Cephalopods are widely distributed throughout the entire ocean and many of them are commercially important species. Although studies on the distribution of paralarvae provide the useful information for their effective resource management, their analysis has been difficult mainly due to their low numbers in the mixed zooplankton net samples and difficulty in the morphological identification. As one of the alternative methods, we here developed a cephalopod-specific universal (CPD) primer set targeting mitochondrial cytochrome b and ND6 genes after the multiple alignment of 36 cephalopod complete mitogenome sequences. The expected amplicon sizes were between 465 bp and 471 bp, which showed high degree of sequence variations enough to discriminate cephalopod species. Reliability of CPD primer set was tested with the individual and mixed zooplankton net samples. Finally, total 739 zooplankton net samples collected from Korean waters for 8 months in 2016 were analyzed by Illumina Miseq platform.
Results/Conclusions
CPD primer set successfully amplified the expected PCR products from both individual and zooplankton net samples supporting its quality. As the result of the MiSeq sequencing, total 1,246,517 paired-end contigs were obtained and 53 genotypes were identified at 98 % nucleotide sequence identity. Among them, 85 % of total contigs (29 genotypes) were matched with reference databases at 98 % sequence identity. Watasenia scintillans (56 %), Todarodes pacificus (16 %), and Sepiola birostrata (11 %) were among the most frequently identified species in Korean waters in 2016. 13 % of the total contigs (8 genotypes) exhibited lower than 90 % identity to the currently deposited GenBank database suggesting the additional CPD sequence supplementation. Assigned genotypes included 6 families and 10 genera. Due to its length, we were also able to estimate the genetic diversity of cephalopod species. For example, genotypes of T. pacificus in November was clearly distinct from the others indicating different population. Those data supported the idea that metabarcoding analysis by CPD universal primer set is a good alternative method for the planktonic cephalopod survey. The long-term study by the metabarcoding analysis with CPD primers would provide useful ecological information of the cephalopod species such as spawning, migration, and recruitment, which will be further used for its scientific management in Korean waters.