Development and validation of 107 SNP markers in Todarodes pacificus (Ommastrephidae)

Todarodes pacificus is a commercially important squid species; however, there is little information available regarding the population genetics of this species and effective molecular markers are still unavailable. In the present study, 107 novel single nucleotide polymorphism (SNP) markers were developed using Genotyping-by-Sequencing (GBS). The observed and expected heterozygosities ranged from 0.0345 to 0.5862 and 0.0339–0.4994, respectively. The polymorphism information content ranged from 0.0333 to 0.3747. Among these SNPs, all loci have been confirmed to fit the Hardy–Weinberg equilibrium (p > 0.05). The novel SNPs developed in this study will be helpful for the genetic conservation and scientific management of Todarodes pacificus.


Introduction
The Japanese common squid, Todarodes pacificus (T. pacificus), is a commercially important squid species, which is mainly distributed around the Northwest Pacific Ocean (Arkhipkin et al. 2015;Sakurai 2003). The abundance of this species has fluctuated widely, exhibiting a gradual increase in recent years (Yeong and Ho 2008). It is necessary to enrich the current knowledge of T. pacificus, especially in terms of population genetics and diversity. To date, the majority of research on this species has focused on fisheries hydrography, stock assessment and biology (Fang and Chen 2018), with only a few studies isolating polymorphic loci using microsatellites (An et al. 2014;Iwata et al. 2008). A comprehensive understanding of T. pacificus population genetics remains limited, and effective molecular markers are still unavailable.
With the rapid development of NGS technologies, large numbers of sequencing-based single nucleotide polymorphisms (SNPs) have been discovered and are widely used in population genetic studies, due to their broad genome coverage and automatic detection in high throughput sequencing (Van Tassell et al. 2008;Xiao et al. 2019). Until now, there has been a gap in the application of SNP molecular markers in T. pacificus population genetics. Thus, it is necessary to develop a set of meaningful and readily available SNPs and apply them to the population conservation and sustainable utilization of this species. In this study, we analyzed and characterized a number of SNP markers in T. pacificus using Genotyping-by-Sequencing (GBS).

* Huajie Lu
hjlu@shou.edu.cn * Congcong Wang ccwang@shou.edu.cn  TpSNP 1  F: ACG TTG GAT GCA CAA AGA CTG ATA TTC GCC  R: ACG TTG GAT GTC TGC ATA GCT GAA AGG GTG  EXT: caTGA AAG GGT GCA TTT TGA T   T/A  104  54  0.2278 0.1724 0.2622 0.0652   TpSNP 2  F:ACG TTG GAT GTC TGA CGC ATG TTT CAT CCG  R: ACG TTG GAT GTT GGC CCA TTC TCA TTA CCC  EXT: ccacCTT TCG CTT TCA AAG CTC TGA   A/C  99  49  0.2278 0.1724 0.2622 0.0652   TpSNP 3  F: ACG TTG GAT GTC TTA GAA TGG TGT AAT CCC  R: ACG TTG GAT GCC AGT CTC TAG TTC AGT TGG  EXT: CAG TTG GTA CAG ATG AGT   G/T  110 TpSNP 20 F: ACG TTG GAT GCG AAA ATA TTA GTC TGA ATA TG  R: ACG TTG GAT GTG TGG AGA AAT GAC AAG TpSNP 39 F: ACG TTG GAT GCA ACC GTA TGA CAA AAT AGC  R: ACG TTG GAT GGT CTG CTG CTT TTG TAT GCC  EXT: ctcCAC CTT TAT TGT TTC       1 3 and expected heterozygosity were calculated using Stacks pipeline populations program (Catchen et al. 2013). Polymorphism information content and Hardy-Weinberg equilibrium were estimated using PowerMarker software version V3.25 (Liu and Muse 2005). The Linkage Disequilibrium (LD) was tested using SHEsis (Shi and He 2005). Among the 110 random primer pairs, 107 SNP loci were found to be polymorphic and biallelic in 50 T. pacificus individuals (Supplementary Table 1). The summary statistics of genetic diversity are shown in Table 1. The observed heterozygosity and expected heterozygosity ranged from 0.0345 to 0.5862 and 0.0339-0.4994, respectively. The polymorphism information content ranged from 0.0333 to 0.3747. All loci have been confirmed to fit the Hardy-Weinberg equilibrium after Bonferroni correction (p > 0.05). No significant linkage disequilibrium was detected. To our knowledge, this is the first study to develop SNP markers in T. pacificus. These novel genetic markers will expand the knowledge of this species and provide a useful exploration tool for genetic conservation and population genetics, which is beneficial for the sustainable utilization of T. pacificus resources.
Author contributions XL and XH carried out most of the experiments; CW designed the methods and experiments, interpreted the results and finished the discussion. YL prepared the materials and partly worked on the experiments. HL was responsible for overall supervision, and participated in coordination. All authors read and approved the final manuscript.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval Experimental protocols involving live animals were approved by the Ethics Committee for the Use of Animal Subjects of Shanghai Ocean University.