Let's take an ancestral karyotype as an example to explain in detail how to construct a fusion positions database and test for shared positions across different species.
We begin by using the WGDI toolkit (Sun et al., 2022) with the parameter -d to generate a homologous gene dotplot between Cardamine hirsuta and the ancestral karyotype (ACBK). By employing the WGDI parameter -km, we mapped the protochromosomes of the ancestral karyotype onto the query genome based on collinearity, resulting in the ancestor file, Chi2.ancestor.txt.
From the dotplot, we observe a reciprocal chromosome translocation involving chromosomes ACBK7 and ACBK8 (denoted ACBK/7_8/RCT). Reading the file Chi2.ancestor.txt reveals the fusion breakpoints at Chr3:1948 and Chr8:653.
This information is then recorded in a document, as detailed in the Fusion Positions File section of the WGDI documentation. An example of such a file is Chi2.txt. By executing the WGDI command with the parameter -fpd (e.g., wgdi -fpd Chi2.conf), the corresponding fusion positions database is generated. This process generates the outputs, including the essential files .gff, .lens, .pep, and the ancestor file (e.g., fusion_breakpoints.ancestor.txt). For more details and additional files,such as Chi2.conf, please refer to the Shared_fusion_positions/ directory.
For cases with multiple fusion breakpoints from different species, ensure that the output file remains consistent and that the last column of each input record (e.g., Chi2.txt) has unique markers. Repeatedly executing wgdi -fpd *.conf will append additional fusion positions to the same output.
To test whether the identified fusion positions are shared in other species, compare their genomes against the generated fusion positions database using the WGDI parameters -d, -icl, -bi, and -fd.
Note that the fusion positions database may involve regions that are extracted multiple times. Therefore, collinearity extraction should be performed through interchromosomal comparisons. When running the -icl parameter, set comparison = chromosomes and adjust repeat_number to a smaller value, such as repeat_number = 5.
The criterion for determining shared fusion positions is that a synteny block spans the fusion breakpoint. Specifically, both sides of the breakpoint must contain collinear genes, controlled by the parameter min_genes_per_side.
The final output from -fd will list shared fusion breakpoints. For example, we used Capsella rubella for testing, and the output from the -fd parameter was as follows:
The following are the shared fusion breakpoints: AK1_1, AK1_2
This output indicates that Capsella rubella possesses these two shared fusion breakpoints: AK1_1 and AK1_2. If no such output is generated, it implies that these fusion breakpoints are not present in the tested species.
The following are the shared fusion breakpoints: AK17
This output indicates that Thlaspi arvense possesses these two shared fusion breakpoints: AK17.