a midparent heterosis around 10% in early studies, which is comparable to the midparent heterosis found in wheat and barley. Yield stability of CHA-based wheat as well as CMS-based hybrids of barley and triticale was compared with yield stability of parental and commercial inbred lines on group level. The wheat and barley hybrids showed on average significantly higher dynamic yield stability compared to inbred lines, but the triticale hybrids did not. In the barley registration trials, hybrids had the highest dynamic yield stability on average. The CMS-based triticale hybrids, however, showed on average significantly lower dynamic yield stability as their female parents and the commercial inbred lines across 20 environments. In conclusion, hybrids of wheat and barley possessed an increased yield potential as well as an enhanced dynamic yield stability. In contrast, the CMS-based triticale hybrids showed only marginal yield advantages coupled with low dynamic yield stability. Further research is required to increase economical competitiveness of hybrids in all three crops, to identify and eliminate the reasons for poor performance of CMS-based triticale hybrids and to investigate the suitability of dynamic yield stability measures to identify vigorous and stress tolerant genotypes.

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