Molecular characterization of nineteen cytokine receptor family B (CRFB) members, CRFB1, CRFB2, CRFB4-17, with three CRFB9 and two CRFB14 in a cyprinid fish, the blunt snout bream Megalobrama amblycephala.

The class II cytokine receptor family members are receptors of class 2 helical cytokines in mammals, and are named cytokine receptor family B (CRFB) in fish. In zebrafish, sixteen members, including CRFB1, CRFB2 and CRFB4-17 were reported. With the availability of genome sequence, a total of nineteen CRFBs was identified in the blunt snout bream (Megalobrama amblycephala), including CRFB1, CRFB2, CRFB4-17 with the presence of three CRFB9 isoforms, and two CRFB14 isoforms. These CRFB molecules contain well conserved features, such as fibronectin type III (FNIII) domain, transmembrane and intracellular domains as other class II cytokine receptors, and are phylogenetically grouped into thirteen clades with their homologues from other species of fish. The CRFB genes were constitutively expressed in organs/tissues examined in the fish. The finding of more CRFB members in the bream may provide clues to understand possible receptor-ligand interaction and their diversity from an evolutionary point of view.

Molecular cloning and functional analysis of polymeric immunoglobulin receptor, pIgR, gene in mandarin fish Siniperca chuatsi.

Polymeric immunoglobulin receptor (pIgR) can bind and transport immunoglobulins (Igs), thus playing a role in mucosal immunity. In this study, pIgR gene was cloned in mandarin fish, Siniperca chuatsi, with the open reading frame (ORF) of 1011 bp, encoding 336 amino acids. The pIgR protein consists of a signal peptide, an extracellular domain, a transmembrane domain and an intracellular region, with the presence of two Ig-like domains (ILDs) in the extracellular domain, as reported in other species of fish. The pIgR gene was expressed in all organs/tissues of healthy mandarin fish, with higher level observed in liver and spleen. Following the immersion infection of Flavobacterium columnare, pIgR transcripts were detected in immune related, especially mucosal tissues, with significantly increased transcription during the first two days of infection. Through transfection of plasmids expressing pIgR, IgT and IgM, pIgR was found to be interacted with IgT and IgM as revealed by co-immunoprecipitation and immunofluorescence.

RAD genotyping reveals fine-scale population structure and provides evidence for adaptive divergence in a commercially important fish from the northwestern Pacific Ocean.

Exploring factors shaping genetic structure of marine fish is challenging due to fewer barriers to gene flow in the ocean. However, genome-wide sequence data can greatly enhance our ability to delineate previously unidentified population structure as well as potential adaptive divergence. The small yellow croaker (Larimichthys polyactis) is a commercially important fish species with high gene flow and its overwintering populations experience heterogeneous environment, suggesting possible population differentiation and adaptive divergence. To delineate patterns of population structure as well as test for signatures of local adaptation, a total of 68,666 quality filtered SNP markers were identified for 80 individuals from four overwintering populations by using restriction site-associated DNA sequencing (RAD-seq). Significant genetic differentiation among overwintering populations from the Central Yellow Sea, the South Yellow Sea and the North East China Sea were detected (Pair-wise F ST: 0.00036-0.00390), which were consistent with population division of overwintering groups inferred from traditional ecological approaches. In addition, a total of 126 unique SNPs were detected to be significantly associated with environmental parameters (temperature, salinity and turbidity). These candidate SNPs were involved in multiple pathways such as energy metabolism and phagocytosis, suggesting they may play key roles in growth and innate immunity. Our results suggested the existence of hitherto unrecognized cryptic population structure and local adaptation in this high gene flow marine fish and thus gain new insights into the design of management strategies.

Population Genomic Signatures of Genetic Structure and Environmental Selection in the Catadromous Roughskin Sculpin Trachidermus fasciatus.

Understanding the patterns of genetic diversity and adaptation across species' range is crucial to assess its long-term persistence and determine appropriate conservation measures. The impacts of human activities on the genetic diversity and genetic adaptation to heterogeneous environments remain poorly understood in the marine realm. The roughskin sculpin (Trachidermus fasciatus) is a small catadromous fish, and has been listed as a second-class state protected aquatic animal since 1988 in China. To elucidate the underlying mechanism of population genetic structuring and genetic adaptations to local environments, RAD tags were sequenced for 202 individuals in nine populations across the range of T. fasciatus in China. The pairwise FST values over 9,271 filtered SNPs were significant except that between Dongying and Weifang. All the genetic clustering analysis revealed significant population structure with high support for eight distinct genetic clusters. Both the minor allele frequency spectra and Ne estimations suggested extremely small Ne in some populations (e.g., Qinhuangdao, Rongcheng, Wendeng, and Qingdao), which might result from recent population bottleneck. The strong genetic structure can be partly attributed to genetic drift and habitat fragmentation, likely due to the anthropogenic activities. Annotations of candidate adaptive loci suggested that genes involved in metabolism, development, and osmoregulation were critical for adaptation to spatially heterogenous environment of local populations. In the context of anthropogenic activities and environmental change, results of the present population genomic work provided important contributions to the understanding of genetic differentiation and adaptation to changing environments.

An optimized approach for local de novo assembly of overlapping paired-end RAD reads from multiple individuals.

Restriction site-associated DNA (RAD) sequencing is revolutionizing studies in ecological, evolutionary and conservation genomics. However, the assembly of paired-end RAD reads with random-sheared ends is still challenging, especially for non-model species with high genetic variance. Here, we present an efficient optimized approach with a pipeline software, RADassembler, which makes full use of paired-end RAD reads with random-sheared ends from multiple individuals to assemble RAD contigs. RADassembler integrates the algorithms for choosing the optimal number of mismatches within and across individuals at the clustering stage, and then uses a two-step assembly approach at the assembly stage. RADassembler also uses data reduction and parallelization strategies to promote efficiency. Compared to other tools, both the assembly results based on simulation and real RAD datasets demonstrated that RADassembler could always assemble the appropriate number of contigs with high qualities, and more read pairs were properly mapped to the assembled contigs. This approach provides an optimal tool for dealing with the complexity in the assembly of paired-end RAD reads with random-sheared ends for non-model species in ecological, evolutionary and conservation studies. RADassembler is available at https://github.com/lyl8086/RADscripts.

Genome-Wide SNP Discovery, Genotyping and Their Preliminary Applications for Population Genetic Inference in Spotted Sea Bass (Lateolabrax maculatus).

Next-generation sequencing and the collection of genome-wide single-nucleotide polymorphisms (SNPs) allow identifying fine-scale population genetic structure and genomic regions under selection. The spotted sea bass (Lateolabrax maculatus) is a non-model species of ecological and commercial importance and widely distributed in northwestern Pacific. A total of 22 648 SNPs was discovered across the genome of L. maculatus by paired-end sequencing of restriction-site associated DNA (RAD-PE) for 30 individuals from two populations. The nucleotide diversity (π) for each population was 0.0028±0.0001 in Dandong and 0.0018±0.0001 in Beihai, respectively. Shallow but significant genetic differentiation was detected between the two populations analyzed by using both the whole data set (FST = 0.0550, P < 0.001) and the putatively neutral SNPs (FST = 0.0347, P < 0.001). However, the two populations were highly differentiated based on the putatively adaptive SNPs (FST = 0.6929, P < 0.001). Moreover, a total of 356 SNPs representing 298 unique loci were detected as outliers putatively under divergent selection by FST-based outlier tests as implemented in BAYESCAN and LOSITAN. Functional annotation of the contigs containing putatively adaptive SNPs yielded hits for 22 of 55 (40%) significant BLASTX matches. Candidate genes for local selection constituted a wide array of functions, including binding, catalytic and metabolic activities, etc. The analyses with the SNPs developed in the present study highlighted the importance of genome-wide genetic variation for inference of population structure and local adaptation in L. maculatus.

Population Structure and Adaptive Divergence in a High Gene Flow Marine Fish: The Small Yellow Croaker (Larimichthys polyactis).

The spatial distribution of genetic diversity has been long considered as a key component of policy development for management and conservation of marine fishes. However, unraveling the population genetic structure of migratory fish species is challenging due to high potential for gene flow. Despite the shallow population differentiation revealed by putatively neutral loci, the higher genetic differentiation with panels of putatively adaptive loci could provide greater resolution for stock identification. Here, patterns of population differentiation of small yellow croaker (Larimichthys polyactis) were investigated by genotyping 15 highly polymorphic microsatellites in 337 individuals of 15 geographic populations collected from both spawning and overwintering grounds. Outlier analyses indicated that the locus Lpol03 might be under directional selection, which showed a strong homology with Grid2 gene encoding the glutamate receptor δ2 protein (GluRδ2). Based on Lpol03, two distinct clusters were identified by both STRUCTURE and PCoA analyses, suggesting that there were two overwintering aggregations of L. polyactis. A novel migration pattern was suggested for L. polyactis, which was inconsistent with results of previous studies based on historical fishing yield statistics. These results provided new perspectives on the population genetic structure and migratory routes of L. polyactis, which could have significant implications for sustainable management and utilization of this important fishery resource.

Development and preliminary evaluation of a genomewide single nucleotide polymorphisms resource generated by RAD-seq for the small yellow croaker (Larimichthys polyactis).

Recent advances in high-throughput sequencing technologies have offered the possibility to generate genomewide sequence data to delineate previously unidentified genetic structure, obtain more accurate estimates of demographic parameters and to evaluate potential adaptive divergence. Here, we identified 27 556 single nucleotide polymorphisms for the small yellow croaker (Larimichthys polyactis) using restriction-site-associated DNA (RAD) sequencing of 24 individuals from two populations. Significant sources of genetic variation were identified, with an average nucleotide diversity (π) of 0.00105 ± 0.000425 across individuals, and long-term effective population size was thus estimated to range between 26 172 and 261 716. According to the results, no differentiation between the two populations was detected based on the SNP data set of top quality score per contig or neutral loci. However, the two analysed populations were highly differentiated based on SNP data set of both top FST value per contig and the outlier SNPs. Moreover, local adaptation was highlighted by an FST -based outlier tests implemented in LOSITAN and a total of 538 potentially locally selected SNPs were identified. blast2go annotation of contigs containing the outlier SNPs yielded hits for 37 (66%) of 56 significant blastx matches. Candidate genes for local adaptation constituted a wide array of biological functions, including cellular response to oxidative stress, actin filament binding, ion transmembrane transport and synapse assembly. The generated SNP resources in this study provided a valuable tool for future population genetics and genomics studies of L. polyactis.