Complete cox 1 gene and ITS sequence of Hirudinaria manillensis and molecular evolution analysis

Hirudinaria manillensis is an important blood-sucking medicinal leech widely distributed in Southeast Asia and southern China. It has been shown to treat cardiovascular and cerebrovascular diseases. Molecular identification technology has developed rapidly, and the results are accurate and stable, which can help us to obtain rich biological information of samples for genetic evolution analysis. This study designed 3 pairs of primers for complete cox 1 gene. After sequencing by PCR products and splicing by SeqMan software, the full-length sequence of cox 1 gene was obtained by annotation of open reading frame (ORFs) on MITOS2 platform. The cox 1 gene with a length of 1536nt was obtained, and the species was identified as Hirudinaria manillensis. The Hd value and FST value of cox 1 gene were 0.858 and 0.10938, which were higher than the Hd value of ITS (0.314) and the FST value close to 0. The nucleotide diversity and GST values of the two genes were very low, and the Tajima's D values of the neutral test were negative, without statistical significance. The genetic distance calculated by the Kimura-2 parameter method was very low. According to the biological information provided by the samples in this study, the population size of Hirudinaria manillensis in southern China was on the rise, but stable genetic diversity was not formed, and there was no obvious population differentiation and geographical isolation. This may be due to the mature artificial breeding technology in recent years, which enabled Hirudinaria manillensis to migrate from the traditional gathering places and breed in a large number in the new environment and migration site. At present, there are many molecular barcoding data of cox 1 gene in the NCBI database, but there is no complete cox 1 gene sequence as a reference, and the related ITS sequence information is more limited, indicating that there is still a lot of space for gene research of Hirudinaria manillensis.