Tapping into yeast diversity

Domesticated organisms demonstrate our capacity to influence wild species but also provide us with the opportunity to understand rapid evolution in the context of substantially altered environments and novel selective pressures. Recent advances in genetics and genomics have brought unprecedented insights into the domestication of many organisms and have opened new avenues for further improvements to be made. Yet, our ability to engineer biological systems is not without limits; genetic manipulation is often quite difficult. The budding yeast, Saccharomyces cerevisiae, is not only one of the most powerful model organisms, but is also the premier producer of fermented foods and beverages around the globe. As a model system, it entertains a hefty workforce dedicated to deciphering its genome and the function it encodes at a rich mechanistic level. As a producer, it is used to make leavened bread, and dozens of different alcoholic beverages, such as beer and wine. Yet, applying the awesome power of yeast genetics to understanding its origins and evolution requires some knowledge of its wild ancestors and the environments from which they were derived. A number of surprisingly diverse lineages of S. cerevisiae from both primeval and secondary forests in China have been discovered by Wang and his colleagues. These lineages substantially expand our knowledge of wild yeast diversity and will be a boon to elucidating the ecology, evolution and domestication of this academic and industrial workhorse.     

Genetic engineering of crops as potential source of genetic hazard in the human diet.

The benefits of genetic engineering of crop plants to improve the reliability and quality of the world food supply have been contrasted with public concerns raised about the food safety of the resulting products. Debates have concentrated on the possible unforeseen risks associated with the accumulation of new metabolites in crop plants that may contribute to toxins, allergens and genetic hazards in the human diet. This review examines the various molecular and biochemical mechanisms by which new hazards may appear in foods as a direct consequence of genetic engineering in crop plants. Such hazards may arise from the expression products of the inserted genes, secondary or pleiotropic effects of transgene expression, and random insertional mutagenic effects resulting from transgene integration into plant genomes. However, when traditional plant breeding is evaluated in the same context, these mechanisms are no different from those that have been widely accepted from the past use of new cultivars in agriculture. The risks associated with the introduction of new genes via genetic engineering must be considered alongside the common breeding practice of introgressing large fragments of chromatin from related wild species into crop cultivars. The large proportion of such introgressed DNA involves genes of unknown function linked to the trait of interest such as pest or disease resistance. In this context, the potential risks of introducing new food hazards from the applications of genetic engineering are no different from the risks that might be anticipated from genetic manipulation of crops via traditional breeding. In many respects, the precise manner in which genetic engineering can control the nature and expression of the transferred DNA offers greater confidence for producing the desired outcome compared with traditional breeding.     

Next-generation sequencing for understanding and accelerating crop domestication

Next generation Sequencing (NGS) provides a powerful tool for discovery of domestication genes in crop plants and their wild relatives. The accelerated domestication of new plant species as crops may be facilitated by this knowledge. Re-sequencing of domesticated genotypes can identify regions of low diversity associated with domestication. Species-specific data can be obtained from related wild species by whole-genome shot-gun sequencing. This sequence data can be used to design species specific polymerase chain reaction (PCR) primers. Sequencing of the products of PCR amplification of target genes can be used to explore genetic variation in large numbers of genes and gene families. Novel allelic variation in close or distant relatives can be characterized by NGS. Examples of recent applications of NGS to capture of genetic diversity for crop improvement include rice, sugarcane and Eucalypts. Populations of large numbers of individuals can be screened rapidly. NGS supports the rapid domestication of new plant species and the efficient identification and capture of novel genetic variation from related species.     

Modelling the origins of legume domestication and cultivation

Ladizinsky’s (1987) mathematical model of the domestication of lentils and other Near Eastern legumes is invalid. Ladizinsky believed that the model predicts that high rates of seed harvesting would lead to fixation, in wild populations, of genes conferring lack of seed dormancy (a domesticated trait). In fact, however, the model gives rapid fixation of alleles for non-dormancy underall circumstances; gathering by humans has no effect on allelic frequencies. It appears that in these species cultivation must have preceded the morphological changes that distinguish domesticated from wild plants. The addition of more realistic assumptions to the model does not alter this conclusion. I suggest several scenarios that could explain Near Eastern legume domestication: the most plausible of these postulates that cultivation of cereals led to scheduling conflicts which necessitated the abandonment of harvesting of wild legumes, and hence the initiation of their cultivation. Mathematical modelling may be able to contribute to our understanding of agricultural origins, but it must be carried out with greater rigor and closer attention to the theoretical literature.     

DNA amplification fingerprinting: A strategy for genome analysis

A novel strategy to detect genetic differences among organisms, DNA amplification fingerprinting (DAF), uses a thermostable DNA polymerase directed by usually one short (≥5 bp) oligonucleotide primer of arbitrary sequence to amplify short segments of genomic DNA and generate a range of DNA extension products. These products can be analyzed by polyacrylamide gel electrophoresis and silver staining. DAF is rapid and sensitive and is independent of cloning and prior genetic characterization. Here we describe this new methodology, its application to plant genotyping, and its perspectives in DNA fingerprinting and genome mapping.     

Genetic analysis of wheat domestication and evolution under domestication

Wheat is undoubtedly one of the world's major food sources since the dawn of Near Eastern agriculture and up to the present day. Morphological, physiological, and genetic modifications involved in domestication and subsequent evolution under domestication were investigated in a tetraploid recombinant inbred line population, derived from a cross between durum wheat and its immediate progenitor wild emmer wheat. Experimental data were used to test previous assumptions regarding a protracted domestication process. The brittle rachis (Br) spike, thought to be a primary characteristic of domestication, was mapped to chromosome 2A as a single gene, suggesting, in light of previously reported Br loci (homoeologous group 3), a complex genetic model involved in spike brittleness. Twenty-seven quantitative trait loci (QTLs) conferring threshability and yield components (kernel size and number of kernels per spike) were mapped. The large number of QTLs detected in this and other studies suggests that following domestication, wheat evolutionary processes involved many genomic changes. The Br gene did not show either genetic (co-localization with QTLs) or phenotypic association with threshability or yield components, suggesting independence of the respective loci. It is argued here that changes in spike threshability and agronomic traits (e.g. yield and its components) are the outcome of plant evolution under domestication, rather than the result of a protracted domestication process. Revealing the genomic basis of wheat domestication and evolution under domestication, and clarifying their inter-relationships, will improve our understanding of wheat biology and contribute to further crop improvement.     

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.     

Building a genome engineering toolbox in nonmodel prokaryotic microbes

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO₂, and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop “design rules” for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems.     

The role of recombination and RAD52 in mutation of chromosomal DNA transformed into yeast.

While transformation is a prominent tool for genetic analysis and genome manipulation in many organisms, transforming DNA has often been found to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast chromosome III composed primarily of unique DNA. Wild-type and rad52 Saccharomyces cerevisiae strains were transformed with either a homologous chromosome III or a diverged chromosome III from S. carlsbergensis. The host strain chromosome III had a conditional centromere allowing it to be lost on galactose medium so that recessive mutations in the transformed chromosome could be identified. Following transformation of a RAD+ strain with the homologous chromosome, there were frequent changes in the incoming chromosome, including large deletions and mutations that do not lead to detectable changes in chromosome size. Based on results with the diverged chromosome, interchromosomal recombinational interactions were the source of many of the changes. Even though rad52 exhibits elevated mitotic mutation rates, the percentage of transformed diverged chromosomes incapable of substituting for the resident chromosome was not increased in rad52 compared to the wild-type strain, indicating that the mutator phenotype does not extend to transforming chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning of mammalian DNA into a rad52 as compared to a RAD+ strain, a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained.     

The Multifaceted Origin of Taurine Cattle Reflected by the Mitochondrial Genome

A Neolithic domestication of taurine cattle in the Fertile Crescent from local aurochsen (Bos primigenius) is generally accepted, but a genetic contribution from European aurochsen has been proposed. Here we performed a survey of a large number of taurine cattle mitochondrial DNA (mtDNA) control regions from numerous European breeds confirming the overall clustering within haplogroups (T1, T2 and T3) of Near Eastern ancestry, but also identifying eight mtDNAs (1.3%) that did not fit in haplogroup T. Sequencing of the entire mitochondrial genome showed that four mtDNAs formed a novel branch (haplogroup R) which, after the deep bifurcation that gave rise to the taurine and zebuine lineages, constitutes the earliest known split in the mtDNA phylogeny of B. primigenius. The remaining four mtDNAs were members of the recently discovered haplogroup Q. Phylogeographic data indicate that R mtDNAs were derived from female European aurochsen, possibly in the Italian Peninsula, and sporadically included in domestic herds. In contrast, the available data suggest that Q mtDNAs and T subclades were involved in the same Neolithic event of domestication in the Near East. Thus, the existence of novel (and rare) taurine haplogroups highlights a multifaceted genetic legacy from distinct B. primigenius populations. Taking into account that the maternally transmitted mtDNA tends to underestimate the extent of gene flow from European aurochsen, the detection of the R mtDNAs in autochthonous breeds, some of which are endangered, identifies an unexpected reservoir of genetic variation that should be carefully preserved.     

Synthetic biology of secondary metabolite biosynthesis in actinomycetes: Engineering precursor supply as a way to optimize antibiotic production

Actinomycetes are a rich source for the synthesis of medically and technically useful natural products. The genes encoding the enzymes for their biosynthesis are normally organized in gene clusters, which include also the information for resistance (in the case of antibacterial compounds), regulation, and transport. This facilitates the manipulation of such pathways by molecular genetic techniques. Recent advances in DNA sequencing and analytical chemistry revealed that not only new strains isolated from yet unexplored habitats, but also already known strains possess a large potential for the synthesis of novel compounds. Synthetic Biology now offers a new perspective to exploit this potential further by generating novel pathways, and thereby novel products, by combining different biosynthetic steps originating from different bacteria. The supply of precursors, which are subsequently incorporated into the final product, is often already organized in a modular manner in nature and may directly be exploited for Synthetic Biology. Here we report examples for the synthesis of building blocks and possibilities to modify and optimize antibiotic biosynthesis, exemplary for the synthesis of the manipulation of the synthesis of the glycopeptide antibiotic balhimycin.     

Microbial genomics for the improvement of natural product discovery

The quest for the discovery of novel natural products has entered a new chapter with the enormous wealth of genetic data that is now available. This information has been exploited by using whole-genome sequence mining to uncover cryptic pathways, or biosynthetic pathways for previously undetected metabolites. Alternatively, using known paradigms for secondary metabolite biosynthesis, genetic information has been 'fished out' of DNA libraries resulting in the discovery of new natural products and isolation of gene clusters for known metabolites. Novel natural products have been discovered by expressing genetic data from uncultured organisms or difficult-to-manipulate strains in heterologous hosts. Furthermore, improvements in heterologous expression have not only helped to identify gene clusters but have also made it easier to manipulate these genes in order to generate new compounds. Finally, and perhaps the most crucial aspect of the efficient and prosperous use of the abundance of genetic information, novel enzyme chemistry continues to be discovered, which has aided our understanding of how natural products are biosynthesized de novo, and enabled us to rework the current paradigms for natural product biosynthesis.     

Plasmid uptake by bacteria: a comparison of methods and efficiencies

The ability to introduce individual molecules of plasmid DNA into cells by transformation has been of central importance to the recent rapid advancement of plasmid biology and to the development of DNA cloning methods. Molecular genetic manipulation of bacteria requires the development of plasmid-mediated transformation systems that include (1) chemical transformation, (2) electro-transformation, (3) biolistic transformation, and (4) sonic transformation, leading to the introduction of exogenous plasmid DNA into bacterial cells. In this review, the manipulation properties and transformation efficiencies of these techniques are described. In addition to these methods, a conceptually novel transformation technique, namely the hydrogel exposure method, was developed. The hydrogel exposure method, based on the Yoshida effect, provides a significant advance over chemical means for transforming many strains of Escherichia coli and a variety of other bacterial species. The new term “tribos transformation” has been proposed for this novel technique. We also determined that, compared to conventional methods, the hydrogel exposure method is a novel and convenient method by which to transform bacteria.     

Targeted genome engineering via zinc finger nucleases

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants.     

Biodiversity as a source for synthetic domestication of useful specific traits

Plant and animal domestication form the foundation of agriculture. Currently, there are considerable efforts and hypotheses to understand adaptation and regulatory processes involving domestication and biodiversity organisms. Here, we propose the use of recombinant DNA as a foundation for the synthetic domestication of biodiversity traits. For example, we commented on current studies involving synthetic spider‐like fibres production in bacteria and mimicking oil seed species in genetically manipulated soybean. We suggest that this approach constitutes a sustainable and viable option for conservation and development of value‐added processes and products from biodiversity.     

Genetic consequences of domestication and mass rearing of pest fruit fly Bactrocera tryoni (Diptera: Tephritidae)

Tephritid fruit flies, an important pest of horticulture worldwide, are increasingly targeted for control or eradication by large-scale releases of sterile flies of the same species. For each species treated, strains must be domesticated for mass rearing to provide sufficiently large numbers of individuals for releases. Increases in productivity of domesticated tephritid strains are well documented, but there have been few systematic studies of the genetic consequences of domestication in tephritids. Here, we used nine DNA microsatellite markers to monitor changes in genetic diversity during the early generations of domestication in replicated lines of the fruit fly Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). The observed changes in heterozygosity and allelic richness were compared with the expected changes in heterozygosity generated by a stochastic simulation including genetic drift but not selection. The results showed that repeatable genetic bottlenecks occur in the early generations and that selection occurs in the later generations. Furthermore, using the same simulation, we show that there is inadvertent selection for increased productivity for the entire life on a mass-rearing colony, in addition to intentional selection for increased productivity. That additional selection results from the common practice of establishing the next generation of the breeding colony from a small proportion of one day's pupae collection (the pupal raffle). That selection occurs during all generations and acts only on fecundity variation. Practical methods to counter that unavoidable loss of genetic diversity during the domestication process in B. tryoni are discussed.     

A loss-of-function mutant allele of a glycosyl hydrolase gene has been co-opted for seed weight control during soybean domestication

The resultant DNA from loss-of-function mutation can be recruited in biological evolution and development. Here, we present such a rare and potential case of “to gain by loss” as a neomorphic mutation during soybean domestication for increasing seed weight. Using a population derived from a chromosome segment substitution line of Glycine max(SN14) and Glycine soja(ZYD06), a quantitative trait locus(QTL) of 100-seed weight(q HSW) was mapped on chromosome 11, corresponding to a truncated β-1, 3-gl...     

Global investigation of the co‐evolution of MIRNA genes and microRNA targets during soybean domestication

Although the selection of coding genes during plant domestication has been well studied, the evolution of MIRNA genes (MIRs) and the interaction between microRNAs (miRNAs) and their targets in this process are poorly understood. Here, we present a genome‐wide survey of the selection of MIRs and miRNA targets during soybean domestication and improvement. Our results suggest that, overall, MIRs have higher evolutionary rates than miRNA targets. Nonetheless, they do demonstrate certain similar evolutionary patterns during soybean domestication: MIRs and miRNA targets with high expression and duplication status, and with greater numbers of partners, exhibit lower nucleotide divergence than their counterparts without these characteristics, suggesting that expression level, duplication status, and miRNA–target interaction are essential for evolution of MIRs and miRNA targets. Further investigation revealed that miRNA–target pairs that are subjected to strong purifying selection have greater similarities than those that exhibited genetic diversity. Moreover, mediated by domestication and improvement, the similarities of a large number of miRNA–target pairs in cultivated soybean populations were increased compared to those in wild soybeans, whereas a small number of miRNA–target pairs exhibited decreased similarity, which may be associated with the adoption of particular domestication traits. Taken together, our results shed light on the co‐evolution of MIRs and miRNA targets during soybean domestication.     

Making mitochondrial mutants.

Mitochondrial DNA (mtDNA) encodes a mere 13 polypeptides, all with well-defined cellular functions in mitochondrial energy metabolism. It was first sequenced over two decades ago, yet our understanding of the wider physiological role of mtDNA is surprisingly sketchy. Partly, this reflects the fact that the mitochondrial gene products are essential for life; that is, most mtDNA mutations are expected to be lethal. The technical difficulty of engineering mtDNA mutations has been a major handicap in furthering our understanding of the mitochondrial genetic system. Recent developments now offer some possibilities for the genetic manipulation of mtDNA and for elucidating its contribution to human development, physiology and disease.