Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers in swamp eel (Monopterus albus).

OBJECTIVES: Swamp eel is one model species for sexual reversion and an aquaculture fish in China. One local strain with deep yellow and big spots of Monopterus albus has been selected for consecutive selective breeding. The objectives of this study were characterizing the Simple Sequence Repeats (SSRs) of M. albus in the assembled genome obtained recently, and developing polymorphic SSRs for future breeding programs. METHODS: The genome wide SSRs were mined by using MISA software, and their types and genomic distribution patterns were investigated. Based on the available flanking sequences, primer pairs were batched developed, and Polymorphic SSRs were identified by using Polymorphic SSR Retrieval tool. The obtained polymorphic SSRs were validated by using e-PCR and capillary electrophoresis, then they were used to investigate genetic diversity of one breeding population. RESULTS: A total of 364,802 SSRs were identified in assembled M. albus genome. The total length, density and frequency of SSRs were 8,204,641 bp, 10,259 bp/Mb, and 456.16 loci/Mb, respectively. Mononucleotide repeats were predominant among SSRs (33.33%), and AC and AAT repeats were the most abundant di- and tri-nucleotide repeats motifs. A total of 287,189 primer pairs were designed, and a high-density physical map was constructed (359.11 markers per Mb). A total of 871 polymorphic SSRs were identified, and 38 SSRs of 101 randomly selected ones were validated by using e-PCR and capillary electrophoresis. Using these 38 polymorphic SSRs, 201 alleles were detected and genetic diversity level (Na, PIC, HO, and He) was evaluated. CONCLUSIONS: The genome-wide SSRs and newly developed SSR markers will provide a useful tool for genetic mapping, diversity analysis studies in swamp eel in the future. The high level of genetic diversity (Na = 5.29, PIC = 0.5068, HO = 0.4665, He = 0.5525) but excess of homozygotes (FIS = 0.155) in one breeding population provide baseline information for future breeding program.

A high-quality de novo genome assembly of one swamp eel (Monopterus albus) strain with PacBio and Hi-C sequencing data.

The swamp eel (Monopterus albus) is one economically important fish in China and South-Eastern Asia and a good model species to study sex inversion. There are different genetic lineages and multiple local strains of swamp eel in China, and one local strain of M. albus with deep yellow and big spots has been selected for consecutive selective breeding due to superiority in growth rate and fecundity. A high-quality reference genome of the swamp eel would be a very useful resource for future selective breeding program. In the present study, we applied PacBio single-molecule sequencing technique (SMRT) and the high-throughput chromosome conformation capture (Hi-C) technologies to assemble the M. albus genome. A 799 Mb genome was obtained with the contig N50 length of 2.4 Mb and scaffold N50 length of 67.24 Mb, indicating 110-fold and ∼31.87-fold improvement compared to the earlier released assembly (∼22.24 Kb and 2.11 Mb, respectively). Aided with Hi-C data, a total of 750 contigs were reliably assembled into 12 chromosomes. Using 22,373 protein-coding genes annotated here, the phylogenetic relationships of the swamp eel with other teleosts showed that swamp eel separated from the common ancestor of Zig-zag eel ∼49.9 million years ago, and 769 gene families were found expanded, which are mainly enriched in the immune system, sensory system, and transport and catabolism. This highly accurate, chromosome-level reference genome of M. albus obtained in this work will be used for the development of genome-scale selective breeding.

Genetic and codon usage bias analyses of major capsid protein gene in Ranavirus.

The Ranavirus (one genus of Iridovidae family) is an emerging pathogen that infects fish, amphibian, and reptiles, and causes great economical loss and ecological threat to farmed and wild animals globally. The major capsid protein (MCP) has been used as genetic typing marker and as target to design vaccines. Herein, the codon usage pattern of 73 MCP genes of Ranavirus and Lymphocystivirus are studied by calculating effective number of codons (ENC), relative synonymous codon usage (RSCU), codon adaptation index (CAI), and relative codon deoptimization index (RCDI), and similarity index (SiD). The Ranavirus are confirmed to be classified into five groups by using phylogenetic analysis, and varied nucleotide compositions and hierarchical cluster analysis based on RSCU. The results revealed different codon usage patterns among Lymphocystivirus and five groups of Ranavirus. Ranavirus had six over-represented codons ended with G/C nucleotide, while Lymphocystivirus had six over-represented codons ended with A/T nucleotide. A comparative analysis of parameters that define virus and host relatedness in terms of codon usage were analyzed indicated that Amphibian-like ranaviruses (ALRVs) seem to possess lower ENC values and higher CAIs in contrast to other ranaviruses isolated from fishes, and two groups (FV3-like and CMTV-like group) of them had received higher selection pressure from their hosts as having higher relative codon deoptimization index (RCDI) and similarity index (SiD). The correspondence analysis (COA) and Spearman's rank correlation analyses revealed that nucleotide compositions, relative dinucleotide frequency, mutation pressure, and natural translational selection shape the codon usage pattern in MCP genes and the ENC-GC3S and neutrality plots indicated that the natural selection is the predominant factor. These results contribute to understanding the evolution of Ranavirus and their adaptions to their hosts.

Evolution and multiple origins of zona pellucida genes in vertebrates.

Animal egg coats are composed of different glycoproteins collectively named zona pellucida (ZP) proteins. The characterized vertebrate genes encoding ZP proteins have been classified into six subfamilies, and exhibit low similarity to the ZP genes characterized in certain invertebrates. The origin and evolution of the vertebrate ZP genes remain obscure. A search against 97 representative metazoan species revealed various numbers (ranging from three to 33) of different putative egg-coat ZP genes in all 47 vertebrates and several ZP genes in five invertebrate species, but no putative ZP gene was found in the other 45 species. Based on phylogenetic and synteny analyses, all vertebrate egg-coat ZP genes were classified into eight ZP gene subfamilies. Lineage- and species-specific gene duplications and gene losses occurred frequently and represented the main causes of the patchy distribution of the eight ZP gene subfamilies in vertebrates. Thorough phylogenetic analyses revealed that the vertebrate ZP genes could be traced to three independent origins but were not orthologues of the characterized invertebrate ZP genes. Our results suggested that vertebrate egg-coat ZP genes should be classified into eight subfamilies, and a putative evolutionary map is proposed. These findings would aid the functional and evolutionary analyses of these reproductive genes in vertebrates.