Integration of trinucleotide microsatellites into a linkage map of Citrus

 We report the successful assignment of the first seven microsatellite markers to the Citrus RFLP and isozyme map. A total of 14 microsatellite primer pairs were developed and tested for amplification and product-length polymorphism within a population of plants previously used for linkage-map construction. In each case, the successfully assigned microsatellite mapped to the termini of a different linkage group indicating a widespread distribution throughout the genome. Analysis of allele segregation revealed that two of nine microsatellites displayed a significant deviation from expected ratios (P>0.5). This was compared with other marker types within Citrus and a similar proportion of skewed loci was also found to be present. The analysis of two markers was complicated by the non-amplification of an inherited null allele within the mapping population. The successful integration of microsatellites into the genetic map of Citrus demonstrates the utility of this marker type for genetic analysis within wide intergeneric plant crosses. Received: 16 September 1996 / Accepted: 18 October 1996     

Isolation and characterization of trinucleotide microsatellites in African malaria mosquito Anopheles funestus

We developed microsatellite markers for an important African malaria mosquito Anopheles funestus Giles. The microsatellite‐enriched genomic library was constructed and screened with single‐strand oligonucleotides [(CCT)₁₇, (AAT)₁₇, (CAG)₁₇ and (GA)₂₅] as probes. Among the 47 pairs of polymerase chain reaction primers screened, 31 produced successful and consistent amplification. Although only a few A. funestus individuals from one geographical location were used to screen microsatellite marker polymorphism, 27 markers were found polymorphic and four markers monomorphic. Most polymorphic markers are trinucleotide markers. Isolation of polymorphic microsatellite markers provide useful tools for A. funestus population genetic studies and genome mapping.     

PCR primers for trinucleotide and tetranucleotide microsatellites in greater amberjack,Seriola dumerili

Eighteen nuclear‐encoded microsatellites from a genomic DNA library of greater amberjack, Seriola dumerili, were isolated and characterized. The microsatellites include 13 perfect (five tetranucleotide and eight trinucleotide) and five imperfect (three tetranucleotide, one trinucleotide and one combination dinucleotide/trinucleotide) repeat motifs. The number of alleles at the 18 microsatellites among a sample of 29 fish ranged from two to 20; gene diversity (expected heterozygosity) ranged from 0.068 to 0.950, whereas observed heterozygosity ranged from 0.069 to 0.966. Following Bonferroni correction, genotypes at all 18 microsatellites fit expectations of Hardy–Weinberg equilibrium, and all pairwise comparisons of microsatellites did not deviate significantly from genotypic equilibrium. Greater amberjack support commercial and recreational fisheries along both the Atlantic and the Gulf coasts of the USA and represent a species with potential for worldwide aquaculture. The microsatellites developed will be useful for population genetic studies of ‘wild’ populations and breeding studies of domesticated populations.     

Length polymorphism and allele structure of trinucleotide microsatellites in natural accessions of Arabidopsis thaliana

 The objective of this work was to assess the degree of trinucleotide microsatellite length polymorphism in the selfing species Arabidopsis thaliana. PCR amplifications of 12 microsatellite loci among 49 natural populations revealed between one to eight length variants (alleles) for each locus. The average number of alleles per locus was four and the average genetic diversity index was 0.43. Divergence between length variants was investigated at the nucleotide level. Several observations emerge from the sequence data: (1) for most loci, length polymorphism results only from variations in the number of trinucleotide repeats; (2) for a few others, some variability was noted in the flanking sequences; (3) for compound and interrupted loci containing two arrays of trinucleotide repeats, length variations preferentially affect the longest one. Five of the Arabidopsis thaliana accessions were clearly composed of two sublines. In 2 other accessions, some heterozygous individual plants, probably resulting from recent outcrosses, were found. A phylogenetic tree constructed on the basis of trinucleotide microsatellite allelic diversity shows that genetic relationships among the accessions are not correlated with their geographic origin. Received: 4 November 1997 / Accepted: 3 March 1998     

The effect of insertion of the maize transposable elementMutator is dependent on genetic background

A secondary mutant, derived from an allele of maize alcohol dehydrogenase 1 (Adh1) carrying a Mutator transposable element (Mu1) in its first intron, was reported to exhibit a threefold decrease in ADH enzymatic activity and steady-state RNA levels compared to the original mutant. The original mutant,Adh1-S3034 (abbreviatedS3034), was previously characterized at the molecular level. The derivative, abbreviatedS3034b, has now been cloned; at the DNA sequence level the insertion and surroundingAdh1 sequences are indistinguishable fromS3034. Furthermore, in our lines there is no difference in relative ADH activities between products of the two putative alleles. A comparison of gene expression in heterozygotes obtained by crossing to different tester lines reveals a correlation between the measured decrease in levels of ADH polypeptide produced by the mutant allele and the background in which it is measured; this effect is distinct from any background-related variation in the expression of the progenitor allele. It does not appear to be attributable to alternative patterns of DNA modification. It appears to reflect a background-associated difference in the level of normalAdh1-RNA produced. Thus the previously reported distinction betweenS3034 andS3034b may be due to differences in the extent to which the mutant allele and a given genetic background interact to produce functionalAdh1-RNA.This research was supported by United States Public Health Service Grant GM38616 and United States Department of Agriculture Grant 87-CRCR-1-2500 to J.S. D.O. was supported by an NIH predoctoral training grant to the Department of Genetics.     

Isolation and characterization of di‐ and trinucleotide microsatellites in the freshwater snail Potamopyrgus antipodarum

The freshwater prosobranch snail Potamopyrgus antipodarum is an important model system for studying the maintenance of sexual reproduction in its native New Zealand. Since introduction to the UK in the 1850s, the species has spread throughout Europe and recently the USA. We present the first microsatellites for P. antipodarum. Using two isolation protocols, which are qualitatively compared, we have developed PCR primers for three di‐ and four trinucleotide microsatellites. All loci proved to be polymorphic, when screened for variability in several UK populations. These markers should be of considerable utility in future population and ecological genetics studies of this species.     

Sequence analysis of trinucleotide repeat microsatellites from an enrichment library of the equine genome.

Microsatellites are useful tools for the construction of a linkage map and parentage testing of equines, but only a limited number of equine microsatellites have been elucidated. Thus, we constructed the equine genomic library enriched for DNA fragments containing (CAG)n repeats. The enriched method includes hybridization-capture of repeat regions using biotin-conjugated oligonucleotides, nucleotide substrate-biased polymerase reaction with the oligonucleotides and subsequent PCR amplification, because these procedures are useful for the cloning of less abundant trinucleotide microsatellites. Microsatellites containing (CAG)n repeats were obtained at the ratio of one per 3-4 clones, indicating an enrichment value about 10(4)-fold, resulting in less time consumption and less cost for cloning. In this study, 66 different microsatellites, (CAG)n repeats, were identified. The number of complete simple CAG repeats in our clones ranged 4-33, with an average repeat length of 8.8 units. The microsatellites were useful as sequence-tagged site (STS) markers. In addition, some clones containing (CAG)n repeats showed homology to human (CAG)n-containing genes, which have been previously mapped. These results indicate that the clones might be a useful tool for chromosome comparison between equines and humans.     

A model of weak selection in the infinite alleles framework

Ewens (1972) proposed a model in the infinite allele framework for populations with neutrality of all alleles at a particular locus. This paper proposes a generalisation of Ewens' result for situations where there is a form of weak selection. The models considered here are continuous time, discrete state space Markov processes.     

Developing microsatellites when they are rare: trinucleotide repeat loci in the northern mockingbird Mimus polyglottos

The great value of microsatellite loci to population studies spurs their development. However, finding loci is difficult when microsatellites are uncommon in the genome. Because trinucleotide-repeat loci are rare in northern mockingbirds Mimus polyglottos, we sought a new method for developing a suite of loci. Here we show that a bacteriophage cloning vector, Lambda Zap Express (Stratagene, La Jolla, CA, USA) has several features which make it suitable for this purpose. Using this vector, we made a library of 150 000 size-selected clones and screened with an AAT₁₀ probe; 97 positives were identified. From these, 12 pairs of PCR primers were developed, nine of which amplify polymorphic loci. Certain combinations of these primer pairs enable simultaneous amplification of up to three loci.     

The physiology of weak selection

Reaction rates in metabolic pathways typically exhibit a kind of diminishing returns in which small variations in the activities of the individual enzymes have very little effect on overall flux. These effects are measured by the control coefficients of the enzymes, and most systems are governed by the summation theorem stating that all control coefficients must sum to unity. One implication is that complex systems will not usually contain single rate limiting steps, but rather be controlled to a greater or lesser extent by many enzymes, each exerting relatively small control. Wright understood this principle in 1934 and used it for his physiological theory of dominance. With respect to small variations in enzyme activity, the principle implies that many small variations should have only mild effects on fitness. Analysis of nucleotide polymorphisms in the genes for glucose-6-phosphate dehydrogenase and alkaline phosphatase in Escherichia coli implies that most amino acid replacements are harmful, and that the average selection coefficient against amino acid replacements that are polymorphic in natural populations is 1 x 10(-7) to 5 x 10(-7). In experiments to determine the a priori distribution of selection coefficients among random amino acid replacements, 25 replacements in beta-galactosidase were created by genetic means, and 22 of these produced selective effects too small to be detected in chemostat competition experiments (s less than 0.004 per generation).     

Isolation and characterization of polymorphic trinucleotide microsatellites of the polyploid crucian carp (Carassius auratus)

In this study, we isolated and characterized nine polymorphic trinucleotide microsatellites (CAG and CCT) from the crucian carp (Carassius auratus). The number of alleles per locus ranged from two to six. Five loci showed a significantly excess homozygosity, and a genetic linkage between CAL0102 and CAL0495 was strongly suggested. Our results confirmed the triploidy of Korean individuals, and the microsatellites were found to be useful for analysing the allelic state of the polyploid crucian carp.     

Male gametophytic selection in maize

Summary There is evidence that male gametophyte selection is a widespread phenomenon in higher plants. The pollen tube growth rate is one of the main components of gametophyte selective value; genetic variability for this trait, due to the effect of single genes or to quantitative variation, has been described in maize. However, indication of gametophytic selection has been indirectly obtained; its effect was revealed by the positive relation observed between gametophyte competitive ability and sporophyte metrical traits.This paper considers the results of selection applied to gametophyte populations produced from single plants. The competitive ability of the lines was evaluated in comparison with that of a standard line by means of the pollen mixture technique. Sporophytic traits were measured in the hybrid progeny obtained by crossing selected S₃ and S₄ families with an unrelated single cross and an inbred line. Gametophyte selection produced inbred lines with high gametophyte competitive ability. In view of the selection procedure adopted, this result was interpreted as an indication of haploid expression of genes involved in the control of pollen tube growth. Moreover, this gametophytic trait was positively correlated with sporophytic traits (seedling weight, kernel weight and root tip growth in vitro), indicating that both groups of characters have a common genetic basis.     

The Hill-Robertson effect: evolutionary consequences of weak selection and linkage in finite populations

The 'Hill-Robertson (HR) effect' describes that linkage between sites under selection will reduce the overall effectiveness of selection in finite populations. Here we discuss the major concepts associated with the HR effect and present results of computer simulations focusing on the linkage effects generated by multiple sites under weak selection. Most models of linkage and selection forecast differences in effectiveness of selection between chromosomes or chromosomal regions involving a number of genes. The abundance and physical clustering of weakly selected mutations across genomes, however, justify the investigation of HR effects at a very local level and we pay particular attention to linkage effects among selected sites of the same gene. Overall, HR effects caused by weakly selected mutations predict differences in effectiveness of selection between genes that differ in exon-intron structures and across genes. Under this scenario, introns might play an advantageous role reducing intragenic HR effects. Finally, we summarize observations that are consistent with local HR effects in Drosophila, discuss potential consequences on population genetic studies and suggest future lines of research.     

Convergence of multilocus systems under weak epistasis or weak selection

 The convergence of multilocus systems under viability selection with constant fitnesses is investigated. Generations are discrete and nonoverlapping; the monoecious population mates at random. The number of multiallelic loci, the linkage map, dominance, and epistasis are arbitrary. It is proved that if epistasis or selection is sufficiently weak (and satisfies a certain nondegeneracy assumption whose genericity we establish), then there is always convergence to some equilibrium point. In particular, cycling cannot occur. The behavior of the mean fitness and some other aspects of the dynamics are also analyzed. Received: 15 November 1997 / Revised version: 25 May 1998     

A coalescent model of background selection with recombination,demography and variation in selection coefficients

There is increasing evidence that background selection, the effects of the elimination of recurring deleterious mutations by natural selection on variability at linked sites, may be a major factor shaping genome-wide patterns of genetic diversity. To accurately quantify the importance of background selection, it is vital to have computationally efficient models that include essential biological features. To this end, a structured coalescent procedure is used to construct a model of background selection that takes into account the effects of recombination, recent changes in population size and variation in selection coefficients against deleterious mutations across sites. Furthermore, this model allows a flexible organization of selected and neutral sites in the region concerned, and has the ability to generate sequence variability at both selected and neutral sites, allowing the correlation between these two types of sites to be studied. The accuracy of the model is verified by checking against the results of forward simulations. These simulations also reveal several patterns of diversity that are in qualitative agreement with observations reported in recent studies of DNA sequence polymorphisms. These results suggest that the model should be useful for data analysis.     

An analysis of genetic diversity across the maize genome using microsatellites

How domestication bottlenecks and artificial selection shaped the amount and distribution of genetic variation in the genomes of modern crops is poorly understood. We analyzed diversity at 462 simple sequence repeats (SSRs) or microsatellites spread throughout the maize genome and compared the diversity observed at these SSRs in maize to that observed in its wild progenitor, teosinte. The results reveal a modest genome-wide deficit of diversity in maize relative to teosinte. The relative deficit of diversity is less for SSRs with dinucleotide repeat motifs than for SSRs with repeat motifs of more than two nucleotides, suggesting that the former with their higher mutation rate have partially recovered from the domestication bottleneck. We analyzed the relationship between SSR diversity and proximity to QTL for domestication traits and observed no relationship between these factors. However, we did observe a weak, although significant, spatial correlation for diversity statistics among SSRs within 2 cM of one another, suggesting that SSR diversity is weakly patterned across the genome. Twenty-four of 462 SSRs (5%) show some evidence of positive selection in maize under multiple tests. Overall, the pattern of genetic diversity at maize SSRs can be explained largely by a bottleneck effect with a smaller effect from selection.     

Apostatic selection on prey that match the background

There is good experimental evidence that predators often remove disproportionately more of the common prey types. This 'apostatic selection' could maintain colour polymorphisms within prey species. In nature, morphs of many species appear to match components in the background, but most of the experiments that have tested apostatic selection have used prey that were conspicuous. In work described in this paper, wild birds at one site were presented a random order of 51 experiments. Seventeen frequencies of orange and grey pastry prey were presented on each of three types of background: a hessian sheet scattered with either orange and grey stones (the 'matching' background), or lilac and yellow stones, or green stones (two sorts of 'control' background). Each experiment consisted of four trials in succession and the numbers of the two colours eaten in each trial were recorded when about half the total prey had been eaten. Two methods of graphical analysis revealed that apostatic selection occurred on the prey on all three backgrounds, but was strongest in the grey/orange one. This last result must have been caused by some effect of the match between prey and background colour, and behavioural explanations are suggested. It is unclear whether the prey were exhibiting 'crypsis' or 'masquerade'.     

Contrasted patterns of selection on MHC-linked microsatellites in natural populations of the Malagasy plague reservoir

Plague (Yersinia pestis infection) is a highly virulent rodent disease that persists in many natural ecosystems. The black rat (Rattus rattus) is the main host involved in the plague focus of the central highlands of Madagascar. Black rat populations from this area are highly resistant to plague, whereas those from areas in which the disease is absent (low altitude zones of Madagascar) are susceptible. Various lines of evidence suggest a role for the Major Histocompatibility Complex (MHC) in plague resistance. We therefore used the MHC region as a candidate for detecting signatures of plague-mediated selection in Malagasy black rats, by comparing population genetic structures for five MHC-linked microsatellites and neutral markers in two sampling designs. We first compared four pairs of populations, each pair including one population from the plague focus and one from the disease-free zone. Plague-mediated selection was expected to result in greater genetic differentiation between the two zones than expected under neutrality and this was observed for one MHC-class I-linked locus (D20Img2). For this marker as well as for four other MHC-linked loci, a geographic pattern of genetic structure was found at local scale within the plague focus. This pattern would be expected if plague selection pressures were spatially variable. Finally, another MHC-class I-linked locus (D20Rat21) showed evidences of balancing selection, but it seems more likely that this selection would be related to unknown pathogens more widely distributed in Madagascar than plague.