Here and there: the double-side transgene localization

Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identification of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact filtration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s flanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two flanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the field of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to efficiently localize genomic transposon integrations, which was confirmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

Rewiring Meiosis for Crop Improvement

Meiosis is a specialized cell division that contributes to halve the genome content and reshuffle allelic combinations between generations in sexually reproducing eukaryotes. During meiosis, a large number of programmed DNA double-strand breaks (DSBs) are formed throughout the genome. Repair of meiotic DSBs facilitates the pairing of homologs and forms crossovers which are the reciprocal exchange of genetic information between chromosomes. Meiotic recombination also influences centromere organization and is essential for proper chromosome segregation. Accordingly, meiotic recombination drives genome evolution and is a powerful tool for breeders to create new varieties important to food security. Modifying meiotic recombination has the potential to accelerate plant breeding but it can also have detrimental effects on plant performance by breaking beneficial genetic linkages. Therefore, it is essential to gain a better understanding of these processes in order to develop novel strategies to facilitate plant breeding. Recent progress in targeted recombination technologies, chromosome engineering, and an increasing knowledge in the control of meiotic chromosome segregation has significantly increased our ability to manipulate meiosis. In this review, we summarize the latest findings and technologies on meiosis in plants. We also highlight recent attempts and future directions to manipulate crossover events and control the meiotic division process in a breeding perspective.

Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

Small “Nested” Introgressions from Wild Thinopyrum Species, Conferring Effective Resistance to Fusarium Diseases, Positively Impact Durum Wheat Yield Potential

Today wheat cultivation is facing rapidly changing climate scenarios and yield instability, aggravated by the spreading of severe diseases such as Fusarium head blight (FHB) and Fusarium crown rot (FCR). To obtain productive genotypes resilient to stress pressure, smart breeding approaches must be envisaged, including the exploitation of wild relatives. Here we report on the assessment of the breeding potential of six durum wheat-Thinopyrum spp. recombinant lines (RLs) obtained through chromosome engineering. They are characterized by having 23% or 28% of their 7AL chromosome arm replaced by a “nested” alien segment, composed of homoeologous group 7 chromosome fractions from Th. ponticum and Th. elongatum (=7el1L + 7EL) or from different Th. ponticum accessions (=7el1L + 7el2L). In addition to the 7el1L genes Lr19 + Yp (leaf rust resistance, and yellow pigment content, respectively), these recombinant lines (RLs) possess a highly effective QTL for resistance to FHB and FCR within their 7el2L or 7EL portion. The RLs, their null segregants and well-adapted and productive durum wheat cultivars were evaluated for 16 yield-related traits over two seasons under rainfed and irrigated conditions. The absence of yield penalties and excellent genetic stability of RLs was revealed in the presence of all the alien segment combinations. Both 7el2L and 7EL stacked introgressions had positive impacts on source and sink yield traits, as well as on the overall performance of RLs in conditions of reduced water availability. The four “nested” RLs tested in 2020 were among the top five yielders, overall representing good candidates to be employed in breeding programs to enhance crop security and safety.

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.

Induction of spontaneous human neocentromere formation and long-term maturation

Human centromeres form primarily on α-satellite DNA but sporadically arise de novo at naive ectopic loci, creating neocentromeres. Centromere inheritance is driven primarily by chromatin containing the histone H3 variant CENP-A. Here, we report a chromosome engineering system for neocentromere formation in human cells and characterize the first experimentally induced human neocentromere at a naive locus. The spontaneously formed neocentromere spans a gene-poor 100-kb domain enriched in histone H3 lysine 9 trimethylated (H3K9me3). Long-read sequencing revealed this neocentromere was formed by purely epigenetic means and assembly of a functional kinetochore correlated with CENP-A seeding, eviction of H3K9me3 and local accumulation of mitotic cohesin and RNA polymerase II. At formation, the young neocentromere showed markedly reduced chromosomal passenger complex (CPC) occupancy and poor sister chromatin cohesion. However, long-term tracking revealed increased CPC assembly and low-level transcription providing evidence for centromere maturation over time.

A review of studies on Oryzias dancena

Oryzias dancena (Beloniformes; Teleostei), is a euryhaline teleost that mainly inhabits the brackish or freshwater of river mouths and estuaries around the Bay of Bengal and the Malay Peninsula. It also has a short interval between generations, with spawning possibilities just 60 days after hatching. The aim of this paper is to provide a review for the study of O. dancena based on the studies collected so far, and could form the basis for a wide category of researches including zootoxy, cytogenetics, anesthesia, tagging, salinity tolerance, reproduction, fish disease, chromosome engineering, and trnasgenesis in order to highlight the recent progress in different fields of study using this species.

Development and Cytomolecular Identification of Monosomic Alien Addition and Substitution Lines of Triticale (×Triticosecale Wittmack) With 2Sk Chromosome Conferring Leaf Rust Resistance Derived From Aegilops kotschyi Boiss

Alien chromosome introgression has become a valuable tool to broaden the genetic variability of crop plants via chromosome engineering. This study details the procedure to obtain monosomic addition and monosomic substitution lines of the triticale carrying 2Sk chromosome from Aegilops kotchyi Boiss., which harbors Lr54 + Yr37 leaf and stripe rust-resistant gene loci, respectively. Initially, A. kotschyi × Secale cereale artificial amphiploids (2n = 6x = 42 chromosomes, UUSSRR) were crossed with triticale cv. “Sekundo” (2n = 6x = 42, AABBRR) in order to obtain fertile offspring. Cyto-molecular analyses of five subsequent backcrossing generations revealed that 2Sk chromosome was preferentially transmitted. This allowed for the selection of monosomic 2Sk addition (MA2Sk) lines of triticale. Finally, the 2Sk(2R) substitution plants were obtained by crossing MA2Sk with the nullisomic (N2R) plants of triticale. The presence of 2Sk chromosome in subsequent generations of plants was evaluated using SSR markers linked to Lr54 + Yr37 loci. Disease evaluation of the monosomic 2Sk(2R) substitution plants for the reaction to leaf and stripe rust infection were carried out under controlled conditions in a growth chamber. The results showed significant improvement of leaf rust resistance severity of monosomic substitution plants compared with control (“Sekundo”). In contrast, the introgression of the Lr54 + Yr37 loci did not lead to improvement of stripe rust resistance. In summary, the creation of monosomic addition and monosomic substitution lines of triticale is the starting point for the precise and guided transfer of Lr54 + Yr37 loci. The results showed that the developed materials could be exploited for the development of triticale varieties with resistance to leaf rust.

CRISPR/Cas-mediated chromosome engineering: opening up a new avenue for plant breeding

The advent of powerful site-specific nucleases, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, which enables precise genome manipulation, has revolutionized plant breeding. Until recently, the main focus of researchers has been to simply knock-in or knock-out single genes, or to induce single base changes, but constant improvements of this technology have enabled more ambitious applications that aim to improve plant productivity or other desirable traits. One long-standing aim has been the induction of targeted chromosomal rearrangements (crossovers, inversions, or translocations). The feasibility of this technique has the potential to transform plant breeding, because natural rearrangements, like inversions, for example, typically present obstacles to the breeding process. In this way, genetic linkages between traits could be altered to combine or separate favorable and deleterious genes, respectively. In this review, we discuss recent breakthroughs in the field of chromosome engineering in plants and their potential applications in the field of plant breeding. In the future, these approaches might be applicable in shaping plant chromosomes in a directed manner, based on plant breeding needs.