Browsing by Person "Schmid, Karl J."

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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.
Identification and analysis of a transcriptome of Douglas-fir (Pseudotsuga menziesii) and population structure inference using different next-generation sequencing techniques
(2015) Müller, Thomas; Schmid, Karl J.
Predictions assume severe changes in the climatic conditions in Central Europe in the coming decades. Longer periods of drought and less precipitation during summer are expected. Plants cannot change their habitat and have to adapt to the new conditions or their offspring has to colonize new ecological niches. Due to the long generation times in trees it is important to know if and how trees can cope with the expected climatic conditions. Forest managers already give thought to the composition of future forests, because they have to choose species and populations which have no or only few problems with the changed climate. Douglas-fir (Pseudotsuga menziesii) is a promising tree species for this purpose, because it is adapted to different habitats and climate zones in its natural distribution range in North America. The two main varieties, coastal and interior Douglas-fir, differ genotypically and phenotypically, e.g. in drought tolerance. Douglas-fir trees, mainly of the coastal variety, showed good growth performances in field trials in Germany. Hence, a research project called "DougAdapt" was designed to analyze and to link genotypic and phenotypic differences in several coastal and interior Douglas-fir provenances. In this project, trees from field trials and from greenhouse experiments were sampled. To analyze the genetic diversity of the provenances we first generated reference sequences. Even with modern and cost-efficient next-generation sequencing technologies it would be very expensive to decipher the ~ 19 gigabases of the Douglas-fir genome completely. An alternative to whole genome sequencing is transcriptome sequencing, in which only the coding regions of a genome are sequenced. The transcriptome sequencing, which was performed for the first time in Douglas-fir, resulted in a large number of putative unique transcripts (PUTs). Comparisons with published transcriptomes of other plant species showed that the PUTs represented the transcriptome of Douglas-fir comprehensively. As the sampled seedlings were part of a drought stress experiment and grew under controlled conditions, we were able to identify drought related candidate PUTs, which may be part of the trees response to drought. Furthermore, more than 27,000 previously unknown single nucleotide polymorphisms (SNPs) in Douglas-fir could be identified. SNPs can influence the phenotype of individuals, and they can be used for instance as markers or to analyze genetic diversity. The analysis of genetic diversity of Douglas-fir provenances and the search for genes which may be part of local adaptation were performed with a sequence capture experiment. In sequence capture only predefined regions of a genome are sequenced. We showed that sequence capture based on PUTs as target regions is applicable in species with large and mostly unknown genomes. The polymorphic drought related candidate PUTs showed higher genetic differentiation than the remaining genes. Nevertheless, none of them was among the candidate PUTs for positive selection, which in turn are probably part of the local adaptation of the trees. Despite a high level of gene flow between coastal and interior provenances, the SNP data showed genetic differentiation between both varieties but only very low differentiation between the coastal provenances. We also investigated if genotyping-by-sequencing (GBS) is a suitable method to detect polymorphisms in Douglas-fir and compared the results of two GBS experiments with the sequence capture. The genome is digested with one or several restriction enzymes in GBS. Afterwards, only fragments with a specific length are sequenced, which considerably reduces the part of the genome that is sequenced as well as the costs. The advantage compared to sequence capture is the possibility to sample more individuals at the same time with less effort and costs. We showed that a digestion with two restriction enzymes results in more SNPs with less missing data, compared to using only one restriction enzyme. Both GBS methods returned considerably less SNPs than the sequence capture. Nevertheless, it was possible to distinguish between southern interior, northern interior, and coastal provenances using SNP data of the GBS experiments. GBS, especially with two restriction enzymes, seems to be a promising approach to genotype a large number of Douglas-fir trees and to obtain SNPs at low costs, which can be used in several tasks like genome-wide association studies. A large amount of sequence data and SNPs were analyzed in this thesis. Together with phenotypic information, these data will be crucial for the analysis of useful traits in Douglas-fir, like drought tolerance. Furthermore, the results concerning the Douglas-fir genome and the genetic diversity of different provenances will be beneficial in breeding programs and association studies, which in turn can be helpful to choose the optimal provenances for a given location.
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.
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.
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.