Figure 1.
Schematic diagram of six saltwater crocodile gene clusters representing MHC class I and II.
Arrows indicate annotated genes and their strands (plus and minus); lines with names in the boxes below the annotation indicate BAC clones corresponding to the MHC gene clusters; and sticky ends show restriction sites of Hind III enzyme, and therefore BAC end sequencing. For retrotransposon sequences and endogenous retrovirus (ERV) sequences, asterisks indicate retrotransposon reverse transcriptase (RT) proteins; hashes indicate Gag-Pol precursor polyproteins; and pluses indicate non-LTR retrotransposon LINE-1 (L1).
Table 1.
List of annotated MHC gene clusters of saltwater crocodile genome from a total of nine BACs.
Figure 2.
Comparison of the saltwater crocodile, American alligator and Indian gharial MHC class I and II.
Scaffold ID is indicated on the left of each genomic scaffold with S (an abbreviation of a scaffold) or GC (an abbreviation of a gene cluster) followed by number. MHC gene clusters identified in the current saltwater crocodile genome assembly are illustrated in Fig. 1. Scaffolds from the American alligator (unmasked v0.2.1, id 19558) and Indian gharial (unmasked v0.2, id 19547) are retrieved from CoGe database. Annotation for each row of genes across these three species is indicated on the last column. Dark areas within the MHC class I region indicates ambiguous sites. Plus and minus signs indicate sequence strand. A question mark suggests uncertainty of identifying a single gene or separate genes of MHC class I due to a sequence gap; asterisks indicate genes of which only α or β domains are available to assess intact open reading fragments.
Figure 3.
Comparative MHC organisations of the fugu, chicken, saltwater crocodile and human.
MHC mapping in the fugu, chicken, and human is generated using data from Clark et al. [87], AL023516 (plus Shiina et al. [29] for a framework region), and NT_007592, respectively. Gene cluster 2 of the saltwater crocodile, where a gene model of coding sequences is absent, is omitted in this figure. Graphics in the first row of each vertebrate represent genes in the MHC based on schematic representation in GEvo [88], where all graphics are automatically created if applicable. Unlinked MHC genes and regions in the fugu and saltwater crocodile presented by the absence of line connection indicate that their order is arbitrary and is not based on the current data. Gray arrows indicate gene models; green arrows indicate protein coding sequences (CDS); blue arrows (on top of gray genes) indicate mRNA; and yellow arrows indicate approximately 50% GC content in codon wobble positions. Scales above the graphics show different sizes of MHC regions in kilo base pairs (K) or mega base pairs (M).
Figure 4.
Bayesian phylogenetic tree of MHC class I genes.
The fish MHC class I sequence (Onmy-UBA; AF287487) is used as an outgroup. Brackets on the right show Clades 1 to 4 of the MHC genes/pseudogenes from Crocodylia identified in the current study and previous publications as described in Materials and Methods. For Clades 1 and 3, gene lineages are named with ‘U’ for unknown families of MHC class I and then the locus name, following Klein et al. [86]. Support on branches is indicated by posterior probabilities (PP = 0–1).
Figure 5.
Bayesian phylogenetic tree of MHC class II A genes.
The fish MHC class II A sequence (Onmy-DAA; AFP94173) is used as an outgroup. A bracket on the right shows the DAA gene lineage of the MHC genes from Crocodylia identified in the current study and previous publications as described in Materials and Methods. This gene lineage is named with ‘DAA’ (an abbreviation for MHC class II A), following Klein et al. [86]. Support on branches is indicated by posterior probabilities (PP = 0–1).
Figure 6.
Bayesian phylogenetic analysis of MHC class II B sequences.
The fish MHC class II B sequence (Onmy-DAB; FR688148) is used as an outgroup. Brackets on the right show Clades 1 and 2 of the MHC variants from Crocodylia, and six subclades (A–F) for Clade 1. For Subclades A and B, gene lineages are named with ‘DAB’ (an abbreviation for MHC class II B) and then the identification number, following Klein et al. [86]. Support on branches is indicated by posterior probabilities (PP = 0–1).
Figure 7.
Model of the evolution of the saltwater crocodile MHC.
The MHC of crocodiles is compared with that of fowl, eutherians, and anoles (A) or opossums (B). Each coloured box indicates different genes consistent with the legend in Fig. 2. Broken lines indicate absence of linkage between genes; and dashed boxes indicate unknown linkage as a result of unmapped scaffolds in the saltwater crocodile and green anole. MHC gene mapping in fowl (chicken, quail, black grouse, golden pheasant, and turkey), eutherians (human, chimpanzee, gorilla, rat, mouse, dog, cat, cattle, sheep, pig, and horse), green anoles, and opossums is generated using data from Kelley et al. [5], Wang et al. [31], Ye et al. [13], Chaves et al. [12], Wilming et al. [61], Yuhki et al. [92], Gao et al. [14], Gonidez et al. (manuscript in revision), and Belov et al. [15].
Table 2.
Four pairs of forward and reverse overgos used for BAC library screening of MHC-associated BACs.