DNA Barcodes Construction Of Some Anguilliformes Fishes In The East China Sea And Analysis Of Genome Characteristics Of Ophichthus Evermanni

Anguilliformes belongs to Osteichthyes,Actinopterygii and Anguillomorpha in taxonomy.This fish group has a large number of species and a wide variety of morphological features.They are distributed in the Atlantic Ocean,Indian Ocean and Pacific Ocean including cold sea in the polar and near polar areas,and some species can even enter freshwater.In this study,morphological characteristics of 9 Anguilliformes species in the East China Sea were described and measured by traditional morphological methods.Besides,DNA barcoding method was also conducted to identify and compare these fish species.The average pairwise genetic distances between species,genera and families were calculated,and the phylogenetic relationships between them were also discussed.Based on the Illumina paired-end sequencing platform,the small fragment library was constructed at a low sequencing depth,which was used to obtain the genome survey information and microsatellite composition characteristics of Ophichthus evermanni,a representative species of Ophichthidae family in the coastal waters of the East China Sea.The complete mitochondrial genome sequence of this fish was assembled,and its structure and characteristics were analyzed.The results would lay foundation for the development of the whole genome De novo sequencing strategy of this species in the future,provide molecular evidences for the classification and phylogenetic studies of Anguilliformes in the East China Sea,and also provided and theoretical references for the protection and sustainable utilization of fishery resources in Anguilliformes fishes.The detailed research contents are as follows.1.Morphological characteristics and DNA barcoding analysis of some Anguilliformes fishes in the East China SeaIn this study,the following nine Anguilliformes species were collected from the East China Sea:Dysomma anguillaris,Ophichthus brevicaudatus,Ophichthus evermanni,Muraenesox cinereus,Rhynchoconger ectenurus,Neenchelys parvipectoralis,Moringua macrocephalus,Ariosoma meeki,and Conger myriaster.The mitochondrial cytochrome c oxidase subunit 1(COI)gene fragments were amplified by polymerase chain reaction and then sequenced.The homologous sequences of five Anguilliformes downloaded from the GenBank database were analyzed and compared.The maximum likelihood phylogenetic tree of 14 species,11 genera,and 6 families in Anguilliformes was constructed with Muraena argus and Muraena augusti as outgroups.The results indicated that a total of 43 haplotypes were detected in 72 sequences,and the contents of the four types of bases were 27.4%(T),28.2%(C),25.8%(A),and 18.6%(G),respectively.The average content of A+T(53.2%)was higher than that of G+C(46.8%),suggesting an evident bias of the base composition.Based on the Kimura 2-parameter(K2P)model,the average genetic distances between the species,genera,and families were 0.2188,0.2250,and 0.2327,respectively,indicating that the higher the taxonomic order,the greater the genetic distance.The phylogenetic tree indicated that the Ophichthyidae species can form independent clades and be effectively distinguished.However,the clustering of other genera and families produced a mixed phenomenon.The results suggest that,because of the species diversity and wide distribution of Anguilliformes fishes,the mitochondrial COI gene may be more suitable for species identification and classification in the lower taxonomic category(such as being coordinal or congeneric),and the phylogenetic relationships of Anguilliformes species warrant further exploration in combination with multiple DNA barcodes.2.Genome survey and genetic characteristics analysis of Ophichthus evermanniOphichthus evermanni Jordan&Richardson(1909)is a representative fish of Ophichthidae distributed in the East China Sea,the South China Sea and the coastal waters of southern Japan.In the present study,we conducted a genome survey sequencing(GSS)analysis of Ophichthus evermanni by Illumina sequencing platform to briefly reveal its genomic characteristics and phylogenetic relationship.The first De novo assembled 1.97 Gb draft genome of Ophichthus evermanni was predicted based on K-mer analysis without obvious nucleotide bias.The heterozygosity ratio was 0.70%,and the sequence repeat ratio was calculated to be 43.30%.A total of 9016 putative coding genes were successfully predicted,in which 3587 unigenes were identified by gene ontology(GO)analysis and 4375 unigenes were classified into cluster of orthologous groups for enkaryotic complete genomes(KOG)functional categories.About 2,812,813 microsatellite motifs including mono-,di-,tri-,tetra-,penta-and hexanucleotide motifs were identified,with an occurrence frequency of 23.32%.The most abundant type was dinucleotide repeat motifs,accounting for 49.19%of the total repeat types.The mitochondrial genome,as a byproduct of GSS,was assembled to investigate the evolutionary relationships between Ophichthus evermanni and its relatives.Bayesian inference(BI)phylogenetic tree inferring from concatenated 12 protein-coding genes(PCGs)showed complicated relationships among Ophichthidae species,indicating a polyphyletic origin of the family.3.The complete sequence structure and phylogenetic analysis of the mitochondrial genome of Ophichthus evermanniOphichthidae fishes are widely distributed in tropical and subtropical inshore waters and prefer to live on muddy substrates or coral reefs.Although it is one of the most divergent families in the Anguilliformes,the molecular genetic researches on these fishes are very limited.In this study,the complete mitochondrial genome of Ophichthus evermanni was obtained by high-throughput sequencing.The results of structure and characteristics analysis showed that the total length of the complete mitochondrial DNA was 17,759 bp,with the base composition of A(31.27%),G(16.19%),C(26.22%)and T(26.32%).The A+T content(57.59%)was greater than G+C content(42.41%),showing an obvious AT bias.Unlike other typical teleosts,the gene arrangement was identified in the mitogenome of Ophichthus evermanni.ND6 gene and the conjoint tRNA-Glu were translocated between tRNA-Thr and tRNA-Pro,and another highly homologous D-loop region was located in the upstream of the ND6 gene.Nine genes were located in the L-strand,while the rests were located in the H-strand.Except for tRNA-Ser(AGC),The other 21 tRNAs were typical clover secondary structures.The phylogenetic relationships of Ophichthidae fishes were constructed based on 12 protein-coding genes(excluding ND6)using the Neighbour-joining and Bayesian methods,respectively.The results inferred that Ophichthus evermanni was closely related to Ophichthus brevicaudatus and Pisodonophis cancrivorus,and the genetic divergent occurred in genus Ophichthus was relatively late among family Ophichthidae.