Comparative genomics : a case study of genome, chromosome and gene family evolution

Abstract

Comparisons between the genomes have provided invaluable resources and tools for the annotation of DNA sequences. Comparative genomics is widely used to identify function of DNA sequences, understand dynamics of the evolutionary processes and provide framework for genome organization. Particularly useful for comparative analyses are the genomes of distantly related organisms such as marsupials and human. When the DNA sequence data was scarce and expensive to acquire, comparative analyses of genomes were performed by comparative gene mapping studies. However, with the advent of cheaper and faster DNA sequencing technology, genome scale comparisons at a single nucleotide level were made possible. This cheaper and faster DNA sequencing has resulted in an avalanche of information available for comparative analysis. A major challenge of comparative analysis is to explore genome organization and evolution by locating the markers on the chromosome. This has traditionally been achieved by genetic linkage and physical maps, which are generated by different experiments and not always easy to align. I have pioneered a strategy to characterize the low coverage genome sequences, which were subsequently used for identifying microsatellite markers for the tammar wallaby linkage map. My strategy allowed for easy integration of the physical map and linkage map to construct an integrated map of the tammar wallaby genome. Specifically, I first identified and used conserved blocks of synteny between opossum and human, which were reconstructed for tammar wallaby by overlapping sequence searches and assembly process. These reconstructed conserved blocks of synteny were then assigned to tammar wallaby chromosomes using the physical mapping data. Since tammar wallaby linkage groups have previously been assigned to chromosomes, the gap regions and corresponding conserved blocks of synteny were easily identified from the karyotype. Subsequently microsatellite markers were sought in the conserved blocks of synteny and tested for polymorphism in the mapping families. 26 targeted markers were finally used for linkage analysis. This systematic use and characterization of the low coverage genome sequences led to a great saving in cost and effort for obtaining high quality linkage map (150 markers), which will be used for the assembly of the low coverage sequences. Once the assemblies of the genomes are produced, they can be used to infer fine scale karyotype rearrangements that occurred during the evolution of chromosomes. For instance, comparisons of the sex chromosomes between the three major groups of mammals (placental, marsupial and monotreme) has led to a new understanding of how human sex chromosomes evolved from two genome blocks, one representing a conserved therian X (XCR) and one a region added in the placental lineage (XAR). A recent study involving genome assembly comparisons, in which the human Xchromosome genes were compared with the chicken genome, concluded that the human X chromosome is composed of three evolutionary layers. This conclusion was inconsistent with the widely accepted model proposing only two evolutionary layers, the X-conserved region and the X-added region. I therefore investigated the origins of the genome blocks making up the human X chromosome to identify the cause of this inconsistency and resolve it. My comparative analysis of the location and order of the human X chromosome homologous genes in rat, opossum and chicken genomes revealed that the problem arose because of inaccurate assignment of chicken paralogs as the orthologs. I identified the true orthologs of the human X genes in the chicken/zebrafinch EST database and showed that paralogs were incorrectly identified because the orthologs were missing from an incomplete chicken genome assembly. I then mapped the orthologs of human X genes in tammar wallaby by using the fluorescent in situ hybridization, and compared them with platypus, chicken, lizard and frog genomes to conclude that the human X chromosome is composed of only two evolutionary layers, the X-added region and the X-conserved region, as originally proposed. The assignment of orthologous or paralogous relationships in order to track gene evolution is particularly difficult for genes that belong to large families. In the last part of my research I analysed a huge and rather unusual gene family, the olfactory receptor gene (ORG) family. Comparative analysis of ORGs is difficult because the family is extremely large (~1000 genes in mammals). No comprehensive analyses have yet been performed to identify and characterize members of this gene family in a systematic fashion since the advent of large-scale genome sequence data. Therefore, I performed exhaustive searches to first identify olfactory receptor genes in all vertebrates. There are approximately 1000 olfactory receptor genes in mammals and frog, 500 in birds and 150 in lizards and fish. I also classified this gene family in 101 evolutionarily related groups of genes to provide a framework for dissecting evolutionary pathways. I also proposed a systematic nomenclature for this gene family based on the classification. This specialist data mining and classification strategy for olfactory receptor genes will provide unique opportunities to advance our understanding of this gene family in the future.