Evolutionary engineering by genome shuffling

An upsurge in the bioeconomy drives the need for engineering microorganisms with increasingly complex phenotypes. Gains in productivity of industrial microbes depend on the development of improved strains. Classical strain improvement programmes for the generation, screening and isolation of such mutant strains have existed for several decades. An alternative to traditional strain improvement methods, genome shuffling, allows the directed evolution of whole organisms via recursive recombination at the genome level. This review deals chiefly with the technical aspects of genome shuffling. It first presents the diversity of organisms and phenotypes typically evolved using this technology and then reviews available sources of genetic diversity and recombination methodologies. Analysis of the literature reveals that genome shuffling has so far been restricted to microorganisms, both prokaryotes and eukaryotes, with an overepresentation of antibiotics- and biofuel-producing microbes. Mutagenesis is the main source of genetic diversity, with few studies adopting alternative strategies. Recombination is usually done by protoplast fusion or sexual recombination, again with few exceptions. For both diversity and recombination, prospective methods that have not yet been used are also presented. Finally, the potential of genome shuffling for gaining insight into the genetic basis of complex phenotypes is also discussed.     

Whole genome amplification--applications and advances

The concept of whole genome amplification is something that has arisen in the past few years as the polymerase chain reaction (PCR) has been adapted to replicate regions of genomes that are of biological interest. The applications are many--forensic science, embryonic disease diagnosis, bioterrorism genome detection, "immortalization" of clinical samples, microbial diversity, and genotyping. Several recent papers suggest that whole genomes can be replicated without bias or non-random distribution of the target, these findings open up a new avenue to molecular biology.     

Whole genome association mapping by incompatibilities and local perfect phylogenies

Background  

With current technology, vast amounts of data can be cheaply and efficiently produced in association studies, and to prevent data analysis to become the bottleneck of studies, fast and efficient analysis methods that scale to such data set sizes must be developed.     

Actinomycetes genome engineering approaches

This review provides an overview of new technologies for DNA manipulations in actinomycetes exploiting recombinogenic engineering (Flp-FRT, Cre-loxP, Dre-rox, Tn5, GusA and I-SceI systems). We will describe some new vectors recently developed for engineering of complex phenotypes in actinomycetes. Several site-specific recombinases, transposons, reporter genes and I-SceI endonuclease have been utilized for genome manipulation in actinomycetes. Novel molecular tools will help to overcome many technical difficulties and will encourage new efforts to address the function of actinomycete genes.     

Whole genome approaches to quantitative genetics

Apart from parent-offspring pairs and clones, relative pairs vary in the proportion of the genome that they share identical by descent. In the past, quantitative geneticists have used the expected value of sharing genes by descent to estimate genetic parameters and predict breeding values. With the possibility to genotype individuals for many markers across the genome it is now possible to empirically estimate the actual relationship between relatives. We review some of the theory underlying the variation in genetic identity, show applications to estimating genetic variance for height in humans and discuss other applications.     

Whole genome amplification using single-primer PCR

Comprehensive genomic molecular analyses require relatively large DNA amounts that are often not available from forensic, clinical and other crucial biological samples. Numerous methods to amplify the whole genome have been proposed for cancer, forensic and taxonomic research. Unfortunately, when using truly random primers for the initial priming step, all of these procedures suffer from high background problems for sub-nanogram quantities of input DNA. Here we report an approach to eliminate this problem for PCR-based methods even at levels of DNA approaching that of a single cell.     

Whole genome plasticity in pathogenic bacteria

The exploitation of bacterial genome sequences has so far provided a wealth of new general information about the genetic diversity of bacteria, such as that of many pathogens. Comparative genomics uncovered many genome variations in closely related bacteria and revealed basic principles involved in bacterial diversification, improving our knowledge of the evolution of bacterial pathogens. A correlation between metabolic versatility and genome size has become evident. The degenerated life styles of obligate intracellular pathogens correlate with significantly reduced genome sizes, a phenomenon that has been termed "evolution by reduction". These mechanisms can permanently alter bacterial genotypes and result in adaptation to their environment by genome optimization. In this review, we summarize the recent results of genome-wide approaches to studying the genetic diversity of pathogenic bacteria that indicate that the acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content.     

Whole genome duplication of intra- and inter-chromosomes in the tomato genome

Whole genome duplication(WGD)events have been proven to occur in the evolutionary history of most angiosperms.Tomato is considered a model species of the Solanaceae family.In this study,we describe the details of the evolutionary process of the tomato genome by detecting collinearity blocks and dating the WGD events on the tree of life by combining two different methods:synonymous substitution rates(Ks)and phylogenetic trees.In total,593 collinearity blocks were discovered out of 12 pseudo-chromosomes constructed. It was evident that chromosome 2 had experienced an intra-chromosomal duplication event.Major inter-chromosomal duplication occurred among all the pseudo-chromosome.We calculated the Ks value of these collinearity blocks.Two peaks of Ks distribution were found,corresponding to two WGD events occurring approximately 36-82 million years ago(MYA)and 148-205 MYA.Additionally, the results of phylogenetic trees suggested that the more recent WGD event may have occurred after the divergence of the rosidasterid clade,but before the major diversification in Solanaceae.The older WGD event was shown to have occurred before the divergence of the rosid-asterid clade and after the divergence of rice-Arabidopsis(monocot-dicot).     

Whole genome transcriptome polymorphisms in Arabidopsis thaliana

ArrayPlex is a software package that centrally provides a large number of flexible toolsets useful for functional genomics, including microarray data storage, quality assessments, data visualization, gene annotation retrieval, statistical tests, genomic sequence retrieval and motif analysis. It uses a client-server architecture based on open source components, provides graphical, command-line, and programmatic access to all needed resources, and is extensible by virtue of a documented application programming interface. ArrayPlex is available at http://sourceforge.net/projects/arrayplex/.     

Whole genome sequences of four Brucella strains

Brucella melitensis and Brucella suis are intracellular pathogens of livestock and humans. Here we report four genome sequences, those of the virulent strain B. melitensis M28-12 and vaccine strains B. melitensis M5 and M111 and B. suis S2, which show different virulences and pathogenicities, which will help to design a more effective brucellosis vaccine.     

Organoid technologies meet genome engineering

Three‐dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two‐dimensional (2D) cell culture models. Organoids, the 3D self‐organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease‐associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.     

Whole genome searching with shotgun proteomic data: applications for genome annotation

High-throughput genome sequencing continues to accelerate the rate at which complete genomes are available for biological research. Many of these new genome sequences have little or no genome annotation currently available and hence rely upon computational predictions of protein coding genes. Evidence of translation from proteomic techniques could facilitate experimental validation of protein coding genes, but the techniques for whole genome searching with MS/MS data have not been adequately developed to date. Here we describe GENQUEST, a novel method using peptide isoelectric focusing and accurate mass to greatly reduce the peptide search space, making fast, accurate, and sensitive whole human genome searching possible on common desktop computers. In an initial experiment, almost all exonic peptides identified in a protein database search were identified when searching genomic sequence. Many peptides identified exclusively in the genome searches were incorrectly identified or could not be experimentally validated, highlighting the importance of orthogonal validation. Experimentally validated peptides exclusive to the genomic searches can be used to reannotate protein coding genes. GENQUEST represents an experimental tool that can be used by the proteomics community at large for validating computational approaches to genome annotation.     

应用基因组工程构建新型大肠杆菌细胞工厂

基因组工程（genome engineering）是指为了实现某一目标对复制系统进行有意地、广泛地遗传修饰。大肠杆菌作为常用细胞工厂之一,在生物制造领域应用广泛,已成为合成生物学的主要研究对象。应用基因组工程改造大肠杆菌可以进一步拓展其应用范围。介绍了基因组工程最新技术发展,如基因组整合、染色体进化、多元自动化基因组工程、可寻迹多元重组工程等,及其在改造大肠杆菌提高其生产能力、稳定性及环境耐受能力等方面的研究进展。     

Whole genome associated studies for age at menarche

Puberty, the transition from childhood to adult body size and sexual maturity, is a complex multi-staged process involving growth acceleration, weight gain and the appearance of secondary sexual physical features over a 2- to 3-year period. Recent genome-wide association (GWA) studies for age at menarche, the onset of the first menstrual period in girls, have identified the first common genetic variants to be robustly associated with pubertal timing. The findings indicate a novel link between microRNA pre-processing and the timing of whole organism growth and development. Our studies also demonstrate that the use of easily measured phenotypic markers, such as age at menarche in girls, in GWA studies can uncover genetic variants with wider relevance to more complex phenotypes, such as pubertal onset and the tempo of growth in both sexes.