Development of the cephalopod-specific universal primer set and its application for the metabarcoding analysis of planktonic cephalopods in Korean waters

PeerJ

article

Eun-Bi Kim¹  Soo Rin Lee¹  Chung Il Lee²  Hyun Park³  Hyun-Woo Kim¹ 

[1] Interdisciplinary Program of Biomedical, Mechanical, and Electrical Engineering, Pukyong National University;Department of Marine Bioscience, Gangneung-Wonju National University;Unit of Polar Genomics, Korea Polar Research Institute, Incheon;Department of Marine Biology, Pukyong National University

关键词: Cephalopods;    Metabarcoding;    Next-generation sequencing;    Paralarvae;    Korean waters;   

DOI  :  10.7717/peerj.7140

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Although spatiotemporal analysis of the cephalopod larvae provides the useful information for the effective management of their resources, it has been difficult mainly due to their low numbers in the mixed zooplankton net samples and difficulty in morphological identification. In order to analyze the planktonic cephalopods using next-generation sequencing (NGS), we have designed a cephalopod-specific universal (CPD) primer set targeting a region covering mitochondrial cytochrome b and ND6 genes based on the currently identified 36 complete cephalopod mitochondrial genome sequences in the GenBank database. The expected amplicon sizes by CPD primers were between 465 and 471 bp, which was applicable to the MiSeq system (Illumina, San Diego, CA, USA). NGS results of pooled DNAs from 8 months (including 739 zooplankton net samples) collected from Korean waters in 2016 showed the exclusive cephalopod sequences with little contaminant sequences supporting the specificity of CPD primer set. Total 47 representative cephalopod haplotypes (seven families and 10 genera) were obtained from 1,439,414 merged reads. Among the total analyzed haplotypes, Watasenia scintillans, Todarodes pacificus, and Sepiola birostrata were the most abundant species in Korean waters. Two “unidentified” clades in order Oegopsida were identified, which was showed less than 90% sequence identity but closely related to Enoploteuthidae and Idiosepiidae, respectively. Monthly changes in proportions of each haplotype were also identified, which may reflect its reproduction and spawning period. The larvae of W. scintillans was dominant from February to June, while high proportions of other cephalopod taxa were also identified from August to November. Only single haplotype was dominant in W. scintillans (Type 2) throughout the year, while two distinct haplotypes showed seasonal differences in T. pacificus.

【 授权许可】

CC BY