supplementary files for manuscript “Intraspecific variation of four serpin gene families of the Asian buffalo leech (Hirudinaria manillensis)”

Objective: To observe the characterization of intraspecific variation in serine protease inhibitors (serpin) and their encoding genes. Methods: We performed genome resequencing and transcriptome sequencing on a total of 70 samples from seven populations of the Asian buffalo leech (Hirudinaria manillensis) in Guangdong and Guangxi, China. We identified six genes of four serpin gene families (antistasin, therostasin, bdellin, and LDTI) and systematically analyzed the intraspecific sequence variation, selection pressure, and gene expression characteristics of these genes. Results: All genes were rich in genetic variation, with a total of 130 and 93 variable sites detected from coding region sequences and protein sequences, respectively. One pseudogene sequence was present in antistasin1 and LDTI, respectively. The genes were driven by purifying selection pressure overall, with highly significant differences in selection pressure indices among the six genes. A large number of potential positive selection sites were present in all genes, with at least one significant positive selection site present in each gene. The overall expression levels varied significantly among the genes, with bdellin2 having the highest mean value, followed by bdellin1, and therostasin having the lowest. Except for antistasin1, the remaining five genes showed significant overall differences among the seven populations. Conclusion: The genetic characteristics (sequence variation, selection pressure, and expression levels) of the intraspecific level of Asian buffalo leech serpin genes differ significantly both among genes and individuals, providing a rich genetic resource for studying the identification and development of antithrombotic agents in Asian buffalo leech.