PrimeRoot: a cutting-edge technology designed to achieve precise and targeted large DNA insertion in plants.

Being the green gold of the future, microalgae and cyanobacteria have recently attracted considerable interest worldwide, for their metabolites such as lipids, protein, pigments, and bioactive compounds have immense potential for sustainable energy and pharmaceutical production capabilities. In the last decades, the efforts attended to enhance the usage of microalgae and cyanobacteria by genetic manipulation, synthetic and metabolic engineering. However, the development of photoautotrophic cell factories have rarely compared to the heterotrophic counterparts due to limited tools, bioinformatics, and multi-omics database. Therefore, recent advances of their genome editing techniques by clustered regularly interspaced short palindromic repeats (CRISPR) technology, and potential applications of their metabolic engineering and regulation approaches are examined in this review. Moreover, the contemporary achievements of synthetic biology approaches of microalgae and cyanobacteria in carbon fixation and sequestration, lipid and triacylglycerol (TAG), and sustainable production of high value-added chemicals, such as carotenoids and docosahexaenoic acid (DHA), have been also discussed. From recent genomic study to trends in metabolic regulation of microalgae and cyanobacteria and a comprehensive assessment of the current challenges and opportunities for microalgae and cyanobacteria is also conducted.

Plants, fungi, and bacteria synthesize a wide range of secondary metabolites that exhibit diverse biological activities. These bioactives, due to their potential benefits in research and therapeutics, have gained immense industrial importance. There is a need to synthesize these bioactives at significantly higher concentrations using cost-effective measures to be economically viable. However, the broader study of industrially important secondary metabolites has been hindered, thus, far due to a shortage of reliable, comparatively easy, and highly effective gene manipulation techniques. With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas), there is a revolution in the field of genetic engineering. CRISPR/Cas system, due to its simplicity and ease of use. This has widened its application in plant breeding, strain improvement, and engineering the metabolic pathways involved in the biochemical synthesis of industrially valuable bioactive. This review briefly introduces the CRISPR/Cas9 system and summarizes the applications of CRISPR/Cas9-mediated editing tools for the production of plant and fungal-derived bioactives.

6 - Plant genome sequencing: Models for developing synteny maps and association mapping

Synthetic biology is an emerging technology that uses the principles of physics, chemistry, mathematics, and biology. Synthetic biology is the technology enabler that could be applied across all fields such as agricultural, food, health, chemical, and industrial manufacturing sectors. In this chapter, several applications of synthetic biology across all disciplines are delineated, and challenges associated with the implementation of synthetic biology technologies are addressed in detail.

Meiosis is a key feature of sexual reproduction. During meiosis homologous chromosomes replicate, recombine, and randomly segregate, followed by the segregation of sister chromatids to produce haploid cells. The unique genotypes of recombinant gametes are an essential substrate for the selection of superior genotypes in natural populations and in plant breeding. In this review we summarize current knowledge on meiosis in diverse monocot and dicot crop species and provide a comprehensive resource of cloned meiotic mutants in six crop species (rice, maize, wheat, barley, tomato, and Brassica species). Generally, the functional roles of meiotic proteins are conserved between plant species, but we highlight notable differences in mutant phenotypes. The physical lengths of plant chromosomes vary greatly; for instance, wheat chromosomes are roughly one order of magnitude longer than those of rice. We explore how chromosomal distribution for crossover recombination can vary between species. We conclude that research on meiosis in crops will continue to complement that in Arabidopsis, and alongside possible applications in plant breeding will facilitate a better understanding of how the different stages of meiosis are controlled in plant species.

In this era of high-throughput genomics, the number of available genome sequences and genomic data sets for many organisms is growing rapidly. Many species are now represented by multiple genomes, rather than a single reference genome. Traditional comparative genomics approaches are bound by the physical constraints of a single genome that are unable to represent all genomic diversity of a species or population. As a result, there is an ongoing shift towards pangenomic approaches, which promise to precisely address the sequence and variation described in large collections of related genomes. However, the lack of practical applications of pangenomes prevents us from understanding the full genetic diversity variation within a species or population. PanTools is a recently developed toolkit for pangenome construction, graph annotation, and homology detection. In this thesis, I develop and employ novel applications utilizing PanTools' annotated pangenome graph representation, mainly focusing on the analysis of plant and bacterial pathogen genomes. In Chapter 1 I introduce the field of comparative genomics and explain the essential principles for comparing genetic blueprints of different organisms. I describe the limitations of traditional methods, highlighting how reference genome bias and computational constraints are driving a transition to pangenomic approaches. A brief historical overview of pangenome studies elucidates the significance of this transition and introduces novel concepts used in pangenomics. The most prevalent pangenome representations are further detailed, discussing their advantages and shortcomings. Finally, I delineate several critical challenges that must be overcome to advance the recently emerged field of pangenomics. Chapter 2 lays the groundwork for comparative genomics in PanTools by introducing a set of functionalities designed to handle large collections of bacterial genomes. Quality assurance to validate the genome quality and annotations before analyzing the data. Characterizing a pangenome's gene content (core, accessory, unique) and pangenome openness estimation. Furthermore, implementation of phylogenomic methods to reconstruct evolutionary relationships across bacterial species. The hierarchical graph representation was extended to enable incorporation of phenotype/metadata and functional annotations. The functionalities are demonstrated in a comparative study of nearly 200 Pectobacterium genomes. We describe the pangenome's large accessory genome, whose genetic repertoire far surpasses that of a single bacterial genome. The gene-based pangenome representation is further utilized as a framework for identifying genetic differences between blackleg causing and non-causing strains. In Chapter 3, the methodology introduced in the previous chapter is updated to enable large-scale comparative genomics in pangenomes throughout all kingdoms of life. To obtain an accurate representation of gene families across different genome collections with varying sizes, complexities, and evolutionary distances, we propose a method that adjusts strictness of the homology grouping based on validated single-copy orthologs. We demonstrate generic applicability and scalability of the software by emphasizing functional annotation, gene-level and phylogenomic analyses on seven use cases from different taxonomic kingdoms: ranging from viruses to human and plant genomes. Recent breakthroughs in sequencing and assembly technologies have delivered the first high quality haplotype-resolved (phased) genome assemblies. Chapter 4 focuses on harnessing the variation in these novel assemblies, which was previously hidden within haploid references. PanTools' methodology is updated, and novel functionalities are introduced specifically to enable the analysis of intragenomic variation, complementing existing intergenomic analyses. A relational synteny layer is incorporated into the hierarchical pangenome graph, linking collinear gene loci across all sequences. The functionalities are employed to pangenomes of diploid apples and tetraploid potatoes. We demonstrate high heterozygosity among homeologous chromosomes, characterized by gene absence/presence variation. Additionally, the analyses shed light on the evolutionary history of apple and potato species by revealing structural chromosomal rearrangements that occurred after whole-genome duplication events. Chapter 5 presents various enhancements to the PanTools toolkit required for integrating pangenomics into plant breeding applications. We address a major bottleneck in the pangenome construction algorithm to increase scalability for a higher number of genomes. Improved construction runtime is demonstrated on various datasets, with the largest being a Solanum (genus-level) pangenome, including a combined total of 29 (haplotype-resolved) tomato and potato genomes. We further introduce functionalities for detecting gene presence/absence variation in WGS samples and integrating them into the pangenome graph, showcasing gene-bases pangenomic analysis of 474 Lactuca resequencing lines. Finally, we introduce a visual analysis browser for exploring sequence variants in the context of 20 tomato references with metadata and 444 resequenced accessions. Chapter 6 serves as the conclusion of this thesis. I explain how the field of pangenomics has evolved during the timeframe of this thesis, detailing its current representations and applications, and how my work contributes to the field. Recognizing pangenomics as an emerging field that requires continuous development, I discuss the key challenges it faces. Finally, I conclude by exploring promising future directions for pangenomics research, with a particular focus on applications in bacteria and plants.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a genetic locus of prokaryotes and contain highly conserved direct repeats, spacers, and CRISPR-associated genes. Spacers in CRISPRs are known as adaptive immune markers and reveal what types of phage or foreign DNA have been introduced in the past. The primary objective of this study was to analyze spacer sequences in CRISPR arrays of 15 Salmonella enterica subspecies and to determine if Salmonella CRISPRs are indeed involved in resistance to foreign DNAs. Using a bioinformatics algorithm, the CRISPR arrays of 15 subspecies of S. enterica were predicted. The transformation efficiencies of the wild-type and mutant strains lacking a space were determined using the plasmid harboring the same sequences with the space. Analysis of the CRISPR arrays indicated that S. Typhimurium encoded three possible CRISPR regions in the genome. Notably, 48 or 55 spacers were predicted in the genomes of S. Typhimurium 14028 and LT2 strains, respectively, and 39 were precisely identical. To confirm this prediction, the predicted CRISPR regions of S. Typhimurium 14028 were sequenced using the specific primers. Interestingly, a homology search of individual spacers found that the 2nd spacer of CRISPR 2 was nearly identical to a partial genome region of phage FSL SP-016. The mutant strain showed two to threefold increased transformation efficiency compared to that of the wild-type strain. These results demonstrate that the spacer sequence is dependent on genetic relations, especially for adaptive immunity against phage or foreign DNAs.

Plant breeding is one of the oldest sustainable agriculture methods used to increase the yield, quality and other biomaterial for human use. Many crops like fruits, vegetables, ornamental flowers, shrubs and trees, have been long cultivated to satisfy human food and aesthetical needs. Conventional breeding technologies like selection, hybridization, mutation through physical and chemical methods, and modern transgenic approaches are often used to improve the desired traits without inducing the pleiotropic effects. But these breeding methods are highly laborious and complicated to enhance crop production. Recently, targeted genome editing through engineered nuclease including zinc finger nuclease, transcription activator like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPRs) have been used to improve various traits in plants. Genome editing has emerged as a novel alternative approach to classical breeding with higher mutagenic efficiency. Here, we briefly cover the strengths of CRISPRs in comparison with other genome editing techniques. We also discuss its potential applications in genetic improvement of various crops and future prospective.

Synthetic biology and metabolic engineering strategies in identifying and producing plant natural products; with emphasis on the CRISPR/Cas systems

Molecular breeding has transformed the ornamental plant industry by enabling breeders to introduce desirable traits with greater precision and speed compared to traditional methods. This review explores recent trends in molecular breeding techniques for ornamental plants, covering the period from 2010 to 2024. Key technologies discussed include marker-assisted selection (MAS), next-generation sequencing (NGS), genomic selection (GS), and genome editing techniques like CRISPR-Cas9. MAS has been instrumental in improving traits such as flower color and disease resistance by using molecular markers linked to specific genes. The use of NGS has expanded the genetic understanding of complex traits, such as floral scent and stress tolerance, by allowing for comprehensive genome and transcriptome analysis.Genomic selection has emerged as a powerful tool for predicting the performance of polygenic traits, such as flower size and vase life, by integrating genome-wide markers and phenotypic data. Furthermore, the advent of CRISPR-Cas9 technology has enabled precise gene editing, allowing for targeted modifications of traits like fragrance and sterility in invasive species. The review highlights specific case studies, such as the application of MAS in roses and petunias, NGS in orchids and gerberas, and CRISPR-Cas9 in jasmine and Japanese knotweed, to demonstrate the practical impact of these technologies.Challenges in molecular breeding include the limited availability of genomic resources for many ornamental species and the complexity of multi-gene traits, which require advanced breeding strategies. Despite these challenges, molecular breeding offers significant potential for improving both the aesthetic and functional traits of ornamental plants. The future of the field lies in expanding genomic resources, enhancing breeding precision through new tools, and integrating molecular techniques into commercial horticultural practices to meet evolving market demands. This review offers insights into these trends, providing a roadmap for future research and commercial applications in ornamental plant breeding.

Catalytic RNA, ribozyme, and its applications in synthetic biology

Chapter 3 - Application of Plant Breeding and Genomics for Improved Sorghum and Pearl Millet Grain Nutritional Quality

CRISPR and microbial genomics have emerged as transformative tools in modern plant breeding, offering unprecedented precision and efficiency in crop improvement. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology enables targeted genome editing by introducing precise modifications at specific DNA sequences, allowing plant breeders to enhance desirable traits such as disease resistance, yield potential, stress tolerance, and nutritional value without introducing foreign DNA. Simultaneously, advancements in microbial genomics have unraveled the complex interactions between plants and their associated microbiomes, including beneficial microbes that promote growth, enhance nutrient uptake, and protect against pathogens. By leveraging CRISPR to edit both plant genomes and the genomes of symbiotic or endophytic microbes, researchers can develop integrated breeding strategies that optimize plant-microbe interactions for improved crop performance. These biotechnological approaches promise to accelerate breeding cycles, reduce reliance on chemical inputs, and contribute to sustainable agriculture, addressing global challenges like food security and environmental conservation.

CRISPR technology, which is widely used for plant genome editing, will accelerate the breeding of food crops beyond what was imaginable before its development. Here we provide a brief overview of CRISPR technology, its most important applications for crop improvement and several technological breakthroughs. We also make predictions of the applications of CRISPR technology to food crops, which we believe would provide the potential for synthetic biology and domestication of crops. We also discuss the implications of regulatory policy for deployment of the technology in the developing world. CRISPR technology has been widely used in plant genome editing and has great potential in precision breeding. The application of CRISPR technology to food crops provides potential for crop synthetic biology and crop domestication. The authors also discuss the implications of regulatory policy for deployment of the technology in the developing world.

Certain new plant breeding techniques and their marketability in the context of EU GMO legislation – recent developments