3.5 Chromosomal breakage and reunion verified by synteny comparison
To verify the hypothesis of chromosome breakage and reunion, comparative chromosome analysis was performed using MUMmer4 to identify genome-scale syntenic regions between female and male O. bidens . The genome sequences corresponding to their chromosomes were highly consistent (Figure 6). Similarly, the chromosomal sequences of female GH14/GH38 exhibited a significant identity with the male LG06 using MUMmer4 with a minimum match length of 1,000 bp and 85% of filter (Figure 7a). The female GH14/GH38 chromosomal sequence alignment to the male LG06 showed identities of 97.93% and 97.72%, respectively, suggesting that the female GH14 and GH38 have fused to form the male LG06. Indeed, structural variations were identified by performing sequence comparisons, and we found that in the male LG06, the deletions included 232 in the intergenic region, 20 in the upstream region, 12 in the downstream area, and 259 in the intronic region. The insertion sites included 218 in the intergenic region, 26 in the upstream region, 23 in the downstream region, and 248 in the intronic area. Finally, the duplication, inversion, and translocation sites lacked a homolog to the female GH14/GH38 (Figure 7b). The deletions and insertions that occurred in the male LG06 were lower. They accounted for 3.4% (1038/30202) of the entire variable sites in all-maleO. bidens chromosomes, suggesting that the male LG06 resulted from the fusion of the female GH14 and GH38 rather than from massive rearrangements.
According to the annotated gene sets in the male O. bidensgenome, using homology searching of the female O. bidens genome, we identified seven male-specific genes in the male LG06 chromosome, including four uncharacterized protein genes; two genes involved in replication, recombination, and repair functions (EVM0022038, EVM0026331); and one gene annotated as DNA polymerase epsilon subunit 2 (EVM0017949), engaged in DNA-dependent DNA replication in biological processes.
To reveal the possible mechanism of chromosome number variation by breakage in Cyprinid fish, the chromosomes of male O. bidens were aligned to a single chromosome of C. idella , exhibiting significant inter-chromosomal conservation (Figure 8a). C. idellashowed a high homology and 269 synteny blocks with 21,628 gene pairs in the genome comparison. Each of the 14 C. idella chromosomes broke into two chromosomes of the male O. bidens , and massive inter-chromosomal rearrangements occurred after the divergence of the two species (Figure 8b). Notably, the fusion of the broken fragments from C. idella Ch08 and Ch17 chromosomes gave rise to the LG06 chromosome of male O. bidens , indicating that chromosome breakages help to increase their number in fish chromosomal evolution.