Genomic evolution during a 10,000-generation experiment with bacteria

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.

Detecting immigration by using multilocus genotypes

Immigration is an important force shaping the social structure, evolution, and genetics of populations. A statistical method is presented that uses multilocus genotypes to identify individuals who are immigrants, or have recent immigrant ancestry. The method is appropriate for use with allozymes, microsatellites, or restriction fragment length polymorphisms (RFLPs) and assumes linkage equilibrium among loci. Potential applications include studies of dispersal among natural populations of animals and plants, human evolutionary studies, and typing zoo animals of unknown origin (for use in captive breeding programs). The method is illustrated by analyzing RFLP genotypes in samples of humans from Australian, Japanese, New Guinean, and Senegalese populations. The test has power to detect immigrant ancestors, for these data, up to two generations in the past even though the overall differentiation of allele frequencies among populations is low.

A neurotransmitter transporter encoded by the Drosophila inebriated gene

Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl−-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron.

High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology

The worldwide threat of tuberculosis to human health emphasizes the need to develop novel approaches to a global epidemiological surveillance. The current standard for Mycobacterium tuberculosis typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Here, we propose a high-resolution typing method based on variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 human minisatellite-like regions of the M. tuberculosis genome. MIRU-VNTR profiles of 72 different M. tuberculosis isolates were established by PCR analysis of all 12 loci. From 2 to 8 MIRU-VNTR alleles were identified in the 12 regions in these strains, which corresponds to a potential of over 16 million different combinations, yielding a resolution power close to that of IS6110-RFLP. All epidemiologically related isolates tested were perfectly clustered by MIRU-VNTR typing, indicating that the stability of these MIRU-VNTRs is adequate to track outbreak episodes. The correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Compared with IS6110-RFLP, high-resolution MIRU-VNTR typing has the considerable advantages of being fast, appropriate for all M. tuberculosis isolates, including strains that have a few IS6110 copies, and permitting easy and rapid comparison of results from independent laboratories. This typing method opens the way to the construction of digital global databases for molecular epidemiology studies of M. tuberculosis.

The role of genetic and genomic attributes in the success of polyploids

In 1950, G. Ledyard Stebbins devoted two chapters of his book Variation and Evolution in Plants (Columbia Univ. Press, New York) to polyploidy, one on occurrence and nature and one on distribution and significance. Fifty years later, many of the questions Stebbins posed have not been answered, and many new questions have arisen. In this paper, we review some of the genetic attributes of polyploids that have been suggested to account for the tremendous success of polyploid plants. Based on a limited number of studies, we conclude: (i) Polyploids, both individuals and populations, generally maintain higher levels of heterozygosity than do their diploid progenitors. (ii) Polyploids exhibit less inbreeding depression than do their diploid parents and can therefore tolerate higher levels of selfing; polyploid ferns indeed have higher levels of selfing than do their diploid parents, but polyploid angiosperms do not differ in outcrossing rates from their diploid parents. (iii) Most polyploid species are polyphyletic, having formed recurrently from genetically different diploid parents. This mode of formation incorporates genetic diversity from multiple progenitor populations into the polyploid “species”; thus, genetic diversity in polyploid species is much higher than expected by models of polyploid formation involving a single origin. (iv) Genome rearrangement may be a common attribute of polyploids, based on evidence from genome in situ hybridization (GISH), restriction fragment length polymorphism (RFLP) analysis, and chromosome mapping. (v) Several groups of plants may be ancient polyploids, with large regions of homologous DNA. These duplicated genes and genomes can undergo divergent evolution and evolve new functions. These genetic and genomic attributes of polyploids may have both biochemical and ecological benefits that contribute to the success of polyploids in nature.

Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers.

A maximum likelihood approach of half tetrad analysis (HTA) based on multiple restriction fragment length polymorphism (RFLP) markers was developed. This procedure estimates the relative frequencies of 2n gametes produced by mechanisms genetically equivalent to first division restitution (FDR) or second division restitution and simultaneously locates the centromere within a linkage group of RFLP marker loci. The method was applied to the diploid alfalfa clone PG-F9 (2n = 2x = 16) previously selected because of its high frequency of 2n egg production. HTA was based on four RFLP loci for which PG-F9 was heterozygous with codominant alleles that were absent in the tetraploid tester. Models including three linked and one unlinked RFLP loci were developed and tested. Results of the HTA showed that PG-F9 produced 6% FDR and 94% second division restitution 2n eggs. Information from a marker locus belonging to one linkage group was used to more precisely locate the centromere on a different linkage group. HTA, together with previous cytological analysis, indicated that in PG-F9, FDR 2n eggs are likely produced by diplospory, a mechanism common among apomictic species. The occurrence of FDR 2n eggs in plant species and their importance for crop evolution and breeding is discussed together with the potential applicability of multilocus HTA in the study of reproductive mutants.

Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families.

In this paper, a reverse-transcriptase PCR-based protocol suitable for efficient expression analysis of multigene families is presented. The method combines restriction fragment length polymorphism (RFLP) technology with a gene family-specific version of mRNA differential display and hence is called "RFLP-coupled domain-directed differential display. "With this method, expression of all members of a multigene family at many different developmental stages, in diverse tissues and even in different organisms, can be displayed on one gel. Moreover, bands of interest, representing gene family members, are directly accessible to sequence analysis, without the need for subcloning. The method thus enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. Here the technique was used to analyze differential expression of MADS-box genes in male and female inflorescences of maize (Zea mays ssp. mays). Six different MADS-box genes could be identified, being either specifically expressed in the female sex or preferentially expressed in male or female inflorescences, respectively. Other possible applications of the method are discussed.

Interactions between quantitative trait loci in soybean in which trait variation at one locus is conditional upon a specific allele at another.

A large recombinant inbred population of soybean has been characterized for 220 restriction fragment-length polymorphism (RFLP) markers. Values for agronomic traits also have been measured. Quantitative trait loci (QTL) for height, yield, and maturity were located by their linkage to RFLP markers. QTL controlling large amounts of trait variation were analyzed for the dependence of trait variation on particular alleles at a second locus by comparing cumulative distributions of the trait for each genotype (four genotypes per pair of loci). Interesting pairs of loci were analyzed statistically with maximum likelihood and Monte Carlo comparison of additive and epistatic models. For each locus affecting height, variation was conditional upon the presence of a particular allele at a second unlinked locus that itself explained little or no trait variation. The results show that interactions between QTL are frequent and control large effects. Interactions distinguished between different QTL in a single linkage group and between QTL that affect different traits closely linked to one RFLP marker--i.e., distinguished between pleiotropy and closely linked genes. The implications for the evolution of inbreeding plants and for the construction of agronomic breeding strategies are discussed.