Roscoe Stanyon

Evolution of mammalian genome organization inferred from comparative gene mapping

Comparative genome analyses, including chromosome painting in over 40 diverse mammalian species, ordered gene maps from several representatives of different mammalian and vertebrate orders, and large-scale sequencing of the human and mouse genomes are beginning to provide insight into the rates and patterns of chromosomal evolution on a whole-genome scale, as well as into the forces that have sculpted the genomes of extant mammalian species.

Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization

A new strategy for analyzing chromosomal evolution in primates is presented using chromosomal in situ suppression (CISS) hybridization. Biotin-labeled DNA libraries from flow-sorted human chromosomes are hybridized to chromosome preparations of catarrhines, platyrrhines, and prosimians. By this approach rearrangements of chromosomes that occurred during hominoid evolution are visualized directly at the level of DNA sequences, even in primate species with pronounced chromosomal shuffles.

Homologies in human and Macasa fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries

We established chromosomal homologies between all chromosomes of the human karyotype and that of an old world monkey (Macaca fuscata) by chromosomal in situ suppression (CISS) hybridization with human chromosome specific DNA libraries. Except for the human chromosome 2 library and limited cross-hybridization of X and Y chromosome libraries all human DNA libraries hybridized to single GTG-banded macaque chromosomes. Only three macaque chromosomes (2, 7, 13) were each hybridized by two separate human libraries (7 and 21, 14 and 15, 20 and 22 respectively). Thus, an unequivocally high degree of synteny between human and macaque chromosomes has been maintained for more than 20 million years. As previously suggested, both Papionini (macaques, baboons, mandrills and cercocebus monkeys, all of which have nearly identical karyotypes) and humans are chromosomally conservative. The results suggest, that CISS hybridization can be expected to become an indispensable tool in comparative chromosome and gene mapping and will help clarify chromosomal phylogenies with speed and accuracy.

Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats

Reciprocal chromosome painting between mouse and rat using complete chromosome probe sets of both species permitted us to assign the chromosomal homology between these rodents. The comparative gene mapping data and chromosome painting have a better than 90% correspondence. The reciprocal painting results graphically show that mouse and rat have strikingly different karyotypes. At least 14 translocations have occurred in the 10–20 million years of evolution that separates these two species. The evolutionary rate of chromosome translocations between these two rodents appears to be up to 10 times greater than that found between humans and cats, or between humans and chimpanzees, where over the last 5–6 million years just one translocation has occurred. Outgroup comparison shows that the mouse genome has incorporated at least three times the amount of interchromosomal rearrangements compared to the rat genome. The utility of chromosome painting was also illustrated by the assignment of two new chromosome homologies between rat and mouse unsuspected by gene mapping: between mouse 11 and rat 20 and between mouse 17 and rat 6. We conclude that reciprocal chromosome painting is a powerful method, which can be used with confidence to chart the genome and predict the chromosome location of genes. Reciprocal painting combined with gene mapping data will allow the construction of large-scale comparative chromosome maps between placental mammals and perhaps other animals.

DEFINING THE ANCESTRAL KARYOTYPE OF ALL PRIMATES BY MULTIDIRECTIONAL CHROMOSOME PAINTING BETWEEN TREE SHREWS, LEMURS AND HUMANS

Abstract.We used multidirectional chromosome painting with probes derived by bivariate fluorescence-activated flow sorting of chromosomes from human, black lemur (Eulemurmacacomacaco) and tree shrew (Tupaiabelangeri, order Scandentia) to better define the karyological relationship of tree shrews and primates. An assumed close relationship between tree shrews and primates also assists in the reconstruction of the ancestral primate karyotype taking the tree shrew as an ”outgroup” species. The results indicate that T.belangeri has a highly derived karyotype. Tandem fusions or fissions of chromosomal segments seem to be the predominant mechanism in the evolution of this tree shrew karyotype. The 22 human autosomal painting probes delineated 40 different segments, which is in the range found in most mammals analyzed by chromosome painting up to now. There were no reciprocal translocations that would distinguish the karyotype of the tree shrew from an assumed primitive primate karyotype. This karyotype would have included the chromosomal forms 1a, 1b, 2a, 2b, 3/21, 4–11, 12a/22a, 12b/22b, 13, 14/15, 16a, 16b, 17, 18, 19a, 19b, 20 and X and Y and had a diploid chromosome number of 2n=50. Of these forms, chromosomes 1a, 1b, 4, 8, 12a/22a, and 12b/22bmay be common derived characters that would link the tree shrew with primates. To define the exact phylogenetic relationships of the tree shrews and the genomic rearrangements that gave rise to the primates and eventually to humans further chromosome painting in Rodentia, Lagomorpha, Dermoptera and Chiroptera is needed, but many of the landmarks of genomic evolution are now known.

Chromosome painting in mammals as an approach to comparative genomics

Chromosome painting has become a routine tool in comparative cytogenetics. The utility of interspecies chromosome painting has been demonstrated in taxa characterized by highly rearranged karyotypes such as in rodents and lesser apes. Chromosome painting also provides a new level of precision in comparative genome analysis for eliminating errors of confounding convergence with homology. Recent results hold promise that molecular cytogenetics will make a significant contribution to the understanding of the major features of genome evolution.

Primate chromosome evolution: Ancestral karyotypes, marker order and neocentromeres

In 1992 the Japanese macaque was the first species for which the homology of the entire karyotype was established by cross-species chromosome painting. Today, there are chromosome painting data on more than 50 species of primates. Although chromosome painting is a rapid and economical method for tracking translocations, it has limited utility for revealing intrachromosomal rearrangements. Fortunately, the use of BAC-FISH in the last few years has allowed remarkable progress in determining marker order along primate chromosomes and there are now marker order data on an array of primate species for a good number of chromosomes. These data reveal inversions, but also show that centromeres of many orthologous chromosomes are embedded in different genomic contexts. Even if the mechanisms of neocentromere formation and progression are just beginning to be understood, it is clear that these phenomena had a significant impact on shaping the primate genome and are fundamental to our understanding of genome evolution. In this report we complete and integrate the dataset of BAC-FISH marker order for human syntenies 1, 2, 4, 5, 8, 12, 17, 18, 19, 21, 22 and the X. These results allowed us to develop hypotheses about the content, marker order and centromere position in ancestral karyotypes at five major branching points on the primate evolutionary tree: ancestral primate, ancestral anthropoid, ancestral platyrrhine, ancestral catarrhine and ancestral hominoid. Current models suggest that between-species structural rearrangements are often intimately related to speciation. Comparative primate cytogenetics has become an important tool for elucidating the phylogeny and the taxonomy of primates. It has become increasingly apparent that molecular cytogenetic data in the future can be fruitfully combined with whole-genome assemblies to advance our understanding of primate genome evolution as well as the mechanisms and processes that have led to the origin of the human genome.

Molecular cytotaxonomy of New World monkeys (Platyrrhini) – comparative analysis of five species by multi-color chromosome painting gives evidence for a classification of Callimico goeldii within the family of Callitrichidae

Chromosome rearrangements are considered as “rare genomic changes” and can provide useful markers and even landmarks for reconstructing phylogenies complementary to DNA sequence data and bio-morphological comparisons. Here, we applied multi-directional chromosome painting to reconstruct the chromosome phylogeny and evolutionary relationships among the New World monkey (Platyrrhini) species Callithrix argentata, Cebuella pygmaea, Saguinus oedipus, Callithrix jacchus and Callimico goeldii. The results clarified several aspects of New Wold monkey phylogeny. In particular the phylogenetic position of C. goeldii was elucidated, which has been controversially discussed and variously classified in the family Callitrichidae, in the family Cebidae or in its own family Callimiconidae. Comparative genome maps were established by multi-color fluorescence in situ hybridization (FISH) with human, S. oedipus and Lagothrix lagothricha chromosome- specific DNA probes. From these data we reconstructed the putative ancestral karyotype of all Callitrichidae. Various derived chromosomal syntenies are shared by all five species and cytogenetically define Callitrichidae – including Callimico goeldii – as a distinctive group within the Platyrrhini. C. pygmaea and C. argentata share identical chromosomal syntenies from which S. oedipus and C. jacchus differ by single independent translocations. A common derived chromosomal change links Callimico with the marmosets to the exclusion of the tamarins, however, it has further diverged from an ancestral marmoset karyotype by at least four apomorphic rearrangements. Saimiri sciureus, representing the Cebinae, exclusively shares a derived syntenic association with all Callithrichidae, defining the genus Saimiri as a sister group.

Centromere repositioning in mammals.

The evolutionary history of chromosomes can be tracked by the comparative hybridization of large panels of bacterial artificial chromosome clones. This approach has disclosed an unprecedented phenomenon: ‘centromere repositioning’, that is, the movement of the centromere along the chromosome without marker order variation. The occurrence of evolutionary new centromeres (ENCs) is relatively frequent. In macaque, for instance, 9 out of 20 autosomal centromeres are evolutionarily new; in donkey at least 5 such neocentromeres originated after divergence from the zebra, in less than 1 million years. Recently, orangutan chromosome 9, considered to be heterozygous for a complex rearrangement, was discovered to be an ENC. In humans, in addition to neocentromeres that arise in acentric fragments and result in clinical phenotypes, 8 centromere-repositioning events have been reported. These ‘real-time’ repositioned centromere-seeding events provide clues to ENC birth and progression. In the present paper, we provide a review of the centromere repositioning. We add new data on the population genetics of the ENC of the orangutan, and describe for the first time an ENC on the X chromosome of squirrel monkeys. Next-generation sequencing technologies have started an unprecedented, flourishing period of rapid whole-genome sequencing. In this context, it is worth noting that these technologies, uncoupled from cytogenetics, would miss all the biological data on evolutionary centromere repositioning. Therefore, we can anticipate that classical and molecular cytogenetics will continue to have a crucial role in the identification of centromere movements. Indeed, all ENCs and human neocentromeres were found following classical and molecular cytogenetic investigations.

Molecular evidence for species-level distinctions in clouded leopards.

Among the 37 living species of Felidae, the clouded leopard (Neofelis nebulosa) is generally classified as a monotypic genus basal to the Panthera lineage of great cats. This secretive, mid-sized (16-23 kg) carnivore, now severely endangered, is traditionally subdivided into four southeast Asian subspecies (Figure 1A). We used molecular genetic methods to re-evaluate subspecies partitions and to quantify patterns of population genetic variation among 109 clouded leopards of known geographic origin (Figure 1A, Tables S1 ans S2 in the Supplemental Data available online). We found strong phylogeographic monophyly and large genetic distances between N. n. nebulosa (mainland) and N. n. diardi (Borneo; n = 3 individuals) with mtDNA (771 bp), nuclear DNA (3100 bp), and 51 microsatellite loci. Thirty-six fixed mitochondrial and nuclear nucleotide differences and 20 microsatellite loci with nonoverlapping allele-size ranges distinguished N. n. nebulosa from N. n. diardi. Along with fixed subspecies-specific chromosomal differences, this degree of differentiation is equivalent to, or greater than, comparable measures among five recognized Panthera species (lion, tiger, leopard, jaguar, and snow leopard). These distinctions increase the urgency of clouded leopard conservation efforts, and if affirmed by morphological analysis and wider sampling of N. n. diardi in Borneo and Sumatra, would support reclassification of N. n. diardi as a new species (Neofelis diardi).