**2.3.2 Mapping and sequencing of the wallaby MHC**

The opossum and wallaby are distantly related species, having diverged from a common ancestor around 60 – 80 mya, making a comparison of these two species similar to the informative human-mouse comparison. Unlike the opossum, the wallaby genome was only lightly sequenced (Renfree et al., 2011), leaving many gaps in the genome assembly. If detailed comparative analysis was to be carried out on the MHC, an alternative approach was required.

Initial comparative analysis of these two MHCs was carried out using gene mapping. BACs containing MHC genes from all three Classes were isolated from a tammar wallaby BAC library. These clones were FISH-mapped to wallaby chromosomes with startling results. All Class II and Class III genes, as well as MHC flanking genes, mapped to the expected location on chromosome 2. Surprisingly, all of the MHC Class I BACs mapped to locations on every chromosome except chromosome 2 and the sex chromosomes (Deakin et al., 2007). This unexpected and unprecedented result made a more thorough analysis of these genes critical. As a result, a concerted effort was made to sequence the entire tammar wallaby MHC, including the 'core' MHC located on chromosome 2 and many of the dispersed Class I genes found elsewhere in the genome. A BAC-based approach was taken, with the idea of constructing a BAC-contig across the core MHC, as well as sequencing the dispersed Class I genes.

After finding Class I genes dispersed across the genome, a thorough screening of the wallaby BAC library was performed in order to isolate as many Class I genes as possible. As a result four additional BAC clones containing Class I genes were isolated, with FISHmapping of these BACs localizing these genes to the core MHC region on chromosome 2 (Siddle et al., 2009). Complete sequencing of these BACs identified six Class I genes within the core MHC, which were interspersed with antigen processing genes and a Class II gene. Sequencing of ten BACs mapping outside this region identified nine Class I genes with open reading frames. In depth sequence analysis of these BACs revealed a tendency for Kangaroo Endogenous Retroviral Element (KERV) to flank these dispersed Class I genes, suggesting that this element may be implicated in the movement of these genes to regions outside the core MHC (Siddle et al., 2009).

A BAC contig across the core MHC on wallaby chromosome 2 was constructed for sequencing purposes (Siddle et al., 2011). Unfortunately, despite extensive library screening with overgo probes designed from BAC end sequence, a single contig spanning the entire region was not obtained. Instead, the isolated BACs assembled into nine contigs and three 'orphaned' BACs. The order of these contigs and orphaned BACs was determined using BAC clones as probes for FISH on metaphase chromosome spreads and interphase nuclei. The resulting 4.7Mb sequence contained 129 predicted genes from all three MHC Classes. A comparison of the gene arrangement between wallaby, opossum and other vertebrates indicated that the wallaby MHC has a novel MHC gene arrangement, even within the core MHC. The wallaby Class II genes have undergone an expansion, residing in two clusters either side of the Class III region. Once again, KERV sequences are prominent in this region and may have contributed to the overall genomic instability of the wallaby MHC region (Siddle et al., 2011).
