Institut für Pflanzenzüchtung, Saatgutforschung und Populationsgenetik

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Studies on flowering time and photoperiod sensitivity in domesticated and wild amaranth species (Amaranthus spp.)
(2023) Baturaygil, Ali; Schmid, Karl J.
Flowering time plays fundamental roles in the local adaptation and agricultural productivity of the crops. Photoperiodic response regulates the time of flowering by adjusting the response of plant circadian rhythm to environmental signals. Amaranth (Amaranthus spp.) is a short-day crop native to Central and South America, and mainly used as grain and vegetable. Hence, photoperiod sensitivity is a pivotal trait for grain amaranths in Central Europe climatic and long-day conditions, as it determines the local adaptability and the cultivation purpose of the crop i.e., grain or biomass production. However, the knowledge on the different aspects such as breeding, domestication history and adaptation genetics is very limited in grain amaranths. In this project, we studied such different aspects of grain amaranths by addressing the elucidative photoperiod sensitivity trait. In the first study, the phenotypic evaluation of biomass yield components revealed two distinct growth types. Of those, our ten biomass genotypes showed mild to high photoperiod sensitivity, flowered late or completely rejected flowering, reached long final plant heights and low dry matter content. In contrast, the only grain type variety showed photoperiod insensitivity, flowered early, and reached a short final plant height and a relatively higher dry matter content. Our results suggested that selection for both high dry matter yield and content requires a trade-off between photoperiod sensitivity and early flowering, due to the negative correlation between these traits. In the second study, characterization of genebank accessions from the three major grain species (A. caudatus, A. cruentus, A. hypochondriacus) and their wild relative species (A. hybridus and A. quitensis) for adaptive traits such as flowering time and seed setting under long-day conditions discovered a larger photoperiodic variation in the Central American accessions ranging from insensitivity to high sensitivity, whereas South American accessions showed a more narrow variation, limited by mild sensitivity. This result suggests the Central American origin of the wild relative A. hybridus, which might have migrated from Central to South America, and potentially has been selected against high photoperiod sensitivity. Moreover, we studied the environmental variables that may influence seed setting. Photoperiod insensitive accessions set seed regardless of their origin. However, mild photoperiod-sensitive accessions set seed, only if they were from warm center of origin. In the third study, we investigated the genetic architecture of photoperiod sensitivity. The bimodal-like flowering time distributions, and the linkage and association mapping studies using three different populations revealed that photoperiod sensitivity trait is controlled in an oligogenic manner. In particular, all three populations consistently found the same ‘consensus region’ that includes a very promising candidate gene called ‘response regulator of two-component system’. The homologs of this candidate gene are responsible for photoperiodic response in a variety of different crops and the model species Arabidopsis thaliana. In addition, the phenotypic analyses, and the marker data (i) showed photoperiod sensitivity guided pleiotropic relationships between the traits, (ii) revealed a potential epistatic behavior of the genomic region controlling photoperiod sensitivity, and (iii) showed the dominance of photoperiod sensitivity over insensitivity in that region.
Utilization of landraces of European flint maize for breeding and genetic research
(2023) Renner, Juliane; Melchinger, Albrecht E.
Mais ist eine der wichtigsten Kulturarten für die Landwirtschaft weltweit. Seit seiner Domestikation bildeten Landrassen den traditionellen Sortentyp. Durch Selektion und genetische Faktoren entstand eine breite Diversität an panmiktisch vermehrten Populationen, die gut an lokale Bedingungen angepasst waren. Dies änderte sich mit der Einführung der Hybridzüchtung, als nahezu alle Landrassen in der landwirtschaftlichen Produktion und als Ausgangsmaterial für die Züchtung verschwanden. Molekulare Analysen zeigen eine enge genetische Basis des Flint Pools im Vergleich zum Dent Pool. Genetische Ressourcen im Mais gehören zu den umfangreichsten aller Nutzpflanzen. Die Nutzung dieses bislang ungenutzten Reservoirs an genetischer Diversität in Landrassen bietet eine Möglichkeit, um der fortschreitenden Einengung der genetischen Basis entgegenzuwirken und somit den Aufgaben der Pflanzenzüchtung im Hinblick auf eine wachsende Weltbevölkerung sowie den Herausforderungen des Klimawandels und reduzierten Inputs im Anbau gerecht zu werden. Übergeordnetes Ziel dieser Studie war die Evaluierung europäischer Flint-Mais Landrassen, um deren genetische Vielfalt nutzen zu können. Im Speziellen waren die Ziele (i) die Variation in Testkreuzungen europäischer Mais-Landrassen zu bestimmen; (ii) die phänotypische und genotypische Variation der Linien innerhalb und zwischen Landrassen zu beurteilen; (iii) die Eigenleistung dieser Linien mit Elite-Linien sowie Founder-Linien aus dem europäischen Flint-Pool zu vergleichen; (iv) das Potential von doppelhaploiden (DH) Linien aus Landrassen im Vergleich zum Elitematerial für die Züchtung zu analysieren, um die enge genetische Basis des Flint-Pools zu erweitern; (v) die Verwendung von DH-Bibliotheken aus Landrassen für die Assoziationskartierung bis hin zur Eingrenzung kausaler Gene zu demonstrieren; und (vi) Schlussfolgerungen und Leitlinien für die Züchtung und Forschung zu erörtern , um DH-Linien aus Landrassen nutzbar zu machen. In einem ersten Versuch wurde eine umfangreiche Kollektion von 70 europäischen Flint-Landrassen mehrortig in Kombination mit zwei Elite Dent-Testern auf ihre Testkreuzungsleistung hin untersucht. Verglichen mit dem Ertrag moderner Hybriden war der Kornertrag der Testkreuzungen der Landrassen im Durchschnitt um 26 % geringer, jedoch wurde eine hohe genotypische Varianz zwischen den Landrassen für alle Merkmale beobachtet. Die Korrelationen waren mittel bis hoch für die meisten Merkmalskombinationen und entsprachen denen im Elitezuchtmaterial. Die genetische Korrelation der beiden Testkreuzungsserien überstieg 0,74 für alle Merkmale. Dies zeigt, dass es ausreicht die Leistung von Testkreuzungen in Kombination mit einem oder zwei Testern - bestehend aus Einfachkreuzungen des anderen Gen-Pools – zu bewerten, um das Potenzial von Landrassen für die Züchtung zu beurteilen. In einem zweiten Versuch produzierten wir Bibliotheken von DH-Linien der vielversprechendsten Landrassen des vorigen Versuches. Insgesamt wurden 389 DH-Linien aus sechs europäischer Flint Landrassen zusammen mit vier Flint Founder-Linien und 53 Elite Flintlinien auf 16 agronomische Merkmale an vier Standorten geprüft. Die genotypische Varianz (σ^2G) innerhalb der DH-Bibliotheken war größer als die zwischen den Bibliotheken und übertraf auch σ^2G der Elite Flintlinien. Darüber hinaus variierten die Mittelwerte und σ^2G zwischen den DH-Bibliotheken, was zu großen Unterschieden im Brauchbarkeits-Kriterium („usefulness“) führte. Der mittlere Kornertrag der Elite Flintlinien übertraf den der Flint Founder-Linien um 25 % und der DH-Bibliotheken um 62 %, was auf den beträchtlichen Zuchtfortschritt im Elitematerial hinweist sowie auf die erhebliche genetische Bürde, welche in den DH-Bibliotheken vorliegt. Die Brauchbarkeit der besten DH-Linien war trotzdem für viele Merkmale, einschließlich dem Kornertrag, mit der von Elite Flintlinien vergleichbar. Dies zeigt das enorme Potenzial, Landrassen zur Verbreiterung des genetisch engen Elite Flint-Pools zu verwenden. In einem dritten Versuch wurden das genetische Material des vorherigen Versuches mit dem MaizeSNP50 BeadChip von Illumina® genotypisiert und Samen aller Genotypen zur Extraktion und Analyse von 288 Metaboliten mit GC-MS verwendet. Sowohl die agronomischen Merkmale als auch die Metabolit-Daten wurden für eine Assoziationskartierung verwendet. Der schnelle Abfall des Kopplungsungleichgewichts benachbarter Marker in den DH-Bibliotheken im Vergleich zu den Elite Flintlinien führte zu einer hervorragenden Auflösung in der QTL-Kartierung, was durch die Feinkartierung eines QTL (= quantitative trait locus) für Ölgehalt bis zur Phenylalanin Insertion F469 in DGAT1-2 als kausale Variante demonstriert werden konnte. Darüber hinaus wurden für den Metaboliten Allantoin, der im Zusammenhang mit abiotischem Stress steht, Promotorpolymorphismen sowie die Expression einer Allantoinase als vermutete Ursache der Variation identifiziert. Dies gelang trotz der moderaten Größe der Kartierungspopulation. Diese Ergebnisse sind ermutigend, um DH-Bibliotheken von Landrassen für die Assoziationskartierung zu verwenden und QTL bis auf die kausalen Varianten zu entschlüsseln. Eine Erweiterung der Populationsgrößen der DH-Bibliotheken, ähnlich wie sie in anderen Versuchsdesigns in der Literatur verwendet wurden, ist hierbei zu empfehlen, um mit diesem Ansatz QTL zu detektieren, welche lediglich einen kleinen Teil der genetischen Varianz erklären. Dies eröffnet neue Wege zur Nutzung natürlicher und/oder neu geschaffener Allele in der Züchtung. Zusammenfassend zeigen die Ergebnisse dieser Arbeit, dass die genetische Variation europäischer Landrassen bei Flint-Mais eine einzigartige Quelle darstellt, um die fortschreitende Verengung der genetischen Basis des Elitematerials in diesem Gen-Pool umzukehren. Um vielversprechende Landrassen zu identifizieren, schlagen wir folgenden zweistufigen Ansatz vor: (i) Basierend auf der Bewertung der molekularen Diversität werden etwa hundert Landrassen in Leistungsprüfungen auf ihre Anpassungsfähigkeit für die Zielregionen evaluiert und ihre Kombinationsfähigkeit mit dem entgegengesetzten heterotischen Gen-Pool in Testkreuzungen mit einer Einfachkreuzung als Tester bewertet. (ii) Für eine geringe Zahl (< 6) von Landrassen wird anschließend eine große Anzahl von DH-Linien erstellt, welche für die Nutzung in der Assoziationskartierung und/oder genomischen Selektion phänotypisiert und genotypisiert werden, um diese „Goldreserven“ für die Maiszüchtung mit innovativen Methoden zugänglich zu machen.
Population genomics of herbicide resistance in Alopecurus myosuroides
(2022) Kersten, Sonja; Schmid, Karl J.
Over the past 50 years, herbicides have often replaced mechanical and manual human weed control, thus representing a major factor in yield productivity in modern agriculture. Herbicide applications, however, exert strong selection pressures on weeds. As a consequence, these species have developed herbicide resistance through adaptive, beneficial alleles that increase in number to ensure the persistence of the populations, a phenomenon known as evolutionary rescue. A major research question is whether herbicide resistance adaptation is more likely to arise from standing genetic variation that was present before the onset of herbicide selection or from de novo mutations that arose after herbicide selection began. To address this question, I focused on target-site resistance (TSR) point mutations, which cause a lower binding affinity to the target protein of the respective herbicides. I first investigated the diversity of TSR haplotypes in populations of the grass species Alopecurus myosuroides (common name: blackgrass), and compared it with the TSR diversity outcome of simulated populations under both evolutionary scenarios. I first conducted a population genetics study of A. myosuroides, which is the most problematic weed in winter cereals across the European continent due to rapid resistance evolution. To obtain genome-wide polymorphic markers, I adapted a restriction site-associated DNA sequencing protocol to this species. I began by analyzing the diversity and population structure in a smaller local South German collection. The fact that I could differentiate populations on a local scale motivated me to extend the study to a European-wide collection, in which I found clear population structure, albeit with low differentiation and some evidence for admixture across Europe. In addition, I generated highly accurate long-read amplicons from single individuals of two loci, ACETYL-COA CARBOXYLASE (ACCase) and ACETOLACTATE SYNTHASE (ALS), which are the targets of the two main herbicide modes of action used in European cereal crops. I obtained completely phased haplotype information, supporting the analysis of haplotype diversity on a population level. I found a remarkable diversity of beneficial TSR mutations at the field level arising from multiple haplotypes of independent origin, so called soft sweeps. I used this information to perform forward simulations to investigate the evolutionary origin of these mutations. I found evidence that a majority of resistance mutations originated from standing genetic variation. While this at first may appear surprising, it is consistent with very large census and effective population sizes in blackgrass. Since long-read amplicon sequencing of single individuals could be costly and time consuming, I extended the analysis to pools of 150 to 200 individuals from Germany, Belgium, France, the Netherlands and the United Kingdom. By combining the power of a more stringent accuracy criterion in our long-reads and a novel clustering software (PacBio amplicon analysis), I was able to preserve individual haplotype information in pooled samples. Furthermore, in a proof of concept experiment, I was able to recover in our pools most haplotypes previously sequenced in individuals. The amplicon study provides a versatile workflow that can be easily adapted to any gene of interest in different species. In conclusion, I found that many A. myosuroides populations likely already have the genetic prerequisites not only for rapid evolution of resistance to currently used herbicides, but also to herbicides that have not yet been brought to market.
Assessment of phenotypic, genomic and novel approaches for soybean breeding in Central Europe
(2022) Zhu, Xintian; Würschum, Tobias
Soybean is the economically most important leguminous crop worldwide and serves as a main source of plant protein for human nutrition and animal feed. Europe is dependent on plant protein imports and the EU protein self-sufficiency, which is an issue that has been on the political agenda for several decades, has recently received renewed interest. The protein imports are mainly in the form of soybean meal, and soybean therefore appears well-suited to mitigate the protein deficit in Europe. This, however, requires an improvement of soybean production as well as an expansion of soybean cultivation and thus breeding of new cultivars that combine agronomic performance with adaptation to the climatic conditions in Central Europe. The objective of this thesis was to characterize, evaluate and devise approaches that can improve the efficiency of soybean breeding. Breeding is essentially the generation of new genetic variation and the subsequent selection of superior genotypes as candidates for new cultivars. The process of selection can be supported by marker-assisted or genomic selection, which are both based on molecular markers. A first step towards the utilization of these approaches in breeding is the characterization of the genetic architecture underlying the target traits. In this study, we therefore performed QTL mapping for six target traits in a large population of 944 recombinant inbred lines from eight biparental families. The results showed that some major-effect QTL are present that could be utilized in marker-assisted selection, but in general the target traits are quantitatively inherited. For such traits controlled by numerous small-effect QTL, genomic selection has proven as a powerful tool to assist selection in breeding programs. We therefore also evaluated the genomic prediction accuracy and found this to be high and promising for the six traits of interest. In conclusion, these results illustrated the potential of genomic selection for soybean breeding programs, but a potential limitation of this approach are the costs required for genotyping with molecular markers. Phenomic selection is an alternative approach that uses near-infrared or other spectral data for prediction instead of the marker data used for its genomic counterpart. Here, we evaluated the phenomic predictive ability in soybean as well as in triticale and maize. Phenomic prediction based on near-infrared spectroscopy (NIRS) of seeds showed a comparable or even slightly higher predictive ability than genomic prediction. Collectively, our results illustrate the potential of phenomic selection for breeding of complex traits in soybean and other crops. The advantage of this approach is that NIRS data are often available anyhow and can be generated with much lower costs than the molecular marker data, also in high-throughput required to screen the large numbers of selection candidates in breeding programs. Soybean is a short-day plant originating from temperate China, and thus adaptation to the climatic conditions of Central Europe is a major breeding goal. In this study, we established a large diversity panel of 1,503 early-maturing soybeans, comprising of European breeding material and accessions from genebanks. This panel was evaluated in six environments, which revealed valuable genetic variation that can be introgressed into our breeding programs. In addition, we deciphered the genetic architecture of the adaptation traits flowering time and maturity. Taken together, the findings of this study show the potential of several phenotypic, genomic and novel approaches that can be integrated to improve the efficiency of soybean breeding and thus hold great promise to assist the expansion of soybean cultivation in Central Europe through breeding of adapted and agronomically improved cultivars.
Optimum strategies to implement genomic selection in hybrid breeding
(2022) Marulanda Martinez, Jose Joaquin; Melchinger, Albrecht E.
To satisfy the rising demand for more agricultural production, a boost in the annual expected selection gain (ΔGa) of traits such as grain yield and especially yield stability has to be rapidly achieved. Hybrid breeding has contributed to a notable increment in performance for numerous allogamous species and has been proposed as a way to match the increased demand for autogamous cereals such as rice, wheat, and barley. An additional tool to increase the rate of annual selection gain is genomic selection (GS), a method to assess the merit of an individual by simultaneously accounting for the effects associated with hundreds to thousands of DNA markers. Successful integration of GS and hybrid breeding should go beyond the study of GS prediction accuracy and focus on the design of breeding strategies, for which GS maximizes ΔGa and optimizes the allocation of resources. The main goal of this thesis was to examine strategies for optimum implementation of GS in hybrid breeding with emphasis on estimation set design to perform GS within biparental populations and on the optimization of hybrid breeding strategies through model calculations. One strategy, GSrapid, with moderate nursery selection, one stage of GS, and one stage of phenotypic selection, reached the greatest ΔGa for single trait selection regardless of the budget, costs, variance components, and accuracy of genomic prediction. GSrapid was also the most efficient strategy for the simultaneous improvement of two traits regardless of the correlation between traits, selection index chosen, and economic weights assigned to each trait. The success of this strategy relies principally on the reduction of breeding cycle length and marginally on the increase in selection intensity. Moving from traditional breeding strategies based on phenotypic selection to strategies using GS for single trait improvement in hybrid breeding could lead not only to increments in ΔGa but also to large savings in the budget. The implementation of nursery selection in breeding strategies boosted the importance of efficient systems for inbred generation accompanied by improvements in the methods of hybrid seed production for experimental tests. When it comes to multiple trait improvement, the choice between optimum and base selection indices had minor impact on the net merit. However, considerable differences for ΔGa of single traits were observed when applying optimum or base indices if the variance components of the traits to be improved differed. The role of the economic weights assigned to each trait was determinant and small variations in the weights led to a remarkable genetic loss in one of the traits. The optimum design of estimation sets to perform GS within biparental populations should be based on phenotypic data, rather than molecular marker data. This finding poses major challenges for GS-based strategies aiming to select the best new inbreds within second cycle breeding populations, as breeding cycle length might not be reduced. Then, the ES design to optimize GS within biparental populations would have a defined application on the exploitation of within-family variation by increasing selection intensity in biparental populations with the largest potential of producing high-performing inbreds. Based on the results of this thesis, future challenges for the optimum implementation of GS in hybrid breeding strategies include (i) reductions in breeding cycle length and increments in selection intensity by refinements of DH technology or implementation of speed breeding, (ii) improvements in the methods for hybrid seed production, facilitating the reallocation of resources to the production of more candidates tested during the breeding cycle, and (iii) precise estimation of economic weights, reflecting the importance of the traits for breeding programs and farmers, and maximizing long term ΔGa for the most relevant traits.
Genomic and phenotypic improvement of triticale (×Triticosecale Wittmack) line and hybrid breeding programs
(2021) Trini, Johannes Philipp; Würschum, Tobias
Triticale (×Triticosecale Wittmack) breeding is a success story as it evolved to a serious alternative in farmer’s crop rotations since the 1970s and is grown globally on around 4 million hectares today. New developments, however, pointed out additional possibilities to improve triticale line and hybrid breeding programs increasing its future competitiveness and were evaluated in this study. In more detail, these were to (i) examine the genetic control and evaluate long term genetic trends of plant height in Central European winter triticale, (ii) evaluate the potential of triticale hybrid breeding and hybrid prediction approaches in triticale with a focus on biomass yield, (iii) introduce and examine a concept bypassing the time and resource consuming evaluation of female candidate lines in cytoplasmatic male sterility (CMS) based hybrid breeding, and (iv) to draw conclusions for the future improvement of triticale line and hybrid breeding programs. The genome wide association study detected markers significantly associated with plant height and developmental stage, respectively. These explained 42,16% and 29,31% of the total genotypic variance of plant height and development stage and are probably related to four and three quantitative trait loci (QTL), respectively. The two major QTL detected for plant height were located on chromosomes 5A and 5R which most likely could be assigned to the known height reducing genes Rht12 from wheat and Ddw1 from rye. The third major QTL detected located on chromosome 4B could not be assigned to a known height reducing gene and it cannot be precluded, that these significantly associated markers are identifying one and the same QTL as the markers located on chromosome 5R, as these showed a high linkage disequilibrium amongst each other. Evaluating the 129 registered cultivars showed that plant height decreased since the 1980’s. Evaluating their genetic constitution revealed that most cultivars carried at least one height reducing QTL and that plant height could be reduced even further in cultivars combining more than one height reducing QTL. It was further observed that the frequency of cultivars carrying one or a combination of height reducing QTL increased since the 1980’s. A considerable amount of heterosis has been observed for biomass related traits in triticale hybrids before. However, the use of hybrid prediction approaches for these traits has not been evaluated. Hybrid prediction based on mid parent values already showed very good results illustrating their potential to preselect the most promising parents as prediction accuracies based on parental general combining ability (GCA) effects were only slightly better. When incorporating molecular markers into GCA based prediction accuracies, prediction accuracies decreased slightly compared to prediction accuracies solely based on phenotypic GCA effects. Predicting hybrids incorporating one or two untested parental lines, imitating a scenario where novel female and/or male candidate lines are introduced into a hybrid breeding program, reduced genomic prediction accuracies even further due to the decreasing amount of information which could be exploited from the parents. Additionally including specific combining ability (SCA) effects in the genomic prediction models did not yield additional use. A high proportion of SCA variance compared to the total genetic variance decreased prediction accuracies for the traits fresh and dry biomass yield. In this study simulation studies were used to demonstrate what a prediction accuracy of a specific value actually means for a hybrid breeding programs. Further, an approach was introduced and evaluated showing great potential to evaluate novel female candidate lines for their use in a CMS based hybrid breeding program by bypassing their time and resource demanding introgression into a male sterile cytoplasm using three way hybrids. Prediction accuracies obtained by this novel approach showed highly promising results for most evaluated traits compared to prediction accuracies based on GCA effects or mid parent performance. Additionally incorporating SCA effects into the prediction models showed only a little increase of the prediction accuracies. Further, the results were supported by simulation studies adjusting different parameters, such as the number of parents or the proportion of SCA variance compared to the total genetic variance.
Assessing the genetic variation of phosphate efficiency in European maize (Zea mays L.)
(2022) Weiß, Thea Mi; Würschum, Tobias
Why should plant breeders in Central Europe care about phosphate efficiency? Soil phosphorus levels have mostly reached high to very high levels over the last decades in intensively farmed, livestock-rich regions. However, the European Union demands a restructuring of the agricultural production systems through setting ambitious goals envisaged in the Farm to Fork Strategy. By 2030, fertilizer use should be reduced by 20 %, nutrient losses by at least 50 %. As a consequence, farmers have to be even more efficient with crop inputs, among them the globally limited resource of phosphorus fertilizers, while maintaining high yields. Plant breeding means thinking ahead. Therefore, phosphate-efficient varieties should be developed to help farmers meet this challenge and reduce the need for additional fertilizers. One prerequisite to reach this target is that genotypic variation for the relevant traits is available. Moreover, approaches that assist selection by accurate but also time- and resource-efficient prediction of genotypes are highly valuable in breeding. Finally, the choice of the selection environment and suitable trait assessment for the improvement of phosphate efficiency under well-supplied conditions, need to be elaborated. In this dissertation, a diverse set of maize genotypes from ancient landraces to modern hybrids was investigated for phosphate efficiency-related traits under well-supplied P soil conditions. Multi-environmental field trials were conducted in 2019 and 2020. The reaction to different starter fertilizer treatments of the 20 commercially most important maize hybrids grown in Germany was studied. In the hybrid trial, the factor environment had a significant effect on the impact of starter fertilizers. Especially in early developmental stages genotypes showed a different response to the application of starter fertilizers. On the overall very well-supplied soils, we observed no significant genotype-by-starter fertilizer interaction. Nonetheless, we identified hybrids, which maintained high yields also if no starter fertilizer was provided. Thus, it seems that sufficient variation is available to select and breed for phosphate efficiency under reduced fertilizer conditions. Furthermore, the concept of phenomic prediction, based on near-infrared spectra instead of marker data to predict the performance of genotypes, was applied to 400 diverse lines of maize and compared to genomic prediction. For this, we used seed-based near-infrared spectroscopy data to perform phenomic selection in our line material, which comprised doubled haploid lines from landraces and elite lines. We observed that phenomic prediction generally performed comparable to genomic prediction or even better. In particular, the phenomic selection approach holds great potential for predictions among different groups of breeding material as it is less prone to artifacts resulting from population structure. Phenomic selection is therefore deemed a useful and cost-efficient tool to predict complex traits, including phosphorus concentration and grain yield, which together form the basis to determine phosphate efficiency. Lastly, 20 different indicators for phosphate efficiency were calculated, the genetic variation of the different measures present in this unique set of lines was quantified, and recommendations for breeding were derived. Of the different measures for phosphate efficiency reported in literature, Flint landraces demonstrated valuable allelic diversity with regard to phosphate efficiency during the seedling stage. Due to the highly complex genetic architecture of phosphate efficiency-related traits, a combination of genomic and phenotypic selection appears best suited for their improvement in breeding. Taken together, phosphate efficiency, including its definition and meaning, is largely dependent on the available phosphorus in the target environment as well as the farm type, which specifies the harvested produce and thereby the entire phosphorus removal from the field. In conclusion, future maize breeding should work in environments that are similar to the future target environments, meaning reduced fertilizer inputs and eventually lower soil P levels. Our results demonstrate that breeding of varieties, which perform well without starter fertilizers is feasible and meaningful under the well-supplied conditions prevalent in Central Europe. For the improvement of the highly complex trait phosphate efficiency through breeding we recommend to apply genomic and phenomic prediction along with classical phenotypic screening of genotypes and by this making our food systems more resilient towards upcoming challenges in agriculture.
Gibberella ear rot resistance in European maize : genetic analysis by complementary mapping approaches and improvement with genomic selection
(2022) Han, Sen; Melchinger, Albrecht E.
During the last decades, implementation of molecular markers such as single nucleotide polymorphisms (SNPs) has transformed plant breeding practices from conventional phenotypic selection to marker-assisted selection (MAS) and genomic selection (GS) that are more precise, faster and less resource-consuming. In this dissertation, we investigated these three selection approaches for improving the polygenic trait Gibberella ear rot (GER) resistance in maize (Zea mays L.), which is an important fungal disease in Europe and North America leading to reduced grain yield and grain contaminated with mycotoxins such as deoxynivalenol (DON) and zearalenone (ZON). Three different sets of materials were evaluated in multiple environments and analyzed for different objectives. In the first study, five flint doubled-haploid (DH) families (with size 43 to 204) inter-connected at various levels through common parents, were generated in an incomplete half-diallel design with four parental lines developed by the University of Hohenheim. Significant genotypic variances and generally high heritabilities were observed for all three traits (i.e., GER, DON and days to silking (DS)) in all families, implying good prospects for resistance breeding and phenotypic selection against GER across different environments in European maize germplasm. Genetic correlations were extremely tight between DON and GER and moderately negative for DS with DON or GER, suggesting that indirect selection against GER would be efficient to reduce DON, but maturity should be considered in GER resistance breeding. Using a high-density consensus map with 2,472 marker loci, we compared classical bi-parental mapping of QTL (quantitative trait locus/loci) with multi-parental QTL mapping conducted with joint families and using four different biometric models. Multi-parental QTL mapping models identified all and even further QTL than the bi-parental QTL mapping model conducted within each family. Interestingly, QTL for DON and GER were mostly family-specific, yet multiple families had several common QTL for DS. Many QTL displayed large additive effects and most favorable alleles originated from the highly resistant parent. Interactions between detected QTL and genetic background (family) were rare and had comparatively small effects. Multi-parental QTL mapping models generally did not yield higher prediction accuracy than the bi-parental QTL mapping model for all traits. In the second study, two diversity panels consisting of 130 elite European dent and 114 flint lines, respectively, from the University of Hohenheim were evaluated and subject to a genome-wide association study within each pool. Similar to the first study, highly significant genotypic and genotype × environment interaction variances were observed for GER, DON and DS. Heritabilities were moderately high for GER and DON and high for DS in both pools. Estimated genomic correlations between pools were close to zero for DON and DS, and slightly higher for GER. The detected QTL for DON were all specific to each heterotic pool and none of them was in common with previously detected QTL. Furthermore, no QTL was detected for GER and DS in both pools. Genomic prediction (GP) across pools yielded low or even negative prediction accuracy for all traits. When the training set (TS) size was increased by combining lines from both heterotic pools, the combined-pool GP approaches had no higher prediction accuracy than the within-pool GP approach. Different from expectation, method BayesB did not outperform genomic best linear unbiased prediction (GBLUP). In the third study, we analyzed two backcross (BC) families derived from a resistant and a susceptible recurrent parent. Both BC populations differed substantially in their means for all traits, suggesting that the two recurrent parents have different QTL alleles for GER resistance. Relatively high correlations were observed between DON and ZON concentrations measured by immunoassays and GER visual severity scoring and NIRS (near-infrared spectroscopy) within each BC population. Thus, the mycotoxin content in grain can reliably be reduced by directional selection for GER severity and NIRS measurements that are less expensive and less laborious. In conclusion, GER resistance in European maize germplasm can be effectively improved through breeding with resistant donor lines. GER visual severity scoring and NIRS measurements were found to be reliable predictors for DON and ZON concentrations in grain. We observed that QTL for GER and DON are mostly specific to a few families or a limited number of materials, whereas QTL for DS are more commonly shared between families. The multi-parental QTL mapping approach is complementary to the classical bi-parental QTL mapping in that the latter has generally higher power to identify rare but large-effect QTL for traits such as GER and DON, whereas the former is superior in detecting common but small-effect QTL for traits such as DS. Composing the TS with materials more closely related to the prediction set and increasing the TS size generally resulted in higher prediction accuracy for MAS and GS, irrespective of the trait and statistical model.
Improvements of the doubled haploid technology in maize
(2019) Molenaar, Willem; Melchinger, Albrecht E.
The in vivo doubled haploid (DH) technology in maize carries many advantages over traditional line development by recurrent selfing and has played an integral role in numerous breeding programs since the early 21st century. A bottleneck in the DH technology is still the success rate of chromosome doubling treatment, which has a strong influence on the costs of DH production. Currently, only a minority (~10%) of treated D0 haploid plants result in DH lines. Improvement in the chromosome doubling step of DH production would not only make DH lines cheaper, but could also change the optimum allocation of resources in hybrid breeding. In addition, the development of treatments using alternative doubling agents to colchicine, which is toxic to humans, would improve worker safety and simplify waste disposal issues for developing countries to benefit from the DH technology. Initiating such developments is the goal of this thesis. In a first step, we evaluated anti-mitotic herbicides with different modes of action as alternatives to colchicine for reducing the toxicity of chromosome-doubling treatment and for potentially increasing the success rates. In a series of experiments, we evaluated anti-mitotic herbicides with different modes of action in different concentrations and combinations. Based on the results of the initial experiments, we chose a specific concentration of amiprophos-methyl for evaluation in combination with varying concentrations of pronamide in a further experiment. This revealed the optimal concentration of pronamide in combination with the chosen concentration of amiprophos-methyl. However, this less-toxic treatment showed slightly lower success rates and slightly higher costs per DH line as compared to the standard colchicine treatment. In a second step after evaluating anti-mitotic herbicides for seedling treatment, we evaluated gaseous treatments using nitrous oxide (N2O), an anti-mitotic gas, in varying concentrations and combinations with air and pure oxygen. In two years of evaluation, we found an N2O treatment which had similar success rates as colchicine. The major benefit of such treatment is that this gas can simply be released into the atmosphere, eliminating the difficulty of proper chemical waste disposal, which is difficult to secure in developing countries. The only requirement is a treatment chamber, in contrast to the laboratory facilities required for handling colchicine. In a third step, we evaluated the potential of spontaneous chromosome doubling (SCD) as an alternative to chemical treatment-based chromosome doubling. Although previous studies found significant genetic variation and high heritability for SCD, a classical quantitative genetic analysis, elucidating the type of gene action governing this trait, and a selection experiment for improving SCD was missing in the literature. We found a predominance of additive genetic effects compared to epistatic effects, and a large selection gain after three cycles of recurrent selection for SCD to levels far beyond those reached by standard colchicine treatment. This indicates the great potential of SCD to improve the DH technology. The approximately ten-fold increase in spontaneous chromosome doubling rate (SDR) reached in our recurrent selection experiment marks a paradigm shift in the chromosome doubling step of DH production in maize. DH production efficiency can be greatly increased by the vast improvement in SDR, and production can be further simplified to enable even higher throughput. Instead of chromosome doubling treatment, which involves much handling of seedlings, haploid seeds from germplasm with a high innate ability to produce seed set without chemical treatment can be simply seeded in the DH nursery, eliminating the most costly production steps. Thus, this thesis has provided new opportunities to increase worker safety and reduce toxic waste in DH production, and further provided a proof of concept for genetic improvement of spontaneous chromosome doubling, which has great prospects for increasing the efficiency of DH production in maize.
Strategies for sustainable pearl millet hybrid breeding in West Africa
(2020) Sattler, Felix; Haussmann, Bettina
Pearl millet [Pennisetum glaucum (L.) R. Br.] is grown by >90 million subsistence farmers, mostly in the drylands of Sub-Saharan Africa and India for human consumption and provides additionally fodder and building material. It is commonly grown in regions with 300 – 500 mm of precipitation, low soil phosphorus levels, and temperatures of >42°C), like its center of origin in West Africa (WA). Pearl millet is a highly heterozygous, diploid (2n = 2x = 14) C4 plant species with outcrossing rates of >70%. Yield levels increased largely in India and the US, while they almost stagnated in WA. Challenging, highly variable environments and a weak seed sector are largely contributing to these differences. To suggest a way forward this thesis was meant to guide heterotic group development for sustainable WA pearl millet breeding. The specific objectives were to (I) facilitate efficient use of pearl millet gene bank accessions, (II) identify diversity patterns, (III) validate the yield superiority and stability of pearl millet population hybrids over OPVs, (IV) derive a more comprehensive picture about combining ability patterns, and (V) develop a unified strategy for heterotic grouping and sustainable hybrid breeding. A total of 81 accessions acquired from the pearl millet reference collection was evaluated for resistance to Striga hermonthica (Del.) Benth. in one artificially infested field in Niger. A subset of 74 accessions was characterized in 2009 in multi-environment trials (MET) under low-input and fertilized conditions. The general superiority of local check varieties compared to the genebank accessions highlighted the importance of local adaptation, possibly lost during the ex situ conservation and regeneration. Nevertheless, the development and preservation of germplasm collections are important to maintain the rich genetic diversity. The MET identified several accessions as sources for specific traits of interest and revealed an immense diversity but also strong admixture. This admixture underlines the need to develop heterotic groups. Therefore, 17 WA open-pollinated varieties (OPVs) were crossed in a diallel mating design and tested together with their offspring in nine environments over two years in Niger and Senegal. Results from these MET verified large panmictic better parent heterosis (PBPH) effects with an average of 18% (1–47%) for panicle yield. A large G × E interaction variance was confirmed and it was not possible to define repeatable mega-environments. Importantly, yield stability was more pronounced in the population hybrids compared to their parental OPVs. Furthermore, a superior combining ability among selected OPVs from Niger vs. Senegal was revealed and the evaluated OPVs were clearly grouped by origin based on genetic information. Nevertheless, there was no significant relationship between genetic distance among OPVs and PBPH. These and earlier studies showed a large diversity, sufficiently large heterosis effects and high yield stability in experimental pearl millet population and topcross hybrids, offering a great opportunity for a regionally coordinated hybrid breeding approach. Therefore, we suggested a unified strategy with a continuous output of different hybrid types, specifically tailored to WA. First, existing diversity and combining ability pattern information on western WA and eastern WA cultivars forming loose groups that combine well with each other should be used. Selected genotypes with high general combining ability (GCA) and per se performance from eastern and western WA, respectively, are promising founder populations. Initiating a reciprocal recurrent selection (RRS) program, possibly supported by modern breeding tools, will diverge the two groups further, while improving the inter-pool per se performance. RRS in combination with continuous diversification of both pools allows distinct female and male pool development, line development and introgression of a cytoplasmic male sterility system. Creating OPVs and population hybrids early and aiming for long-term development of topcross hybrids from improved OPVs and newly derived lines is possible alongside the heterotic pool development. Additionally, the RRS lays the foundation for possible future single-cross hybrid breeding programs. The suggested framework is highly ambitious and requires long-term commitment, vision and financial resources. Considering the flexibility regarding single steps and the possibility to develop different types of varieties at every stage of the pool diversification, it has the potential to enhance gains from selection and, with the continuous output of new high-yielding and stable cultivars, to improve the livelihood of WA subsistence farmers substantially.
Comparison of omics technologies for hybrid prediction
(2019) Westhues, Matthias; Melchinger, Albrecht E.
One of the great challenges for plant breeders is dealing with the vast number of putative candidates, which cannot be tested exhaustively in multi-environment field trials. Using pedigree records helped breeders narrowing down the number of candidates substantially. With pedigree information, only a subset of candidates need to be subjected to exhaustive tests of their phenotype whereas the phenotype of the majority of untested relatives is inferred from their common pedigree. A caveat of pedigree information is its inability to capture Mendelian sampling and to accurately reflect relationships among individuals. This shortcoming was mitigated with the advent of marker assays covering regions harboring causal quantitative trait loci. Today, the prediction of untested candidates using information from genomic markers, called genomic prediction, is a routine procedure in larger plant breeding companies. Genomic prediction has revolutionized the prediction of traits with complex genetic architecture but, just as pedigree, cannot properly capture physiological epistasis, referring to complex interactions among genes and endophenotypes, such as RNA, proteins and metabolites. Given their intermediate position in the genotype-phenotype cascade, endophenotypes are expected to represent some of the information missing from the genome, thereby potentially improving predictive abilities. In a first study we explored the ability of several predictor types to forecast genetic values for complex agronomic traits recorded on maize hybrids. Pedigree and genomic information were included as the benchmark for evaluating the merit of metabolites and gene expression data in genetic value prediction. Metabolites, sampled from maize plants grown in field trials, were poor predictors for all traits. Conversely, root-metabolites, grown under controlled conditions, were moderate to competitive predictors for the traits fat as well as dry matter yield. Gene expression data outperformed other individual predictors for the prediction of genetic values for protein and the economically most relevant trait dry matter yield. A genome-wide association study suggested that gene expression data integrated SNP interactions. This might explain the superior performance of this predictor type in the prediction of protein and dry matter yield. Small RNAs were probed for their potential as predictors, given their involvement in transcriptional, post-transcriptional and post-translational regulation. Regardless of the trait, small RNAs could not outperform other predictors. Combinations of predictors did not considerably improve the predictive ability of the best single predictor for any trait but improved the stability of their performance across traits. By assigning different weights to each predictor, we evaluated each predictors optimal contribution for attaining maximum predictive ability. This approach revealed that pedigree, genomic information and gene expression data contribute equally when maximizing predictive ability for grain dry matter content. When attempting to maximize predictive ability for grain yield, pedigree information was superfluous. For genotypes having only genomic information, gene expression data were imputed by using genotypes having both, genomic as well as gene expression data. Previously, this single-step prediction framework was only used for qualitative predictors. Our study revealed that this framework can be employed for improving the cost-effectiveness of quantitative endophenotypes in hybrid prediction. We hope that these studies will further promote exploring endophenotypes as additional predictor types in breeding.
Characterization of genetic variation among Ethiopian barley (Hoerdeum vulgare L.) genotypes
(2019) Abtew, Wosene Gebreselassie; Knierim, Andrea
Barley (Hordeum vulgare L.) is a major cereal crop in Ethiopia and accounts for 8% of the total cereal production based on cultivation area. Farmers may face unpredictable rainfall and drought stress patterns such as terminal drought where rainfall ends before crops have completed their physiological maturity, which then poses a challenge to crop production. The absence of efficient weather forecasts and a lack of efficient communication channels for resource-poor farmers ask for the development of varieties that are robust to such irregularities. A goal of plant breeding for areas with variable climate and limited resources for agricultural inputs is to produce stable varieties with higher average yield across diverse environments and growing conditions. Genotype by environment (G x E) interactions, however, frequently interfere with the selection of widely adapted genotypes. Knowledge about the yield stability of existing Ethiopian barley varieties and landraces under changing environmental variables is important for the future development of barley varieties with high and stable yields. In addition, yield components are quantitative with substantial influence of environment. Yield components also compensate each other in trait correlation dynamics. Since grain yield is a more complex trait than its components, environmental effects and genotype-by-environment (G x E) interactions for grain yield are stronger than for its components. Therefore, indirect selection of yield components may be more efficient than selection on grain yield per se to obtain higher yielding and stable cultivars. A study, therefore, was initiated to 1) characterize the response of a diverse set of barley genotypes to different locations and variable planting dates and identify genotypes with wide adaptation and stable performance and/or genotypes with specific altitude and planting date 2) determine traits that contribute to high and stable yields across a range of different environments and planting dates 3) determine the pattern of population structure and genetic parameters among genotypes conserved in Ethiopian and German gene banks in for different period of time as well as currently growing in farmers’ field. In order to meet the objectives 18 genotypes were tested at four different sowing dates with 15 days interval in different locations (Ambo and Jimma) and years (2012 and 2013). The tested genotypes revealed a wide variation for both static and dynamic yield stability measures. Compared to improved cultivars, farmers landraces displayed higher average static stability and similar superiority indices (dynamic stability). These landraces are therefore a source of germplasm for breeding resilient barley cultivars. Staggered planting proved to be a useful method for evaluating genotype stability across environmental factors beyond location and season. In addition, we also noticed that compensatory relationship between kernels per spike and thousand kernel weight in landraces. Kernels per spike and number of fertile tillers can be proposed as robust traits in barley breeding for a wider adaptation as they had significant and consistent positive total effects on grain yield. In order to determine the pattern of population structure and genetic parameters among genotypes of different origin and gene banks, DNA samples were subject to double-digest by ApeK1 and Hind III enzymes. After sequencing, raw read was checked for major quality parameters. Sequence reads were then filtered for sequencing artifacts and low quality reads (preprocessing). The pre-processed reads were aligned to genome of barley cultivar Morex to call SNPs. Values of observed heterozygosity (Ho) ranged from 0.250 to 0.337 and were higher than the expected heterozygosity (He) that varied from 0.180 to 0.242 in genotypes of all origins. The inbreeding coefficient (FIS) values that ranged between -0.240 and -0.639 across the regions were also higher and negative suggesting existence of excess outcrossing than expected. Based on the inferred clusters by the ADMIXTURE, high Fst values were observed between clusters suggesting high genetic differentiation among the genotypes tested though differentiation was not based on location. In addition, genetic differentiation computed based on the predetermined location, altitude and source of genotypes suggested weak differentiation among the groups. These results indicate that, in Ethiopia, barley genetic variation between regions and altitudes were less pronounced than within region and altitude variations. This calls for the germplasm collection strategies to be cautious in considering location and altitude as a main factor of variation thus strategies should focus on exploiting the within region variation also for better germplasm conservation and utilization. The static yield stability of landrace has to be utilized by breeders for their wider recommendations for those farmers who cannot afford use of farm inputs and specific cultivars. In addition, the relative robustness as well as plasticity of traits sorted by the current study can be incorporated in the breeding strategy of barley in Ethiopia.
Stirring up sorghum hybrid breeding targeting West African smallholder farmers low input environments
(2019) Kante, Papa Ndiaga Moctar; Haussmann, Bettina
Food supply and income in rural areas of West Africa (WA) depend strongly on the local production, and mostly on farmers’ field production of root and tuber crops, and cereals. To feed an ever-increasing population in a context of climate-change and low-input cultural conditions, breeding for resilient crops can guarantee smallholder farmers food security and cash income for a sustainable rural development. Sorghum hybrids for WA were first explored in the early 1970s and hybrid crosses of Malian landraces with introduced Caudatum-race seed parents were evaluated in the early 80s. Although those hybrids exhibited good heterosis for grain yield, their lack of grain quality made them commercially unsustainable. Efforts by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) and its partners resulted in the first series of Guinea-race based hybrids. The short statured hybrids were evaluated in several on-farm farmer-managed yield trials, and showed satisfactory grain yield and quality under farmers’ cultivation conditions. Although taller- relative to shorter- height sorghum can help reduce risks of panicle loss by grazing transhumant cattle, no indication on the yield potential of the tall statured hybrids is available. The advances achieved by ICRISAT and its partners in hybrid development justified establishing a long-term hybrid breeding program to provide farmers with hybrids with sufficient grain yield and good grain quality under low input conditions. However, the lack of quantitative genetic information about the genetic value of new experimental hybrids and their parents (Guinea-Caudatum to complete Guinea background, from different WA origins), or about the efficiency of alternative selection methods for targeting yield performance in the predominantly low-input and phosphorous-deficient sorghum production conditions hinders sorghum hybrid development for this region. Sorghum hybrid breeding was commercially feasible only after the identification of a heritable and stable cytoplasmic male sterility (CMS) mechanism. Hybrid breeding in WA can benefit from molecular marker, especially for the fertility restoration/sterility maintenance of the predominant A1-type of CMS. The major outcomes of this thesis are presented as follow: Mean yields of tall hybrids were 3 to 17% (ranging from 6 to 28 g m−2) higher than that of the local check across all 37 on-farm farmer-managed environments and were highest (14–47%) averaged across the seven trials with the lowest mean yields. The yields of the new set of experimental hybrids were substantially superior to farmers’ local Guinea-race varieties, with 20 to 80% higher means over all hybrids in both low phosphorus (LP) and high phosphorus (HP) environments. Average mid-parent and better-parent heterosis estimates were respectively 78 and 48% under HP, and 75 and 42% under LP. Direct selection under LP was predicted to be 20 to 60% more effective than indirect selection under HP conditions, for hybrid performance under LP. The combining ability estimates provide initial insights into the potential benefit of germplasm from further east in West and Central Africa for developing a male parental pool that is distinct and complimentary to the Malian female pool. On chromosome SBI-05, we found a major A1 CMS fertility restorer locus (Rf5) explaining 19 and 14% of the phenotypic variation in either population. Minor quantitative trait loci (QTL) were detected in these two populations on chromosomes SBI-02, SBI-03, SBI-04 and SBI-10. In the third population, we identified one major A1 CMS fertility restorer locus on chromosome SBI-02, Rf2, explaining 31% of the phenotypic variation in the F2 mapping population. Pentatricopeptide repeat genes in the Rf2 QTL region were sequenced, and we detected in Sobic.002G057050 a missense mutation in the first exon, explaining 81% of the phenotypic variation in an F2:3 validation population and clearly separating B- from R-lines. The Guinea-race hybrids’ substantial yield superiorities over well adapted local Guinea-race varieties suggests that a strategy of breeding hybrids based on Guinea-germplasm can contribute to improving the livelihood of many smallholder farmers in WA. Although the usefulness of direct selection under LP for hybrid performance in the predominantly P-limited target environments was proven, companion evaluations of hybrids under HP would be desirable to identify also new hybrids that can respond to improved fertility conditions for sustainable intensification. The developed KASP marker stands as a promising tool for routine use in WA breeding programs.
Genomic selection in synthetic populations
(2017) Müller, Dominik; Melchinger, Albrecht E.
The foundation of genomic selection has been laid at the beginning of this century. Since then, it has developed into a very active field of research. Although it has originally been developed in dairy cattle breeding, it rapidly attracted the attention of the plant breeding community and has, by now (2017), developed into an integral component of the breeding armamentarium of international companies. Despite its practical success, there are numerous open questions that are highly important to plant breeders. The recent development of large-scale and cost-efficient genotyping platforms was the prerequisite for the rise of genomic selection. Its functional principle is based on information shared between individuals. Genetic similarities between individuals are assessed by the use of genomic fingerprints. These similarities provide information beyond mere family relationships and allow for pooling information from phenotypic data. In practice, first a training set of phenotyped individuals has to be established and is then used to calibrate a statistical model. The model is then used to derive predictions of the genomic values for individuals lacking phenotypic information. Using these predictions can save time by accelerating the breeding program and cost by reducing resources spent for phenotyping. A large body of literature has been devoted to investigate the accuracy of genomic selection for unphenotyped individuals. However, training individuals are themselves often times selection candidates in plant breeding, and there is no conceptual obstacle to apply genomic selection to them, making use of information obtained via marker-based similarities. It is therefore also highly important to assess prediction accuracy and possibilities for its improvement in the training set. Our results demonstrated that it is possible to increase accuracy in the training set by shrinkage estimation of marker-based relationships to reduce the associated noise. The success of this approach depends on the marker density and the population structure. The potential is largest for broad-based populations and under a low marker density. Synthetic populations are produced by intermating a small number of parental components, and they have played an important role in the history of plant breeding for improving germplasm pools through recurrent selection as well as for actual varieties and research on quantitative genetics. The properties of genomic selection have so far not been assessed in synthetics. Moreover, synthetics are an ideal population type to assess the relative importance of three factors by which markers provide information about the state of alleles at QTL, namely (i) pedigree relationships, (ii) co-segregation and (ii) LD in the source germplasm. Our results show that the number of parents is a crucial factor for prediction accuracy. For a very small number of parents, prediction accuracy in a single cycle is highest and mainly determined by co-segregation between markers and QTL, whereas prediction accuracy is reduced for a larger number of parents, where the main source of information is LD within the source germplasm of the parents. Across multiple selection cycles, information from pedigree relationships rapidly vanishes, while co-segregation and ancestral LD are a stable source of information. Long-term genetic gain of genomic selection in synthetics is relatively unaffected by the number of parents, because information from co-segregation and from ancestral LD compensate for each other. Altogether, our results provide an important contribution to a better understanding of the factors underlying genomic selection, and in which cases it works and what information contributes to prediction accuracy.
Factors influencing the accuracy of genomic prediction in plant breeding
(2017) Schopp, Pascal; Melchinger, Albrecht E.
Genomic prediction (GP) is a novel statistical tool to estimate breeding values of selection candidates without the necessity to evaluate them phenotypically. The method calibrates a prediction model based on data of phenotyped individuals that were also genotyped with genome-wide molecular markers. The renunciation of an explicit identification of causal polymorphisms in the DNA sequence allows GP to explain significantly larger amounts of the genetic variance of complex traits than previous mapping-based approaches employed for marker-assisted selection. For these reasons, GP rapidly revolutionized dairy cattle breeding, where the method was originally developed and first implemented. By comparison, plant breeding is characterized by often intensively structured populations and more restricted resources routinely available for model calibration. This thesis addresses important issues related to these peculiarities to further promote an efficient integration of GP into plant breeding.
Evaluation of association mapping and genomic prediction in diverse barley and cauliflower breeding material
(2018) Thorwarth, Patrick; Schmid, Karl J.
Due to the advent of new sequencing technologies and high-throughput phenotyping an almost unlimited amount of data is available. In combination with statistical methods such as Genome-wide association mapping (GWAM) and Genomic prediction (GP), these information can provide valuable insight into the genetic potential of individuals and support selection and crossing decisions in a breeding program. In this thesis we focused on the evaluation of the aforementioned methods in diverse barley (Hordeum vulgare L.) and cauliflower (Brassica oleracea var. botrytis) populations consisting of elite material and genetic resources. We concentrated on the dissection of the influence of specific parameters such as marker type, statistical models, influence of population structure and kinship, on the performance of GWAM and GP. For parts of this thesis, we additionally used simulated data to support findings based on empirical data. First, we compared four different GWAM methods that either use single-marker or haplotypes for the detection of quantitative trait loci in a barley population. To find out the required population size and marker density to detect QTLs of varying effect size, we performed a simulation study based on parameter estimates of the empirical population. We could demonstrate that already in small populations of about 100 individuals, QTLs with a large effect can be detected and that at least 500 individuals are necessary to detect QTLs with an effect < 10%. Furthermore, we demonstrated an increased power of haplotpye based methods in the detection of very small QTLs. In a second study we used a barley population consisting of 750 individuals as training set to compare different GP models, that are currently used by scientists and plant breeders. From the training set 33 offspring families were derived with a total of 750 individuals. This enabled us to assess the prediction ability not only based on cross-validation but also in a large offspring population with varying degree of relatedness to the training population. We investigated the effects of linkage disequilibrium and linkage phase, population structure and relatedness of individuals, on the prediction ability. We could demonstrate a strong effect of the population structure on the prediction ability and show that about 11,203 evenly spaced SNP markers are necessary to predict even genetically distant populations. This implies that at the current marker density prediction ability is based on the relatedness of the individuals. In a third study we focused on the evaluation of GWAM and GP in cauliflower. We focused on the evaluation of genotyping-by-sequencing and compared the influence of imputation methods on the prediction ability and the number of significant associations. We obtained a total 120,693 SNPs in a random collection of 174 cauliflower genebank accessions. We demonstrated that imputation did not increase prediction ability and that the number of detected QTLs only slightly differed between the imputed and the unimputed data set. GP performed well even in such a diverse gene bank sample, but population structure again influenced the prediction ability. We could demonstrate the usefulness and limitations of Genome-wide association mapping and genomic prediction in two species. Even though a lot of research in the field of statistical genetics has provided valuable insight, the usage of Genomic prediction should still be applied with care and only as a supporting tool for classical breeding methods.
Pearl millet breeding in West Africa : steps towards higher productivity and nutritional value
(2018) Pucher, Anna Ida; Haussmann, Bettina
The enormous human population growth in West Africa (WA) in combination with serious production constraints is very problematic condition for food security. The alarming status of micronutrient deficiency in WA exacerbates this situation. For smallholder farmers improved and nutritious crop varieties derived from plant breeding could be a major contributor to enhancing agricultural productivity and reducing malnutrition. Pearl millet (Pennisetum glaucum (L.) R. Br.) is due to its high tolerance to drought and heat, capable to grow under very harsh environments, and is therefore a staple crop in Sahelian WA. Development of multiple pearl millet breeding approaches will be crucial to exploit the potential of this crop.The main goal of this study was to establish a scientific basis for more efficient pearl millet breeding in WA with a specific focus on achieving higher productivity and nutritional value. In order to accomplish this goal, the following objectives were defined: (I) to characterize a broad set of WA pearl millet accessions and to investigate their diversity and geographic patterns based on their phenotype; (II) to identify the potential and strategies to increase the micronutrient level in WA pearl millet; (III) to evaluate the performance of population hybrids and to derive initial strategies of pearl millet hybrid breeding in WA based on combining ability and heterotic patterns; and (IV) to identify molecular markers for the male-fertility restoration locus (Rf) for the A4 cytoplasmic-genic male-sterility (CMS) system in pearl millet using a genotyping-by-sequencing (GBS) based linkage map. The major results and conclusions of the four studies are summarized in the following:Characterization of a collection of 360 WA pearl millet landraces at six sites in WA identified wide ranges for 12 agro-morphological traits, which indicated a tremendous diversity. Principal component analysis revealed very large diversity within individual countries, and a high genetic admixture among WA pearl millet landraces. The high admixture indicates that heterotic grouping based on morphological distance or geographic distance is not possible. The published data of this study gives national breeders a basis to utilize this germplasm.In the second trial, pearl millet grain iron and zinc densities showed significant genetic variation in a set of 72 WA landraces evaluated at three sites and moderate-to-high heritability, which emphasizes a high potential for biofortification. Identified landraces with moderately high and stable micronutrient densities appear suitable for use in future WA biofortification breeding. Due to significant positive correlations among grain iron, zinc and other mineral densities and non-significant correlations between grain yield and mineral densities, selection for high grain iron and zinc density can be performed simultaneously without a negative effect on grain yield or contents of other micronutrients.The third trial evaluated 100 population hybrids and their 20 parental populations (with four parental open-pollinated varieties from each of five WA countries) at six environments and showed hybrid superiority of, on average, 16.7% compared to their parental populations (with a max. of 73%), reflecting the great potential of hybrid breeding. The mean grain yield of hybrids based on inter-country crosses did not differ significantly from intra-country crosses. Geographic distance between parents was not correlated with panmictic midparent heterosis, indicating that heterotic grouping based on geographic distance is not expedient. However, crosses between accessions from Niger/Nigeria and Senegal were outstanding, thus initial heterotic pools could be based on this information. In the long term, sustainable pearl millet hybrid breeding will require combining ability studies to develop heterotic groups.Within the fourth trial, a high-density linkage map based on single nucleotide polymorphism (SNP) markers produced by GBS was generated using a F2 mapping population, which segregated for fertility restoration of the A4 CMS system. A major Rf locus was found on linkage group 2, which was verified by cross-validation showing a very high quantitative trait locus (QTL) occurrence (97%). The QTL explained 14.5% of the phenotypic variance, which was below expectation because the segregation ratio of male-fertile and male-sterile plants (3:1) indicated monogenic dominant inheritance of this trait. The two KASP markers developed for the QTL will support high-throughput screening for the Rf locus and will facilitate the development of male parental pools exhibiting the fertility restoration, which is an essential step to enable economic pearl millet hybrid seed production. We can conclude that WA pearl millet breeding has the potential to increase the pearl millet productivity and nutritional value by utilizing the enormous pearl millet diversity in hybrid and biofortification breeding programs.
Characterization and management of Jatropha curcas L. germplasm
(2018) Senger, Elisa; Melchinger, Albrecht E.
Jatropha curcas L. (jatropha) is a perennial plant of the Euphorbiaceae family that grows in the tropics and subtropics worldwide. Jatropha is targeted to be grown in marginal environments. The seeds are used mainly for production of food products and bioenergy, amongst others. Jatropha breeding is at an early stage. The first obstacle is to generate competitive cultivars for economically feasible cultivation. Mayor breeding objectives are to increase seed yield and yield stability, to decrease production costs, and to improve product quality adapted to specific markets. Jatropha breeding needs to be optimized in several research areas, such as methods and tools for germplasm characterization and breeding techniques, while considering requirements of the agronomic management and product processing. The germplasm can be separated into two naturally occurring germplasm pools that differ in the presence of phorbol esters (PE). These chemical compounds have antinutritional effects on humans and animals and cannot be inactivated or eliminated from the plant material on an industrial scale yet. Therefore, food production is based on cultivars lacking PE, while bioenergy production is less affected from PE presence. The germplasm needs to be characterized and grouped depending on breeding objectives and strategies. Tools for identification of plants that synthesize PE exist, but bear decisive disadvantages or need to be advanced. These tools are exploited for germplasm management and food safety strategies. The objectives of this study were to i) examine the variation of relevant traits among genotypes and between germplasm pools, ii) estimate phenotypic and genotypic trait correlations, iii) investigate location effects and genotype by environment interactions, iv) investigate parental and heterotic effects of genotypes from different germplasm pools as well as the effect of the mating type on expression of relevant traits, and v) develop recommendations for implementation of the findings in jatropha breeding programs. In the first two publications, stress response was investigated. Leaf chlorophyll content (SPAD) was used as a dynamic trait that can be influenced by e.g. water stress and nutrient deficiency. Different genotypes were screened at several locations and at different time points. High genetic diversity was found not only in stress response but also in SPAD value. The fast and non-destructive method is highly promising to be applied in further screenings or stress response studies. In the second publication, genotypic differences in aluminum tolerance were found among seedlings in a greenhouse trial. The rapid test method is applicable in further screenings. However, it needs to be proven that aluminum tolerance at the seedling stage observed under greenhouse conditions is expressed also at later plant developmental stages in the field. In the consecutive three publications, several traits were assessed on seeds and seedlings to detect significant differences between genotypes and/or between germplasm pools. Such traits would be highly valuable for germplasm management. We found that random variation is a disadvantage of quantitative traits and hinders clear assignment of each experimental unit to the respective germplasm pool. Thus, qualitative traits might be favored, such as the “silver shimmer inside the seed testa” that differentiated toxic from non-toxic seeds with a low error rate. However, these results need to be validated. Another application area of the investigated traits is the identification of self-fertilized material within hybrid progeny. In our study, self-fertilized seeds could be differentiated from cross-fertilized ones in specific genotype combinations. Similarly, many seedling traits showed heterotic effects. In the sixth publication, genotype by environment interactions were investigated and recommendations for breeding programs elaborated. A large set of genotypes was grown for four years at three different locations. We showed that selection at only one testing location is highly risky because cultivars with low yield stability could be selected. Therefore, it is indispensable for breeders to work in a network of testing locations that differ in edapho-climatic conditions and apply appropriate experimental designs and statistical tools. In the final publication, several parameters related to the nutritional value of kernels of non-toxic genotypes grown at two locations were assessed. The high nutritional value of this material was presented and compared to soybean, peanut, hazelnut, and corn. Furthermore, preliminary conclusions related to location effects and product processing were drawn. The findings of this thesis contribute to characterization of this novel crop with regard to stress tolerances, seed and seedling characteristics as well as food quality, and help to increase breeding efficiency by presenting simple methods for fast genotype screening as well as grouping of germplasm and by efficient exploitation of testing facilities.
Speciation and domestication genomics of Amaranthus spp.
(2017) Stetter, Markus; Schmid, Karl J.
The genus Amaranthus consists of 50 to 70 species, including several cultivated and weedy species. The seeds of the three grain amaranth species, A. caudatus, A. cruentus and A. hypochondriacus have a high nutritional value and are gluten free. In this work, three main aspects of amaranth genetics are studied, because previous work was limited to few species and few genetic markers: First, the evolutionary relationship between species in the genus; second, the domestication syndrome of South American grain amaranth; and third, crossing methods and controlled growth conditions for amaranth breeding. The genus has been taxonomically split into three subgenera, A. Amaranthus, A. Albersia and A. Acnida. Together with their two relatives A. hybridus and A. quitensis, the three grain amaranths form the Hybridus complex within the A. Amaranthus subgenus. We used genotyping by sequencing (GBS) of 94 genebank accessions, representing 35 species to infer the phylogeny of Amaranthus. SNPs were called using de novo and reference genome based methods and genome sizes of the species were measured using flow cytometry. The analysis of genome size evolution within the genus revealed that with the exception of two lineages polyploidization played a minor role in the history of the genus. A distancebased neighbor joining tree of individual accessions and a species tree based on the multispecies coalescent were constructed. Both phylogenies supported the previous taxonomic classification into three subgenera, but split the A. Acnida subgenus into two distant groups. Analyzing the Hybridus complex gave insights into the domestication history of grain amaranth. The complex was well separated from the other species in the A. Amaranthus subgenus and included the three grain amaranth species and their wild relatives. Individuals within the Hybridus complex did not cluster by species, but rather by their geographic origin from South and Central America. Geographically separated lineages of A. hybridus appeared to be the common ancestor of the three cultivated grain amaranths, while A. quitensis was involved in the domestication of A. caudatus. The domestication of grain amaranth remains unclear and seems to be complex, because the domestication syndrome that differentiates crops from their wild ancestors is only weakly pronounced. Therefore, the domestication syndrome in South American grain amaranth (A. caudatus) was studied by characterizing genetic and phenotypic diversity of A. caudatus and the two potential wild relatives, A. hybridus and A. quitensis. To investigate the evolutionary relationship of A. caudatus and its potential wild ancestors, 119 amaranth accession from the Andean region were characterized using 9,485 GBS derived SNPs. Additionally, two domestication related phenotypes, seed color and seed size, were analyzed. None of the accessions of wild amaranths had white seeds, while this was the predominant seed color in A. caudatus. The seed size did not significantly differ between species, but a genetically distinct group of A. caudatus from Bolivia had significantly larger seeds than the other groups. The genetic analysis revealed a strong differentiation of A. caudatus from its wild relatives. The two relatives did not cluster according to their species assignment, but rather by their geographic origins from Peru and Ecuador. Surprisingly, A. caudatus had a higher genetic diversity than its two close relatives and shared a high proportion of polymorphisms with them, consistent with the absence of strong bottlenecks or high levels of gene flow between them. Efficient crosses are an essential tool for plant research and breeding. Three different crossing methods (open pollination, hot water emasculation and hand emasculation) were evaluated for their efficiency and validated with low cost genetic markers. We identified controlled growth conditions for amaranth that allow short generation times of only six weeks instead of six months. All three crossing methods successfully produced offspring, but with different success rates. Open pollination had the lowest (10%) and hand emasculation the highest success rate (74%). Hot water emasculation showed an intermediate success rate (26%), but high maximum of 94%. Additionally, this method is easy to perform and suitable for large-scale hybrid production. Eleven PCR-based SNP markers were found to be sufficient for intra- and interspecific hybrid identification. Despite the very small flowers, amaranth crosses can be carried out efficiently and verified with inexpensive SNP markers with short generation times under suitable conditions. The phylogeny and population genetic analysis suggest that amaranth domestication was incomplete. Gene flow from A. quitensis into A. caudatus might have prevented the fixation of domestication related alleles. The genus phylogeny and the over 200 genotyped accessions in this work provide the largest genomic resource for amaranth so far.
The genetic basis of heat tolerance in temperate maize (Zea mays L.)
(2016) Frey, Felix P.; Stich, Benjamin
The global mean temperature and probability of heat waves are expected to increase in the future, which has the potential to cause severe damages to maize production. To elucidate the genetic mechanisms of the response of temperate maize to heat stress and for the tolerance to heat stress, in a first experiment I applied gene expression profiling. Therewith, I investigated the transcriptomic response of temperate maize to linearly increasing heat levels. Further, I identified genes associated with heat tolerance in a set of eight genotypes with contrasting heat tolerance behavior. I identified 607 heat responsive genes, which elucidate the genetic pathways behind the response of maize to heat stress and can help to expand the knowledge of plant responses to other abiotic stresses. Further, I identified 39 genes which were differentially regulated between heat tolerant and heat susceptible inbreds and, thus, are putative heat tolerance candidate genes. Two of these candidate genes were located in genome regions which were associated with heat tolerance during seedling and adult stage that have been detected in QTL studies in the frame of this thesis. Their exact molecular functions, however, are still unknown. The statistical approach to identify heat tolerance genes, presented in my thesis, enables researchers to investigate the transcriptomic response of multiple genotypes to changing conditions across several experiments, considering their natural variation for a quantitative trait. In order to develop more heat tolerant cultivars, knowledge of natural variation for heat tolerance in temperate maize is indispensable. Therefore, heat tolerance was assessed in a set of intra- and interpool Dent and Flint populations on a multi-environment level. Usually, heat stress in temperate Europe occurs during the adult stage of maize. However, as maize is of increasing importance as a biogas crop, farmers can reduce the growth period by postponed sowing after the harvest of the winter cereals in early summer and, thus, sensitive maize seedlings can be exposed to heat stress. Therefore, I aimed to assess heat tolerance in six connected segregating Dent and Flint populations during both developmental stages considering besides multiple environments also multiple traits. At heat stress, I observed an average decrease of 20% of the shoot dry weight during seedling stage and an average of 50% of yield loss, when heat stress was present during adult stage. At the heat locations heat stress was present in the year, when the experiments were conducted as temperatures exceeded 32°C there for more than 400 hours during the growing period in contrast to less than 30 hours at the standard locations. This emphasizes that maize crop production can suffer with the increasing number and intensity of summer heat waves. Furthermore, the study revealed strong differences between genotypes, which was indispensable to differentiate between heat tolerant and heat susceptible inbred lines. The tested genotypes originating from the Flint pool turned out to possess higher heat tolerance during seedling stage, whereas the genotypes derived from the Dent pool possessed higher heat tolerance during adult stage. This fact could be exploited by the maintenance of two pools with contrasting heat tolerance and could be beneficial for hybrid breeding. A direct selection of more heat tolerant genotypes in terms of grain yield is expensive and time-consuming. To facilitate the selection process in order to develop more heat tolerant cultivars, breeders could make use of marker assisted selection. To lay the foundation for this technique, in my thesis, QTL for heat tolerance during adult and during seedling stage were identified with the previously mentioned populations. Two QTL explained 19% of the total variance for heat tolerance with respect to grain yield in a simultaneous fit. Furthermore each two QTL were identified for two principal components, which accounted for heat tolerance during seedling stage. They explained 14 and 12% of the respective variance. The results can be used by breeding companies to develop marker assays in order to select heat tolerant genotypes from their proprietary genetic material during both stages in an initial screening. This would reduce the field capacities considerably, which are needed to test heat tolerance on a field level.

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