Stochastic modelling, Bayesian inference, and new in vivo measurements elucidate the debated mtDNA bottleneck mechanism

Dangerous damage to mitochondrial DNA (mtDNA) can be ameliorated during mammalian development through a highly debated mechanism called the mtDNA bottleneck. Uncertainty surrounding this process limits our ability to address inherited mtDNA diseases. We produce a new, physically motivated, generalisable theoretical model for mtDNA populations during development, allowing the first statistical comparison of proposed bottleneck mechanisms. Using approximate Bayesian computation and mouse data, we find most statistical support for a combination of binomial partitioning of mtDNAs at cell divisions and random mtDNA turnover, meaning that the debated exact magnitude of mtDNA copy number depletion is flexible. New experimental measurements from a wild-derived mtDNA pairing in mice confirm the theoretical predictions of this model. We analytically solve a mathematical description of this mechanism, computing probabilities of mtDNA disease onset, efficacy of clinical sampling strategies, and effects of potential dynamic interventions, thus developing a quantitative and experimentally-supported stochastic theory of the bottleneck.Comment: Main text: 14 pages, 5 figures; Supplement: 17 pages, 4 figures; Total: 31 pages, 9 figure

Cassava root crown phenotyping using three-dimension (3D) multi-view stereo reconstruction

Phenotypic analysis of cassava root crowns (CRCs) so far has been limited to visual inspection and very few measurements due to its laborious process in the feld. Here, we developed a platform for acquiring 3D CRC models using close-range photogrammetry for phenotypic analysis. The state of the art is a low cost and easy to set up 3D acquisition requiring only a background sheet, a reference object and a camera, compatible with feld experiments in remote areas. We tested diferent software with CRC samples, and Agisoft and Blender were the most suitable software for generating high-quality 3D models and data analysis, respectively. We optimized the workfow by testing diferent numbers of images for 3D reconstruction and found that a minimum of 25 images per CRC can provide high quality 3D models. Up to ten traits, including 3D crown volumes, 3D crown surface, root density, surface to-volume ratio, root numbers, root angle, crown diameter, cylinder soil volume, CRC compactness and root length can be extracted providing novel parameters for studying cassava storage roots. We applied this platform to partial-inbred cassava populations and demonstrated that our platform provides reliable 3D CRC modelling for phenotypic analysis, analysis of genetic variances and supporting breeding selection

Disruption of a DUF247 containing protein alters cell wall polysaccharides and reduces growth in Arabidopsis

Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughout Arabidopsis, particularly in vascular and developing tissues, and its disruption affects the expression of other gene members, indicating a regulatory control role within the gene family. Our results demonstrate that DUF247-1 is required for normal cell wall composition and structure and Arabidopsis growth

Maximizing signal-to-noise ratio in the random mutation capture assay

The ‘Random Mutation Capture’ assay allows for the sensitive quantitation of DNA mutations at extremely low mutation frequencies. This method is based on PCR detection of mutations that render the mutated target sequence resistant to restriction enzyme digestion. The original protocol prescribes an end-point dilution to about 0.1 mutant DNA molecules per PCR well, such that the mutation burden can be simply calculated by counting the number of amplified PCR wells. However, the statistical aspects associated with the single molecular nature of this protocol and several other molecular approaches relying on binary (on/off) output can significantly affect the quantification accuracy, and this issue has so far been ignored. The present work proposes a design of experiment (DoE) using statistical modeling and Monte Carlo simulations to obtain a statistically optimal sampling protocol, one that minimizes the coefficient of variance in the measurement estimates. Here, the DoE prescribed a dilution factor at about 1.6 mutant molecules per well. Theoretical results and experimental validation revealed an up to 10-fold improvement in the information obtained per PCR well, i.e. the optimal protocol achieves the same coefficient of variation using one-tenth the number of wells used in the original assay. Additionally, this optimization equally applies to any method that relies on binary detection of a small number of templates

Transmission of Mitochondrial DNA Diseases and Ways to Prevent Them

Recent reports of strong selection of mitochondrial DNA (mtDNA) during transmission in animal models of mtDNA disease, and of nuclear transfer in both animal models and humans, have important scientific implications. These are directly applicable to the genetic management of mtDNA disease. The risk that a mitochondrial disorder will be transmitted is difficult to estimate due to heteroplasmy—the existence of normal and mutant mtDNA in the same individual, tissue, or cell. In addition, the mtDNA bottleneck during oogenesis frequently results in dramatic and unpredictable inter-generational fluctuations in the proportions of mutant and wild-type mtDNA. Pre-implantation genetic diagnosis (PGD) for mtDNA disease enables embryos produced by in vitro fertilization (IVF) to be screened for mtDNA mutations. Embryos determined to be at low risk (i.e., those having low mutant mtDNA load) can be preferentially transferred to the uterus with the aim of initiating unaffected pregnancies. New evidence that some types of deleterious mtDNA mutations are eliminated within a few generations suggests that women undergoing PGD have a reasonable chance of generating embryos with a lower mutant load than their own. While nuclear transfer may become an alternative approach in future, there might be more difficulties, ethical as well as technical. This Review outlines the implications of recent advances for genetic management of these potentially devastating disorders

Priorities to inform research on marine plastic pollution in Southeast Asia

This is the final version. Available from Elsevier via the DOI in this record. Southeast Asia is considered to have some of the highest levels of marine plastic pollution in the world. It is therefore vitally important to increase our understanding of the impacts and risks of plastic pollution to marine ecosystems and the essential services they provide to support the development of mitigation measures in the region. An interdisciplinary, international network of experts (Australia, Indonesia, Ireland, Malaysia, the Philippines, Singapore, Thailand, the United Kingdom, and Vietnam) set a research agenda for marine plastic pollution in the region, synthesizing current knowledge and highlighting areas for further research in Southeast Asia. Using an inductive method, 21 research questions emerged under five non-predefined key themes, grouping them according to which: (1) characterise marine plastic pollution in Southeast Asia; (2) explore its movement and fate across the region; (3) describe the biological and chemical modifications marine plastic pollution undergoes; (4) detail its environmental, social, and economic impacts; and, finally, (5) target regional policies and possible solutions. Questions relating to these research priority areas highlight the importance of better understanding the fate of marine plastic pollution, its degradation, and the impacts and risks it can generate across communities and different ecosystem services. Knowledge of these aspects will help support actions which currently suffer from transboundary problems, lack of responsibility, and inaction to tackle the issue from its point source in the region. Being profoundly affected by marine plastic pollution, Southeast Asian countries provide an opportunity to test the effectiveness of innovative and socially inclusive changes in marine plastic governance, as well as both high and low-tech solutions, which can offer insights and actionable models to the rest of the world.Natural Environment Research CouncilNational Research Foundation, Prime Minister’s Office (Singapore

The complete mitochondrial genome of Dictyostelium intermedium

Dictyostelium intermedium is a member of dictyostelids, the unicellular eukaryotes with a unique life cycle, including a social cycle. Despite the high diversity of dictyostelids, only five species' complete mitochondrial genome sequences were reported. This study aimed to add the D. intermedium mitochondrial genome sequence to the list. The size of this genome is 58,627 bp, with 73.99% A/T, containing 62 genes located on one strand: 41 protein-coding genes, three ribosomal RNA genes, and 18 transfer RNA genes. The 41 protein-coding genes comprised 18 oxidative phosphorylation-related, 16 ribosomal, and seven hypothetical protein-coding genes. The cox1/2 and rnl gene contained introns, similar to other species of Dictyostelium. The phylogenetic tree built based on 34 protein sequences supported the monophyletic clade of Dictyostelium and the dictyostelids' ancestor's position between the two dictyostelids orders: Dictyosteliales and Acytosteliales

The complete chloroplast genome sequence of Asian Palmyra palm (Borassus flabellifer)

Abstract Objective Borassus flabellifer or Asian Palmyra palm is widely distributed in South and Southeast Asia and is horticultural and economic importance for its fruit and palm sugar production. However, its population is in rapid decline, and only a few genetic data are available. We sequenced the complete chloroplast (cp) genome of B. flabellifer to provide its genetic data for further utilization. Results The cp genome was obtained by Illumina sequencing and manual gap fillings providing 160,021 bp in length containing a pair of inverted repeats (IRs) with 27,256 bp. These IRs divide the genome into a large single copy region 87,444 bp and a small single copy region 18,065 bp. In total, 113 unique genes, 134 SSRs and 47 large repeats were identified. This is the first complete cp genome reported in the genus Borassus. A comparative analysis among members of the Borasseae tribe revealed that the B. flabellifer cp genome is, so far, the largest and the cp genomes of this tribe have a similar structure, gene number and gene arrangement. A phylogenetic tree reconstructed based on 74 protein-coding genes from 70 monocots demonstrates short branch lengths indicating slow evolutionary rates of cp genomes in family Arecaceae