Development and application of mammalian molecular cytogenetic tools for genome reconstruction, evolution and reproductive screening

Chromosomal analysis enables a genome-wide overview of an organism, it can provide information when used to study cellular function, the taxonomic relationship between divergent species and disease phenotypes. Consequently, chromosomal analysis is used to identify chromosomal rearrangements in an individual, which can be associated with disease and/or reproductive complications, or within a population, which is associated with speciation and reproductive isolation. The techniques used to examine the chromosomes of an organism have improved considerably over the past four decades. Observations were traditionally achieved through the production of Giemsa stained chromosomes which permitted banding analysis, therefore enabling the detection of differences in chromosome morphology and number, to more specific, molecular cytogenetic approaches (fluorescence in situ hybridisation - FISH) which can be used to identify sub-microscopic differences. Today, genome sequencing facilitates genome-wide analysis at a higher resolution than previously possible; sequence information can be used in a multitude of ways, including identification of specific mutations which result in disease, investigating homologous segments between divergent species and for ascertaining potential drug targets. However, without a physical genetic map it is now apparent that by themselves genome sequence assemblies fail to provide sufficient information regarding certain biological questions, in particular genome organisation throughout times of mammalian evolution. However, it is now apparent that map-based chromosome-level assemblies are required for deeper analysis of the genome.

With this in mind, the purpose of this work was to extend upon, and develop efficient cytogenetic tools to screen for chromosomal rearrangements in mammalian species, in the context evolutionarily events and to examine chromosomal rearrangements that manifest as fertility problems in a range of agricultural and zoological animals.

Using traditional karyotyping techniques, Ducos et al (2007) demonstrated that the translocation incidence rate was 0.47% in unproven boars. In this work, a large number of boars (>1000) were analysed using a FISH-based screening device, whereby 13 unique chromosomal translocations were detected, resulting in an incidence rate of 1%. Therefore, the results in this work demonstrate that the incidence rate is under reported in the current literature.

Before this work, karyotype analysis was the only technique used to identify chromosomal rearrangements in cattle. As a consequence of the success observed in pigs, a FISH-based device was developed to screen for chromosomal translocations in cattle. Using this technology, heterozygous and homozygous 1;29 translocations were identified, and an unreported 12;23 reciprocal translocation.