Population Structure Of Sthenoteuthis Oualaniensis In The East Indian Ocean And South China Sea Based On Molecular Markers

Purpleback flying squid,Sthenoteuthis oualaniensis,widely distributed in tropical and subtropical waters such as the equator in the Indian and Pacific oceans.The species has a fast growth rate,strong reproductive capacity,short life cycle,and occupies an important position in marine ecosystems,not only as a predator of zooplankton,lanternfish and many other organisms,but also as a food source for many marine predators.At the same time,the abundance of Iris Sthenoteuthis oualaniensis resources is relatively high,the resources in the northwest Indian Ocean and the South China Sea are large,the potential commercial value is high,and it is gradually becoming one of the important fishery species.A comprehensive understanding of the genetic structure and genetic diversity of the population of the iris Sthenoteuthis oualaniensis population is of great significance for the evaluation and management of iris Sthenoteuthis oualaniensis fishery resources and the sustainable development and utilization of resources.（1）The DNA quality was assessed by comparing the effects of traditional phenol-chloroform,kit method,and traditional phenol-chloroform method in the genomic DNA extraction of iris Sthenoteuthis oualaniensis muscle tissue,and using characteristic absorbance values OD_260/280,DNA concentration,agarose gel electrophoresis,and PCR amplification of COΙgenes.The results showed that the genomic DNA concentration of iris Sthenoteuthis oualaniensis extracted by the improved phenol-chloroform method was 565.4-625.1 ng/μL,which was significantly higher than that of the other two methods.A₂₆₀/A₂₈₀ values are 1.75-1.81,and the agarose gel electrophoresis bands are clear and there is no drag-and-drop phenomenon.The target band of COΙgene PCR amplification is around 1000 bp,clear and bright,and the quality is stable.At the same time,improved phenol-chloroform achieved the same effect in genomic DNA extraction of Ommastrephidae（Ommastrephes bartrami,Sthenoteuthis pteropus,Dosidicus gigas）.In summary,the improved phenol-chloroform method is suitable for the extraction of cephalopod genomic DNA,and is convenient and fast,low-cost,and can meet the relevant molecular biology studies of PCR or genome-wide analysis.（2）Cytb gene marker design primers were used for PCR amplification and sequencing,and a total of 120 iris Sthenoteuthis oualaniensis samples were genetically analyzed in four populations of the eastern Indian Ocean in the northern equator,southern and northern and southern South China Seas.The results showed that 84 haplotypes were defined by 120 individuals,and the nucleotide diversity index（P_i）and haplotype diversity index（H_d）were 0.27332 and 0.981,respectively,of which the haplotype diversity of the population in the northern part of the South China Sea was 0.989,and the nucleotide diversity of the group in the southern part of the South China Sea was0.30660.Haplotype mesh and phylogenetic trees show significant genetic differentiation between populations in the Eastern Indian Ocean and the South China Sea.The results of molecular variance analysis showed that 54.90%of the genetic variation came from between groups.The F_st（F-Statistics）analysis between populations further showed that the F_st values of the northern and southern populations in the eastern Indian Ocean equator were 0.08588,and the F_st values of the northern and southern populations in the South China Sea were 0.02536,revealing that there was no significant genetic differentiation between the two populations in the South China Sea,and there was a moderate degree of genetic differentiation between the two populations in the eastern Indian Ocean.Among them,the frequent current activity on both sides of the equator in the eastern Indian Ocean may be an important factor leading to the genetic differentiation of the north-south population of the equator,while the geographical isolation between the eastern Indian Ocean and the South China Sea leads to significant genetic differentiation between the two geographical groups.The neutrality test results of the population in the northern Eastern Indian Ocean showed a very significant negative value（F_s=-26.56809,P<0.01）,and the nucleotide unpaired distribution map showed a significant undirected peak,which speculated that the population may have experienced a population expansion event historically.（3）PCR amplification and sequencing of primers using COΙgene marker design to supplement and validate Cytb gene marker sequencing results.The results showed that56 haplotypes were defined in 120 iris Sthenoteuthis oualaniensis individuals,and the haplotype diversity index（H_d）and nucleotide diversity index（P_i）were 0.932 and0.00945,respectively,showing a high haplotype diversity index.The variation index derived from the inter-population was 61.44%,and the F_st analysis between the two groups showed that the F_st value of the northern and southern equatorial groups in the eastern Indian Ocean was 0.06408,and the F_st value of the northern and southern populations in the South China Sea was 0.02081,revealing that there was a small genetic differentiation between the north and south groups of the equator in the eastern Indian Ocean,and there was no genetic differentiation in the South China Sea population.The results shown by the COΙgene marker are consistent with the results of the Cytb gene marker,which proves that there is frequent gene exchange between populations in the South China Sea and the complex population structure,and the equatorial rapids near the equator in the eastern Indian Ocean may be the cause of genetic differentiation between the north and south populations.Therefore,the equatorial and South China Sea groups in the Eastern Indian Ocean are two independent groups,and should also be regarded as two independent management units in fisheries management.