Screening Universal COⅠ Primers for eDNA Metabarcoding of Freshwater Fishes on Hainan Island

Abstract

The known universal primers for environmental DNA (eDNA) metabarcoding of fish are mainly located in the mitochondrial ribosome gene regions. Not only are the reference sequences insufficient but also some relative fish species could not be identified using these 12S and 16S primers. In this study, the freshwater fishes of Hainan Island were selected to meet six investigation targets. (1) Six conserved regions were selected through the cytochrome c oxidase subunit I (COⅠ) sequences of 8 orders, 26 families, 101 genera, and 150 species; (2) Based on the base variation, species identification, eDNA degradation, and read length requirements of high-throughput sequencing, 26 primers were designed from four flanking conserved regions; however, six primers did not meet the Premier score requirements. (3) The first-round PCR results of 72 freshwater fish species on Hainan Island showed that 11 primers had high universality. Among them, there were 5 primers with more than 70 species successfully amplified and brighter PCR bands than marker. The second-round of PCR results showed that the amplification success rate of 3×2 (forward and reverse, respectively) primer combinations generated by the five primers was 100%, and the optimal primer combinations after PCR band length and brightness screening were "HN-A-F4, HN-D-R3" (hereinafter referred to as HN-COⅠ). (4) High-throughput sequencing results of 30 water samples showed that the total number of clean reads, fish sequences, operational taxonomic units (OTUs), and fish OTUs generated by HN-COⅠ were 0.77, 1.22, 0.85, and 1.32 times those of MiFish-U, respectively. (5) The proportion of fish OTUs annotated to species, genus, and family was 81.40%, 11.63%, and 6.98% for HN-COⅠ and 81.54%, 4.62%, and 13.85% for MiFish-U, respectively. The non-metric multidimensional scaling (NMDS) clustering pattern of "primer + water sample" formed two distinct groups with an obvious boundary (stress=0.15). The minimum and average values of pairwise genetic distances of species amplicons within genera of HN-COⅠ were 1.23 and 1.57 times those of MiFish-U, respectively. (6) The correlation coefficient of the linear regression equation of common carp biomass and sequence copies of the six indoor density groups was low. HN-COⅠ and MiFish-U could not accurately reflect the biomass of common carp. In this study, the advantages and disadvantages of HN-COⅠ were screened and compared with those of MiFish-U. The results showed that HN-COⅠ primers have a high targeting ability for freshwater fishes on Hainan Island. HN-COⅠ are advantageous in preventing eDNA contamination from non-target organisms, such as microorganisms and mammals, and is also conducive to the detection and accurate identification of freshwater fishes.

Keywords

• hainan island
• freshwater fish
• environmental dna
• coⅰ
• universal primers