Comparative anchor tagged sequences (CATS) for integrative mapping of mammalian genomes.

Precise comparisons of mammalian gene maps require common anchor loci as landmarks for conserved chromosomal segments. Using a computer script that automates DNA sequence database alignments, we designed 410 evolutionarily conserved primer pair sequences which are specific for anchor locus gene amplification from any mammalian species' DNA. Primer pairs were designed to span introns for polymorphism ascertainment, and to include sufficient exonic sequence (25-400 bp) to allow for gene identification. A total of 318 primer pairs were optimized for domestic cats, and 86% of the sequenced feline PCR products showed homology to the gene of primer origin. A screen of 20 mammals from 11 orders revealed that 35-52% of the 318 primers yielded a single PCR product without further optimization suggesting that nearly 75% can be optimized for any eutherian mammal.

Genetic variation in clonal vertebrates detected by simple-sequence DNA fingerprinting.

The measurement of clonal heterogeneity is central to understanding the evolutionary and population genetics of the roughly 50 species of vertebrates that lack effective genetic recombination. Simple-sequence DNA fingerprinting with oligonucleotide probes (CAC)5 and (GACA)4 is a sensitive and efficient means of detecting this heterogeneity in natural populations of two clonal fishes, Poecilia formosa, an apomictic unisexual, and Rivulus marmoratus, a selfing hermaphrodite. The fingerprints are clonally stable for at least three generations. The technique clearly differentiates allozymically identical laboratory lines of R. marmoratus that were previously distinguishable only by histocompatibility analysis. The technique also reveals apparent cases of shifts in clonal composition of a natural population of each species. Clonal variation in most natural populations is quite high. For example, a sample of 19 specimens of P. formosa from one station on the Rio Soto la Marina contained 16 clones (average clonal frequency = 0.07). This level of clonal diversity implies that mutation, subsequent to the founding of clonal lineages, is an important source of variation in these populations. It also suggests that chance (sampling error) has a previously unappreciated role in determining the clonal composition of populations even though some of the clones may be divergent in biologically significant features.

Extreme clonal diversity and divergence in populations of a selfing hermaphroditic fish.

Recombination is unknown in natural populations of Rivulus marmoratus, a selfing hermaphrodite, and genetic variation is likely due to mutation alone. DNA fingerprinting with an array of microsatellite [e.g., (CT)9] and minisatellite (e.g., the 33.15 core sequence) probes reveals very high clonal diversity within samples of seven Floridian populations, of which five contain about as many clones as there are individuals. There are 42 clones among 58 individuals surveyed (mean, 1.4 individuals per clone), a level of genetic diversity unprecedented among clonal animals. Moreover, all of the probes recognize the same clones even though, at high hybridization stringencies, there is little overlap in the fingerprint patterns they generate. This suggests that most sympatric clones differ by multiple and independent mutational steps. In one population studied in detail, the average number of mutational steps separating two clones is estimated at 9 or 10 and may be substantially higher. The mutational discontinuities among sympatric clones make it unlikely that they evolved by accumulation of neutral mutations in populations that are otherwise genetically uniform. The data argue that the mixing of unrelated individuals from different local populations occurs to an extent previously unappreciated and/or that divergence of clones is mediated by natural selection. If confirmed, the latter would be a serious challenge to current ideas on the predominant role of recombination in promoting the evolution of biological novelty.

Clonal stability and mutation in the self-fertilizing hermaphroditic fish, Rivulus marmoratus.

Previous investigations of natural populations of the hermaphroditic, self-fertilizing fish species Rivulus marmoratus demonstrated a surprising amount of interclonal differentiation among highly polymorphic "DNA fingerprint" loci. The genetic differentiation observed among clones was thought to be the effect of extreme population mixing because of high rates of migration and population extinction. It was demonstrated that mutation rates at hypervariable loci would have to exceed 10(-4) on average to alone account for the observed interclonal differences. The present study reports that, among laboratory lines of this species, mutation rates at the most unstable set of hypervariable loci are not greater than 3.52 x 10(-4), and are probably lower. Mutation rates at several other sets of loci are even lower. A field transplantation study demonstrated complete clonal stability over several generations. These results suggest that the high interclonal differences observed in natural populations of this species is not caused by a generally higher rate of mutation at these specific loci.

Physical assignments of human chromosome 13 genes on pig chromosome 11 demonstrate extensive synteny and gene order conservation between pig and human.

Previous mapping between the human and pig genomes suggested extensive conservation of human chromosome 13 (HSA13) to pig chromosome 11 (SSC11). The objectives of this study were comparative gene mapping of pig homologs of HSA13 genes and examining gene order within this conserved synteny group by physical assignment of each locus. A detailed HSA13 to SSC11 comparison was chosen since the comparative gene map is not well developed for these chromosomes and a rearranged gene order within conserved synteny groups was observed from the comparison between HSA13 and bovine chromosome 12 (BTA12). Heterologous primers for PCR were designed and used to amplify pig homologous fragments. The pig fragments were sequenced to confirm the homology. Six pig STSs (FLT1, ESD, RB1, HTR2A, EDNRB, and F10) were physically mapped using a somatic cell hybrid panel to SSC11, and fluorescent in situ hybridization (FISH) mapping was also applied to improve map resolution and determine gene order. Results from this study increase the comparative information available on SSC11 and suggest a conserved gene order on SSC11 and HSA13, in contrast to human:bovine comparisons of this syntenic group.

A comparative gene map of the horse (Equus caballus).

A comparative gene map of the horse genome composed of 127 loci was assembled based on the new assignment of 68 equine type I loci and on data published previously. PCR primers based on consensus gene sequences conserved across mammalian species were used to amplify markers for assigning 68 equine type I loci to 27 horse synteny groups established previously with a horse-mouse somatic cell hybrid panel (SCHP, UC Davis). This increased the number of coding genes mapped to the horse genome by over 2-fold and allowed refinements of the comparative mapping data available for this species. In conjunction with 57 previous assignments of type I loci to the horse genome map, these data have allowed us to confirm the assignment of 24 equine synteny groups to their respective chromosomes, to provisionally assign nine synteny groups to chromosomes, and to further refine the genetic composition established with Zoo-FISH of two horse chromosomes. The equine type I markers developed in this study provide an important resource for the future development of the horse linkage and physical genome maps.

Equine synteny mapping of comparative anchor tagged sequences (CATS) from human Chromosome 5.

Comparative anchor tagged sequences (CATS) from human Chromosome 5 (HSA5) were used as PCR primers to produce molecular markers for synteny mapping in the horse. Primer sets for 21 genes yielded eight horse-specific markers, which were mapped with the UC Davis horse-mouse somatic cell hybrid panel into two synteny groups: UCD14 and UCD21. These data, in conjunction with earlier human chromosome painting studies of the horse karyotype and synteny mapping of horse microsatellite markers physically mapped by FISH, confirm the assignment of UCD21 to ECA21 and suggest that UCD14 is located on ECA14. In addition, our results can be used to substantiate previously published data which indicate that ECA21 contains material orthologous to HSA5p and HSA5q, and to propose an approximate region for an evolutionary chromosomal rearrangement event.