Using gene synteny to define putative superscaffolds as an aid for physical mapping

A physical map of genome scaffolds on chromosomes greatly increases the value of a genome sequence in studies of genomic organisation,  reconstruction of mutational events that have led to extant chromosomal diversity, and identification of candidate sex determining genes. Such a physical map is typically achieved by anchoring BAC containing target genes associated with a scaffold to particular chromosomes using fluorescent in situ hybridization (FISH). When genomes sequence is highly fragmented, as in species with high repeat content, the number of scaffolds that need to be mapped to achieve satisfactory coverage of the chromosomes is prohibitively high. For the dragon and koala genomes, we reduced the effort involved by using gene synteny to identify putative superscaffolds, that is, by establishing adjacency of genome scaffolds and building daisy chains of scaffolds on the basis of gene content and gene order. For the dragon Pogona vitticeps, tables of chicken genes in physical order were matched, as reference, against their homologues in dragon and their respective assembled scaffolds. A similar table was generated for anole as reference against dragon. Dragon scaffolds that abutted with respect to gene order and with their junction spanned by a contiguous series of consecutive genes in chicken and anole, respectively, were joined to form putative derived 2-scaffolds. The set of 2-scaffolds were then assembled into putative superscaffolds. BACs associated with terminal genes on the superscaffolds were then physically mapped to chromosomes using FISH to confirm contiguity, and establish position and orientation. A similar analysis was undertaken with koala, sandwiched between assemblies for tammar wallaby and opossum. Superscaffolding enabled us to physically anchor, with only  69 BACs, approximately 42% of the dragon genome to each of the 16 chromosome pairs, including all microchromosomes. As a comparison, 405 BACs were required to anchor 60% of the anole genome assembly to chromosomes, but only half the microchromosomes. For koala, the largest super-contig spanned 129 Mbp which equates to approximately half of koala chromosome 7.  Virtual koala chromosome maps have been constructed using conserved synteny information but require verifcation by phyiscal mapping.