agReg-SNPdb-Plants: A Database of Regulatory SNPs for Agricultural Plant Species.

Single nucleotide polymorphisms (SNPs) that are located in the promoter regions of genes and affect the binding of transcription factors (TFs) are called regulatory SNPs (rSNPs). Their identification can be highly valuable for the interpretation of genome-wide association studies (GWAS), since rSNPs can reveal the biologically causative variant and decipher the regulatory mechanisms behind a phenotype. In our previous work, we presented agReg-SNPdb, a database of regulatory SNPs for agriculturally important animal species. To complement this previous work, in this study we present the extension agReg-SNPdb-Plants storing rSNPs and their predicted effects on TF-binding for 13 agriculturally important plant species and subspecies (Brassica napus, Helianthus annuus, Hordeum vulgare, Oryza glaberrima, Oryza glumipatula, Oryza sativa Indica, Oryza sativa Japonica, Solanum lycopersicum, Sorghum bicolor, Triticum aestivum, Triticum turgidum, Vitis vinifera, and Zea mays). agReg-SNPdb-Plants can be queried via a web interface that allows users to search for SNP IDs, chromosomal regions, or genes. For a comprehensive interpretation of GWAS results or larger SNP-sets, it is possible to download the whole list of SNPs and their impact on transcription factor binding sites (TFBSs) from the website chromosome-wise.

Deciphering the Molecular Mechanism Underlying African Animal Trypanosomiasis by Means of the 1000 Bull Genomes Project Genomic Dataset.

African Animal Trypanosomiasis (AAT) is a neglected tropical disease and spreads by the vector tsetse fly, which carries the infectious Trypanosoma sp. in their saliva. Particularly, this parasitic disease affects the health of livestock, thereby imposing economic constraints on farmers, costing billions of dollars every year, especially in sub-Saharan African countries. Mainly considering the AAT disease as a multistage progression process, we previously performed upstream analysis to identify transcription factors (TFs), their co-operations, over-represented pathways and master regulators. However, downstream analysis, including effectors, corresponding gene expression profiles and their association with the regulatory SNPs (rSNPs), has not yet been established. Therefore, in this study, we aim to investigate the complex interplay of rSNPs, corresponding gene expression and downstream effectors with regard to the AAT disease progression based on two cattle breeds: trypanosusceptible Boran and trypanotolerant N'Dama. Our findings provide mechanistic insights into the effectors involved in the regulation of several signal transduction pathways, thereby differentiating the molecular mechanism with regard to the immune responses of the cattle breeds. The effectors and their associated genes (especially MAPKAPK5, CSK, DOK2, RAC1 and DNMT1) could be promising drug candidates as they orchestrate various downstream regulatory cascades in both cattle breeds.

Deciphering Pleiotropic Signatures of Regulatory SNPs in Zea mays L. Using Multi-Omics Data and Machine Learning Algorithms.

Maize is one of the most widely grown cereals in the world. However, to address the challenges in maize breeding arising from climatic anomalies, there is a need for developing novel strategies to harness the power of multi-omics technologies. In this regard, pleiotropy is an important genetic phenomenon that can be utilized to simultaneously enhance multiple agronomic phenotypes in maize. In addition to pleiotropy, another aspect is the consideration of the regulatory SNPs (rSNPs) that are likely to have causal effects in phenotypic development. By incorporating both aspects in our study, we performed a systematic analysis based on multi-omics data to reveal the novel pleiotropic signatures of rSNPs in a global maize population. For this purpose, we first applied Random Forests and then Markov clustering algorithms to decipher the pleiotropic signatures of rSNPs, based on which hierarchical network models are constructed to elucidate the complex interplay among transcription factors, rSNPs, and phenotypes. The results obtained in our study could help to understand the genetic programs orchestrating multiple phenotypes and thus could provide novel breeding targets for the simultaneous improvement of several agronomic traits.

Comparative Investigation of Gene Regulatory Processes Underlying Avian Influenza Viruses in Chicken and Duck.

The avian influenza virus (AIV) mainly affects birds and not only causes animals' deaths, but also poses a great risk of zoonotically infecting humans. While ducks and wild waterfowl are seen as a natural reservoir for AIVs and can withstand most virus strains, chicken mostly succumb to infection with high pathogenic avian influenza (HPAI). To date, the mechanisms underlying the susceptibility of chicken and the effective immune response of duck have not been completely unraveled. In this study, we investigate the transcriptional gene regulation underlying disease progression in chicken and duck after AIV infection. For this purpose, we use a publicly available RNA-sequencing dataset from chicken and ducks infected with low-pathogenic avian influenza (LPAI) H5N2 and HPAI H5N1 (lung and ileum tissues, 1 and 3 days post-infection). Unlike previous studies, we performed a promoter analysis based on orthologous genes to detect important transcription factors (TFs) and their cooperation, based on which we apply a systems biology approach to identify common and species-specific master regulators. We found master regulators such as EGR1, FOS, and SP1, specifically for chicken and ETS1 and SMAD3/4, specifically for duck, which could be responsible for the duck's effective and the chicken's ineffective immune response.

agReg-SNPdb: A Database of Regulatory SNPs for Agricultural Animal Species.

Transcription factors (TFs) govern transcriptional gene regulation by specifically binding to short DNA motifs, known as transcription factor binding sites (TFBSs), in regulatory regions, such as promoters. Today, it is well known that single nucleotide polymorphisms (SNPs) in TFBSs can dramatically affect the level of gene expression, since they can cause a change in the binding affinity of TFs. Such SNPs, referred to as regulatory SNPs (rSNPs), have gained attention in the life sciences due to their causality for specific traits or diseases. In this study, we present agReg-SNPdb, a database comprising rSNP data of seven agricultural and domestic animal species: cattle, pig, chicken, sheep, horse, goat, and dog. To identify the rSNPs, we constructed a bioinformatics pipeline and identified a total of 10,623,512 rSNPs, which are located within TFBSs and affect the binding affinity of putative TFs. Altogether, we implemented the first systematic analysis of SNPs in promoter regions and their impact on the binding affinity of TFs for livestock and made it usable via a web interface.

Unravelling the Complex Interplay of Transcription Factors Orchestrating Seed Oil Content in Brassica napus L.

Transcription factors (TFs) and their complex interplay are essential for directing specific genetic programs, such as responses to environmental stresses, tissue development, or cell differentiation by regulating gene expression. Knowledge regarding TF-TF cooperations could be promising in gaining insight into the developmental switches between the cultivars of Brassica napus L., namely Zhongshuang11 (ZS11), a double-low accession with high-oil- content, and Zhongyou821 (ZY821), a double-high accession with low-oil-content. In this regard, we analysed a time series RNA-seq data set of seed tissue from both of the cultivars by mainly focusing on the monotonically expressed genes (MEGs). The consideration of the MEGs enables the capturing of multi-stage progression processes that are orchestrated by the cooperative TFs and, thus, facilitates the understanding of the molecular mechanisms determining seed oil content. Our findings show that TF families, such as NAC, MYB, DOF, GATA, and HD-ZIP are highly involved in the seed developmental process. Particularly, their preferential partner choices as well as changes in their gene expression profiles seem to be strongly associated with the differentiation of the oil content between the two cultivars. These findings are essential in enhancing our understanding of the genetic programs in both cultivars and developing novel hypotheses for further experimental studies.

In Silico Identification of the Complex Interplay between Regulatory SNPs, Transcription Factors, and Their Related Genes in Brassica napus L. Using Multi-Omics Data.

Regulatory SNPs (rSNPs) are a special class of SNPs which have a high potential to affect the phenotype due to their impact on DNA-binding of transcription factors (TFs). Thus, the knowledge about such rSNPs and TFs could provide essential information regarding different genetic programs, such as tissue development or environmental stress responses. In this study, we use a multi-omics approach by combining genomics, transcriptomics, and proteomics data of two different Brassica napus L. cultivars, namely Zhongshuang11 (ZS11) and Zhongyou821 (ZY821), with high and low oil content, respectively, to monitor the regulatory interplay between rSNPs, TFs and their corresponding genes in the tissues flower, leaf, stem, and root. By predicting the effect of rSNPs on TF-binding and by measuring their association with the cultivars, we identified a total of 41,117 rSNPs, of which 1141 are significantly associated with oil content. We revealed several enriched members of the TF families DOF, MYB, NAC, or TCP, which are important for directing transcriptional programs regulating differential expression of genes within the tissues. In this work, we provide the first genome-wide collection of rSNPs for B. napus and their impact on the regulation of gene expression in vegetative and floral tissues, which will be highly valuable for future studies on rSNPs and gene regulation.

Identification of Age-Specific and Common Key Regulatory Mechanisms Governing Eggshell Strength in Chicken Using Random Forests.

In today's chicken egg industry, maintaining the strength of eggshells in longer laying cycles is pivotal for improving the persistency of egg laying. Eggshell development and mineralization underlie a complex regulatory interplay of various proteins and signaling cascades involving multiple organ systems. Understanding the regulatory mechanisms influencing this dynamic trait over time is imperative, yet scarce. To investigate the temporal changes in the signaling cascades, we considered eggshell strength at two different time points during the egg production cycle and studied the genotype-phenotype associations by employing the Random Forests algorithm on chicken genotypic data. For the analysis of corresponding genes, we adopted a well established systems biology approach to delineate gene regulatory pathways and master regulators underlying this important trait. Our results indicate that, while some of the master regulators (Slc22a1 and Sox11) and pathways are common at different laying stages of chicken, others (e.g., Scn11a, St8sia2, or the TGF- ß pathway) represent age-specific functions. Overall, our results provide: (i) significant insights into age-specific and common molecular mechanisms underlying the regulation of eggshell strength; and (ii) new breeding targets to improve the eggshell quality during the later stages of the chicken production cycle.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species. This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.