Orange-spotted grouper (Epinephelus coioides) is one of the most important marine food species in Southeast Asia and China and has been cultured for decades. In this study, we fully utilized the limited capacity of semiconductor sequencing, the high efficiency of long-range PCR for target enrichment and a non-indexed pooling strategy to screen single-nucleotide polymorphisms (SNPs) in a breeding population of orange-spotted grouper. Forty-one genomic DNA fragments, with a total length of approximately 180 kb, including 22 candidate genes that control growth, and from a DNA pool of 20 heaviest and 22 lightest individuals of the sampled population were successfully sequenced using an Ion Torrent Personal Genome Machine. 3 503 466 clear reads were produced with a length of 192 ± 56 bp, 86.8% of which were mapped to the reference with an average coverage depth of 2567-fold and physical coverage of 98.8%. Finally, 1623 high-quality SNPs were adopted. Compared with Sanger sequencing of three random common regions, the sensitivity and specificity of our approach were 39.4% and 100.0% respectively. A mutation located at the third position of the previously labelled start codon of growth hormone receptor type 1 invalidated the start codon. Furthermore, comparison of the frequencies of genotypes and alleles of this site between the two extreme groups, prediction of signal peptide and identification of conservative mRNA sequences suggested that the functional start codon is likely located at the position of another downstream in-frame ATG in the mutant. These detected SNP markers will provide important tools for the selective breeding of orange-spotted grouper.

The present study represents the first report on the complete mitochondrial genome (mitogenome) of the kawakawa tuna Euthynnus affinis. Illumina next-generation sequencing generated a total of 38,428 reads with an average sequencing depth of 232.83x. The mitogenome of E. affinis was 16,513 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a 849 bp control region, as found in other typical teleosts. With the exception of ND6 and eight tRNA genes, all other mitochondrial genes were encoded on the heavy strand, whose nucleotide composition is 28.11% A, 24.77% T, 17.16% G and 29.96% C. All the protein-coding genes shared the start codon ATG except for COXI gene (start with GTG), and appeared to be terminated with four types of stop codons including two complete codons (TAA and TAG) and two incomplete codons (T and TA).

Recently, the next-generation sequencing (NGS) technology has become a powerful tool for sequencing the teleost mitochondrial genome (mitogenome). Here, we used this technology to determine the mitogenome of the yellowfin tuna (Thunnus albacares). A total of 41,378 reads were generated by Illumina platform with an average depth of 250x. The mitogenome (16,528 bp in length) contained 37 mitochondrial genes with the similar gene order to other typical teleosts. These mitochondrial genes were encoded on the heavy strand except for ND6 and eight tRNA genes. The result of phylogenetic analysis supported two distinct clades dividing the genus Thunnus, but the tuna species of these two genetic clades were different from that of two recognized subgenus based on anatomical characters and geographical distribution. Our results might help to understand the structure, function, and evolutionary history of the yellowfin tuna mitogenome and also provide valuable new insights for phylogenetic affinity of tuna species.

Using Ion Torrent next-generation sequencing (NGS) technology, we sequenced the complete mitochondrial genome (mitogenome) of black and reddish morphs of the coral trout Plectropomus leopardus. High-throughput sequencing generated a total of 958,614 sequence reads covering 164.80 Mb of two mitogenomes with a coverage of 4800X. Thirty-seven mitochondrial genes and gene order of P. leopardus was quite similar to that of other teleostean fishes. Most genes were either abutted or overlapped, and all the protein-coding genes began with an ATG start codon except for COX1 and ATP6. The number of stop codon was different for the black and reddish P. leopardus. Comparisons between the mitochondrial sequences of the two morphs revealed a total of 74 variable sites and one indel. Nucleotide diversity across protein-coding gene varied from 0.0006 (16s rRNA) to 0.0070 (Cytochrome b). As expected, the highest level of nucleotide diversity (0.0291) was detected in the control region. Our results demonstrate the NGS technology based on Ion torrent platform can be used to assemble the mitogenome of fish species.

The aim of this study was to investigate single nucleotide polymorphisms (SNPs) in genes that are presumed to control muscle growth and to determine their potential association with growth in a cultured population of orange-spotted grouper, Epinephelus coioides. Seven genes, myogenic regulatory factor 4 (MRF4), Myf5, MSTN-1, MSTN-2, MyoD1, MyoD2, and myogenin, were selected for the investigation, covering approximately 26kbp. First, the two clades for the genes MSTN (myostatin) and MyoD were confirmed in this species using Bayesian inference analysis of the phylogenetic relationships. Then, the seven genes were enriched by polymerase chain reaction and sequenced using an Ion Torrent Personal Genome Machine. A total of 586 SNPs were discovered. Linkage disequilibrium was decayed by 50% within 250bp based on the combined data, which means that there was high resolution in the association mapping. A mixed linear model considering the population structure and kinship was used to detect the associations between genotypes and phenotypes. Only one site (KR269814.1:g.22T>G) in MSTN-1 was found to be significantly associated with a measured trait, the interorbital distance (false discovery ratio<0.05), and it explained 12.4% of the phenotype variation of this trait. This study provides insight on strategies for molecular marker-assisted breeding in orange-spotted grouper.

Growth hormone-releasing hormone (GHRH) and the receptor, GHRHR, constitute important components of the hypothalamus-pituitary growth axis and act on the downstream growth hormone (GH). PACAP-related peptide/pituitary adenylate cyclase activating polypeptide (PRP-PACAP) is a paralog of GHRH. These genes all play key roles in development and growth patterns. To improve the quality of cultured fish strains, natural genetic variation must be examined and understood. A mixed linear model has been widely used in association mapping, taking the population structures and pairwise kinship patterns into consideration. In this study, a mass cross population of orange-spotted grouper (Epinephelus coioides) was examined. These candidate genes were found to harbor low nucleotide diversity (θw from 0.00154 to 0.00388) and linkage disequilibrium levels (delay of 50% within 2 kbp). Association mapping was employed, and two single-nucleotide polymorphisms (KR269823.1:g.475A>C and KR269823.1:g.2143T>C) were found to be associated with growth (false discovery rate Q < 0.05), explaining 9.0%–17.0% of the phenotypic variance. The association of KR269823.1:g.2143T>C was also found via haplotype-based association (p < 0.05). The identified associations offer new insights into gene functions, and the associated single-nucleotide polymorphisms (SNPs) may be used for breeding purposes.

Previously morphological studies supported the division of the bullet tuna into the two subspecies, Auxis rochei rochei and A. rochei eudorax. As a cosmopolitan species, A. rochei rochei ranges in the Indo-West Pacific and Atlantic oceans, while A. rochei eudorax inhabits in eastern Pacific region. Here, we used the HiSeq next-generation sequencing technique to determine the mitochondrial genome (mitogenome) of A. rochei from Indo-West Pacific collection, and then compared our data with mitogenomic sequences of the Atlantic and eastern Pacific retrieved from NCBI database. Results showed the mitogenome of A. rochei from three geographic collections shared the same genes and gene order, similar to typical teleosts. Also, we examined a low level of nucleotide diversity among these mitogenomic sequences. Interestingly, nucleotide diversity of intra-subspecies (Atlantic versus Indo-West) was higher than that of inter-subspecies (Atlantic versus eastern Pacific, Indo-West versus eastern Pacific).

We firstly reported the complete mitochondrial genome (mitogenome) of the polychaete Tylorrhynchus heterochaetus. Illumina next-generation sequencing generated a total of 19,796 reads with an average depth of 122.91 x. The mitogenome is 16,106 bp in length including the typical structure of 13 protein coding genes, two rRNA genes, 22 tRNA genes and a putative control region. However, T. heterochaetus differs from other Nereididae species in the positions of four genes (tRNA-Met, tRNA-Asp, ATP8, tRNA-Tyr). All 37 genes are encoded on the heavy strand whose nucleotide composition is 32.20 % A, 13.67% C, 21.33% G and 32.80% T.

The study determined the complete mitochondrial genome (mitogenome) of the polychaete, Goniada japonica, which was first reported in the family of Goniadidae. A total of 7162 reads were generated by Illumina HiSeq2500 platform (Illumina Inc., San Diego, CA) with an average depth of 58.41x. The mitogenome of G. japonica was 15,327 bp in size and consists of 37 typical genes (13 protein-coding genes, 2 rRNA genes and 22 tRNA genes) and a putative control region. All the 37 genes were encoded on the heavy strand whose nucleotide compositions were 35.08% of A, 33.69% of T, 11.66% of G, and 19.57% of C, showing a lower content of G + C (31.23%). The gene order of 15 major coding genes was identical to that of the Nereididae species. Phylogenetic analysis showed that G. japonica has a closer relationship with Tylorrhynchus heterochaetus of Nereididae.

The mitochondrial genome of skipjack tuna, Katsuwonus pelamis, was obtained by using HiSeq next-generation sequencing technique. The circular mitogenome is 16,515 bp in length, with the gene number and arrangement being similar to those observed in other teleost fish. Overall base composition is A 28.49%, T 24.58%, C 30.27% and G 16.66%, respectively. Among 13 protein-coding genes, seven of which share the complete stop codon TAA while others terminate with TA or T, all these genes use ATG as start codon except for COI.

The groupers are commercially important marine species over the world , and they are also the important mariculture species of the coastal provinces in south China .The great variety of the species and the lack of morphological characteristics led to much confusion and dispution in their phylogenetic rela-tionships and species identification .In the present study , we try to reveal the phylogenetic relationships of groupers on molecular level .The partial mitochondrial cytochrome b ( Cyt b ) gene and two ribosomal genes (16S and 12S) were combined and applied to species identification and relationship analysis of 13 Epinepheline species from 3 genera distributed in coastal waters of south China .The results showed that:①The mitochondrial genes are appropriate as molecular markers for Epinepheline species identification because considerably high sequence difference among species and very low within species were found in these 3 genera;② Epinephelus was more closely related to Cephalopholis than to Plectropomus; ③ The genetic relationship was very close between E.akaara and E.awoara, E.merra and E.longispinis, E.coioides and E.corallicola.