Genome Survey Analysis And Genetic Marker Identification Of The White-spotted Bamboo Shark Chiloscyllium Plagiosum

Chondrichthyes are one of the oldest extant jawed vertebrates and play an important role in biodiversity conservation and ecosystem stability.The white-spotted bamboo shark（Chiloscyllium plagiosum）,commonly known as lip shark,belongs to the order Orectolobiformes,family Hemiscylliidae.The species is a small benthic shark widely distributed in the Indo-West Pacific Ocean,in China,mainly in the South China Sea,East China Sea,with important economic and ecological value.The white-spotted bamboo shark has become a good material for adaptive evolutionary studies of cartilaginous fishes owing to its wide distribution and strong adaptability.However,due to its biological characteristics,combined with the comprehensive impact of habitat degradation,overfishing and other factors,the resources of this species declined in recent years,and is now listed as "Near Threatened" by the IUCN Red List.Similar to most cartilaginous fishes,the reported molecular genetic information of the white-spotted bamboo shark is relatively limited,which to a certain extent limits the conservation genetics studies.The study of genomics and genetic markers can provide valuable information for the rational resource management and conservation of the white-spotted bamboo shark.In this study,the genome survey analysis of the white-spotted bamboo shark in captivity was carried out based on high-throughput sequencing technology to obtain basic genomic information（including genome size,heterozygous and repeats ratio）of this species.Furthermore,based on whole-genome sequencing data,molecular markers（simple sequence repeat,SSR and single nucleotide polymorphism,SNP）within the genome of the white-spotted bamboo shark were identified,microsatellite distribution patterns were statistically analyzed,SNPs were annotated.And the identified SNPs were used to analyze the demographic history of the white-spotted bamboo shark and assess the effective population size of this species.These genomic data and identified molecular markers in this study may provide valuable fundamental information for in-depth studies on the population genomics and evolutionary biology researches of the white-spotted bamboo shark.1.Genomic characteristics of C.plagiosumThe white-spotted bamboo shark has prominent economic value in food,medicinal and ornamental aspects,and also has an important ecological value as an essential part of the marine ecosystem.To better understand the genomic characteristics of the white-spotted bamboo shark,high-throughput sequencing technology was used to implement the genome survey sequencing.The sample was collected from Haichang Ocean Park,Yantai,China,and one female individual was used for the genome survey analysis.A total of 282.94 Gb raw data and 281.17 Gb clean data were obtained from sequencing.The clean data were used for k-mer analysis to obtain the revised genome size of approximately 4.99 Gb,with heterozygous and repeats ratio of 0.51%and 74.67%,respectively.Comparative analysis showed that the genome size and repeats ratio of the white-spotted bamboo shark sample in this study were higher than the reported genome information（genome size:3.85 Gb,repeats ratio:63.53%）.Given that the reported shark sample was collected from the coast of Fujian,China,and the sample in this study was from an aquarium.Based on this,we hypothesized that the differences in the habitat of the samples might be the reason for the differences in the intraspecific genome size and repeats ratio of the white-spotted bamboo shark.The results of this study provide supplementary data and references for the genomic study of the white-spotted bamboo shark.2.Genome-wide microsatellite identificationIn the present study,the genome survey sequencing data were used to identify genome-wide microsatellites in the white-spotted bamboo shark.A total of 736,562,736,565 and 480,452 genome-wide microsatellites were identified based on preliminary,update（ragtag assembly）,and the reported chromosome-scale assemblies,respectively.These implied that the proportion of simple sequence repeats in the genomic sequences assembled in this study is higher than that reported.From the microsatellite types,the proportion of dinucleotide motif was the highest,followed by mono-and tri-nucleotide.The difference is that the number of tetranucleotide was higher than that of penta-and hexa-nucleotide in the preliminary and updated assembly identification results,while the proportion of pentanucleotide was greater than that of tetra-and hexa-nucleotide in the chromosomal-scale assembly.From the microsatellite motifs,AC/GT,AG/CT,A/T,C/G and AT/AT had high amounts in all three assembly results.From the number of microsatellite repeats,the number of microsatellites was mainly concentrated in 5-19.The genome-wide identification of hundreds of thousands of microsatellites could provide technical support for subsequent population genetics studies of the white-spotted bamboo shark.3.Genome-wide SNP identification and demographic analysisUsing the reported chromosome level genomic information（NCBI accession no.GCF₀04010195.1）as the reference sequence for SNP calling,8,703,211 high-confidence SNPs were finally identified in the whole genome of the white-spotted bamboo shark.The average density was about one SNP per 404 bp,and the density of NC₀57753.1 and NC₀57755.1 was the highest among the 51 chromosomes.The SNPs identified were annotated and most of the mutation types belonged to transition,accounting for 63.74%.Besides,a total of 13,758,432 SNPs were mapped to transcripts,133,720 SNPs were located in exonic regions,771 SNPs were categorized as high-impact mutations,and although relatively lower in number,may play a key role in the genetic and phenotypic evolution of the white-spotted bamboo shark.The effective population size of the white-spotted bamboo shark was also predicted based on these identified SNPs,and the results showed that the species experienced three times of population expansions and two times of population reductions.The effective population of the white-spotted bamboo shark has remained relatively large and constant since the last population expansion（about 300,000 years ago）.Overall,the identification of SNPs and demographic analysis in the study may promote the study of the white-spotted bamboo shark in population genomics,providing valuable molecular data for effective management and conservation of this species.4.Development of marine fish genome survey studiesGenome survey analysis can be used to sequence small fragments at low depth when the genetic characteristics of species are unknown,reducing the time and economic cost associated with blindly undertaking large-scale whole genome deep sequencing.In this section we collected the reported researches on marine fish genome survey to summarize the information on the research subjects and contents.Up to December 1,2022,there are 26 published reports of marine fish genome survey,involving a total of 33 species,mainly focusing on teleost in Perciformes and Scorpaeniformes.Apart from the present study,no related studies with genomic survey analysis of cartilaginous fish have been reported.The contents of genome survey analysis are rich,mainly involving genomic characterization assessment,microsatellites identification,preliminary genome assembly,gene annotation,SNP analysis,mitochondrial genome assembly,phylogenetic analysis,etc.With the accumulation of relevant research reports and genomic data information,there is a trend of more and deeper genomic survey analysis that can be carried out.Considering the large number of marine fish species and the difficulties in sample collection,the genomic studies that have been developed are relatively limited.Therefore,extensive genomic survey of marine fishes will help to further promote the genomics and phylogenetic studies of marine fishes.The comprehensive statistics and assessment of the current study status of marine fish genome survey analysis will help provide basic information for subsequent studies,as well as support the extension of genomic research content in marine fish by using sequencing data more deeply.