Comparative analysis of genome-wide microsatellite distribution characteristics in Sebastiscus species

To investigate the genome-wide distribution of microsatellites in three Sebastiscus species, we identified microsatellites with MISA and performed enrichment analysis of microsatellite-containing genes with clusterProfiler v4.20.0. The results showed that the total genome lengths of S. albofasciatus, S. marmoratus and S. tertius were 813 073 033, 810 035 071 and 798 226 595 bp, comprising 24, 40 and 24 sequences, of which 24, 36 and 24 contained 644 169, 597 324 and 625 694 microsatellite loci, respectively. The total lengths of microsatellite sequences were 13 112 395, 12 749 726 and 13 514 881 bp, accounting for 1.61%, 1.57% and 1.69% of the genomes, with approximately one locus per 1 262.20, 1 356.11 and 1 275.75 bp. S. albofasciatus harbored the largest number of microsatellites and the densest distribution, whereas S. marmoratus harbored the fewest. The proportions of the different microsatellite repeat types ranked as follows: dinucleotides (44.75%-47.68%) > mononucleotides (30.60%-31.93%) > trinucleotides (13.76%-15.40%) > tetranucleotides (4.10%-5.17%) > hexanucleotides (1.39%-2.09%) > pentanucleotides (1.11%-1.14%) in S. albofasciatus and S. marmoratus, whereas in S. tertius hexanucleotides (4.33%) preceded tetranucleotides (4.15%). The dominant repeat units were conserved among the three species: A/T in mono-nucleotides (91.40%-92.97%), AC/GT in di-nucleotides (50.38%-56.39%) and AAT/ATT in tri-nucleotides (29.20%-31.15%), whereas CG/CG accounted for less than 0.1%. Repeat numbers were mostly 5-18 and peaked at 6 (100 357-113 895 loci, 16.80%-17.68%), and microsatellite frequency declined with increasing repeat number and sequence length. The mean length of microsatellite sequences ranged from 20.36-21.60 bp and the total number of microsatellite sequences ranged from 347 645-353 102, of which mono-nucleotide sequences accounted for 41.65%-42.36%. Functionally, the enrichment of microsatellite-containing protein-coding genes differed among the three species: S. tertius and S. albofasciatus were mainly enriched in transcriptional regulation, with S. albofasciatus additionally associated with cytoskeleton organization and protein interactions, whereas S. marmoratus was more associated with cell adhesion, cytoskeleton, axon guidance and the cAMP signaling pathway. Overall, dinucleotide repeats predominated in all three species, reflecting conserved features, whereas microsatellite density and hexanucleotide proportion differed among species. This study comparatively analyzed the genome-wide microsatellite distribution characteristics of three Sebastiscus species, providing baseline data for microsatellite marker development, population genetics and genome evolution research in Sebastiscus fishes.