Hans Ellegren

In order to develop models which appropriately reflect microsatellite evolution, more knowledge is required about the processes by which these simple sequences evolve. In this study, historical mutation events in three avian microsatellite loci belonging to distinct classes of repeat types (one perfect di-, one compound di-, and one perfect tetranucleotide repeat) were examined by sequence analysis of 76 alleles in 39 species spanning the avian phylogeny. The mode and tempo of evolution varied greatly between loci. For the perfect dinucleotide repeat, intraspecific length polymorphism was detected when alleles contained as few as six or seven repeat units, and size expansion over evolutionary timescales was demonstrated for repeats as short as (AG)2. A remarkable level of fragment stability was found for the compound dinucleotide repeat, even in species thought to have diverged over 60 MYA, coinciding with a high level of primer sequence conservation at this locus. In contrast, a hypervariable (AAAG)n locus revealed extraordinary instability and structural heterogeneity in the repeat region, including long arrays of derivative repeat motifs such as (AG)n, (AAGG)n, (AAAAG)n, and even (AAAGAGAG)n. Often, several motifs could be found within the same allele. A large number of cases of allele size homoplasy were detected for all three loci. These findings reinforce the fact that greater attention should be paid to the repeat type and the mutational characteristics of a marker before use in phylogenetic studies.

Among the mechanisms whereby sex is determined in animals, chromosomal sex determination is found in a wide variety of distant taxa. The widespread but not ubiquitous occurrence, not even within lineages, of chromosomal sex determination suggests that sex chromosomes have evolved independently several times during animal radiation, but firm evidence for this is lacking. The most favored model for this process is gradual differentiation of ancestral pairs of autosomes. As known for mammals, sex chromosomes may have a very ancient origin, and it has even been speculated that the sex chromosomes of mammals and birds would share a common chromosomal ancestry. In this study we showed that the two genes, ATP5A1 and CHD1 , so far assigned to the female-specific W chromosome of birds both exist in a very closely related copy on the Z chromosome but are not pseudoautosomal. This indicates a common ancestry of the two sex chromosomes, consistent with the evolution from a pair of autosomes. Comparative mapping demonstrates, however, that ATP5A1 and CHD1 are not sex-linked among eutherian mammals; this is also not the case for the majority of other genes so far assigned to the avian Z chromosome. Our results suggest that the evolution of sex chromosomes has occurred independently in mammals and birds.

The severe nuclear accident at Chernobyl in 1986 resulted in the worst reported accidental exposure of radioactive material to free-living organisms¹. Short-term effects on human populations inhabiting polluted areas include increased incidence of thyroid cancer², infant leukaemia³, and congenital malformations in newborns⁴. Two recent studies⁵,⁶ have reported, although with some controversy⁷,⁸, that germline mutation rates were increased in humans and voles living close to Chernobyl, but little is known about the viability of the organisms affected⁹. Here we report an increased frequency of partial albinism, a morphological aberration associated with a loss of fitness, among barn swallows, Hirundo rustica, breeding close to Chernobyl. Heritability estimates indicate that mutations causing albinism were at least partly of germline origin. Furthermore, evidence for an increased germline mutation rate was obtained from segregation analysis at two hypervariable microsatellite loci, indicating that mutation events in barn swallows from Chernobyl were two- to tenfold higher than in birds from control areas in Ukraine and Italy.

Assuming that new mutations arise mainly during DNA replication, sequence evolution in mammals has been seen as 'male driven' (ref. 1) because of the many more cell divisions in spermatogenesis than in oogenesis. Molecular support for this idea has been obtained from the observation of higher substitution rates in genes on the Y than on the X chromosome of primates and rodents, which are species with male heterogamety, but has not been confirmed by the reciprocal analysis of organisms with female heterogamety. The recent suggestion that an intrinsic reduction in the X-chromosome mutation rate may be confounded with male effects in previous comparisons, and the paradoxical finding of low levels of polymorphism on the primate Y chromosome indicate that the idea of male-biased mutation rate needs to be re-examined. We have analysed the molecular evolution of the gene CHD, which is present on the Z and W sex chromosomes of birds. The substitution rate at synonymous positions, as well as in intron DNA, was considerably higher on the Z chromosome than on the female-specific W chromosome, with an estimated male-to-female bias in mutation rate (alpha m) of 3.9-6.5. Thus, evolution appears to be male driven in birds--a situation that supports a neutral model of molecular evolution.

The application of microsatellites in evolutionary studies requires an understanding of the patterns governing their evolution in different species. The finding that homologous microsatellite loci are longer, i.e., containing more repeat units, in human and in other primates has been taken as evidence for directional microsatellite evolution and for a difference in the rate of evolution between species. However, it has been argued that this finding is an inevitable consequence of biased selection of longer-than-average microsatellites in human, because cloning procedures are adopted to generate polymorphic and, hence, long markers. As a test of this hypothesis, we conducted a reciprocal comparison of the lengths of microsatellite loci in cattle and sheep using markers derived from the bovine genome as well as the ovine genome. In both cases, amplification products were longer in the focal species, and loci were also more polymorphic in the species from which they were originally cloned. The crossing pattern that we found suggests that interspecific length differences detected at homologous microsatellite loci are the result of biased selection of loci associated with cloning procedures. Hence, comparisons of microsatellite evolution between species are flawed unless they are based on reciprocal analyses or on genuinely random selection of loci with respect to repeat length.

A better insight into the occurrence of microsatellites in a range of taxa may help to understand the evolution of simple repeats. Previous studies have found the relative abundance of several repeat motifs to differ among mammals, invertebrates, and plants. Absolute numbers of microsatellites also tend to correlate positively with genome size. We analyzed the occurrence, frequency, and distribution of microsatellites in birds, a taxon with one of the smallest known genome sizes among vertebrates. Dot-blot hybridization revealed that about half of 22 different di-, tri-, and tetranucleotide repeat motifs were clearly more common in human than in three species of birds: chicken, woodpecker, and swallow. For the remaining motifs no clear difference was found. From searching avian database sequences we estimated there to be 30,000–70,000 microsatellites longer than 20 bp in the avian genome. The number of (CA)_⩾10 would be around 7000–9000 and the number of (CA)_⩾14 about 3000. The calculated density of avian microsatellites (total, one every 20–39 kb; (CA)_⩾10, one every 136–150 kb) is much lower than that estimated for the human genome (one every 6 and 30 kb, respectively). This may be explained by the fact that the avian genome contains relatively less noncoding DNA than most mammals and that avian SINE/LINE elements do not terminate in poly(A) tails, which are known to provide a resource for the evolution of simple repeats in mammals. We found no association between microsatellites and SINEs in birds. Primed in situ labeling suggested fairly even distribution of (CA)_n repeats over chicken macrochromosomes and intermediate chromosomes, whereas the microchromosomes, a large part of the Z and W chromosomes, and most telomeres and centromeres had very low concentrations of (CA)_n microsatellites. The scarcity of microsatellites on the microchromosomes is compatible to these regions likely being unusually rich in coding sequences. The low microsatellite density in the genome in general and on the microchromosomes in particular imposes an obstacle for the development of marker-rich genetic maps of chicken and other birds, and for the localization of quantitative trait genes.

The Swedish moose was analysed for genetic variability at major histocompatibility complex (MHC) class I and class II DQA, DQB and DRB loci using restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) techniques. Both methods revealed limited amounts of polymorphism. Since the SSCP analysis concerned an expressed DRB gene it can be concluded that the level of functional MHC class II polymorphism, at least at the DRB locus, is low in Swedish moose. DNA fingerprinting was used to determine if the unusual pattern of low MHC variability could be explained by a low degree of genome‐wide genetic diversity. Hybridizations with two minisatellite probes gave similarity indices somewhat higher than the average for other natural population, but the data suggest that the low MHC variability cannot be explained by a recent population bottleneck. However, since minisatellite sequences evolve more rapidly than MHC sequences, the low levels of MHC diversity may be attributed to a bottleneck of more ancient origin. The selection pressure for MHC variability in moose may also be reduced and we discuss the possibility that its solitary life style may reduce lateral transmission of pathogens in the population.

Eight polymorphic microsatellite markers from the swallow were isolated and characterized. Extraordinary variability was revealed at theHrU6locus with 45 different alleles scored among 46 unrelated individuals. The probability that the same genotype combination would occur in two random and unrelated individuals at six selected loci was as low as 1.3 × 10^‐8and the combined exclusion probability was 0.9996. Stable Mendelian inheritance was observed in about 1000 meioses. No significant linkage was revealed and for almost all combinations of marker‐pairs, linkage closer than 5 cM could be excluded. At two loci, null (nonamplifying) alleles were encountered. Thirteen (30%) extra‐pair offspring were identified in 5 (56%) broods when applying the marker set on a nearly complete swallow colony. We were able to identify a single male from the other families in the colony as the most likely father for nine of the 13 extra‐pair offspring.

Flow-sorted chromosome 18 material from the pig (SSC18) was amplified with SINE-PCR to generate DNA for molecular cloning. An SSC18-enriched library was constructed and subsequently screened with a (CA)15 probe to identify polymorphic microsatellites. Eleven unique micro-satellites were obtained, six of which constituted direct extensions of SINE poly(A) tracts. Eight primer pairs amplified polymorphic loci in unrelated pigs. The polymorphic markers were typed in a Swedish reference pedigree for porcine genome mapping, but only two of them (SO177 and SO179) mapped to SSC18. The remaining markers mapped to chromosomes 3, 4, 7, 9, and 11. Together with two markers from other sources (S0062 and sw787), a four-point linkage group on SSC18 could be established.