Supplementary Material for: High Amplification of CLA-SAT-149 Satellite DNA in Bighead Catfish: Insights into Satellite DNA Evolution in Clariid Lineages

Introduction: Satellite DNA (satDNA) is an important component of eukaryotic genomes that influences chromosomal organization and evolution. This study investigates the sequence diversity, chromosomal distribution, and copy number variation of the CLA-SAT-149 satDNA family (149 bp) in four clariid catfish species, Clarias macrocephalus, C. gariepinus, C. batrachus, and C. nieuhofii, as well as in F1 hybrids and backcross (BC) progeny.
Methods: Fluorescence in situ hybridization (FISH) was used to map CLA-SAT-149 on metaphase chromosomes. Quantitative PCR (qPCR) quantified copy number variation. Amplicon sequencing was applied to characterize sequence variants, and Bayesian clustering analysis was used to assess genetic relationships among subfamilies.
Results: Chromosome mapping showed species-specific signals present in C. macrocephalus and F1 hybrids but absent in C. gariepinus and C. nieuhofii. qPCR revealed significantly higher copy numbers in C. macrocephalus. Two sequence variants were identified: the canonical 149-bp (subfamily A) and an 85-bp derivative (subfamily B). Bayesian analysis indicated multiple genetically distinct subgroups among species. F1 hybrids and BC progeny exhibited unique sequence profiles and copy number patterns, while C. nieuhofii lacked CLA-SAT-149 entirely.
Conclusion: The results support lineage-specific expansion, amplification, and loss of CLA-SAT-149 in clariid catfish, consistent with the satDNA library model. These patterns provide insights into satDNA evolution and have potential applications in aquaculture genetics, hybrid identification, and species differentiation.