Zhaofang Han

Whole-genome single-nucleotide polymorphism (SNP) marker discovery and association analysis with the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content in Larimichthys crocea.

Whole-genome single-nucleotide polymorphism (SNP) markers are valuable genetic resources for the association and conservation studies. Genome-wide SNP development in many teleost species are still challenging because of the genome complexity and the cost of re-sequencing. Genotyping-By-Sequencing (GBS) provided an efficient reduced representative method to squeeze cost for SNP detection; however, most of recent GBS applications were reported on plant organisms. In this work, we used an EcoRI-NlaIII based GBS protocol to teleost large yellow croaker, an important commercial fish in China and East-Asia, and reported the first whole-genome SNP development for the species. 69,845 high quality SNP markers that evenly distributed along genome were detected in at least 80% of 500 individuals. Nearly 95% randomly selected genotypes were successfully validated by Sequenom MassARRAY assay. The association studies with the muscle eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content discovered 39 significant SNP markers, contributing as high up to ∼63% genetic variance that explained by all markers. Functional genes that involved in fat digestion and absorption pathway were identified, such as APOB, CRAT and OSBPL10. Notably, PPT2 Gene, previously identified in the association study of the plasma n-3 and n-6 polyunsaturated fatty acid level in human, was re-discovered in large yellow croaker. Our study verified that EcoRI-NlaIII based GBS could produce quality SNP markers in a cost-efficient manner in teleost genome. The developed SNP markers and the EPA and DHA associated SNP loci provided invaluable resources for the population structure, conservation genetics and genomic selection of large yellow croaker and other fish organisms.

De novo characterization of Larimichthys crocea transcrip-tome for growth-/immune-related gene identification and massive microsatellite (SSR) marker development

The large yellow croaker, Larimichthys crocea is an important marine fish in China with a high economic value. In the last decade, the stock conservation and aquaculture industry of this species have been facing severe challenges because of wild population collapse and degeneration of important economic traits. However, genes contributing to growth and immunity in L. crocea have not been thoroughly analyzed, and available molecular markers are still not sufficient for genetic resource management and molecular selection. In this work, we sequenced the transcriptome in L. crocea liver tissue with a Roche 454 sequencing platform and assembled the transcriptome into 93 801 transcripts. Of them, 38 856 transcripts were successfully annotated in nt, nr, Swiss-Prot, InterPro, COG, GO and KEGG databases. Based on the annotation information, 3 165 unigenes related to growth and immunity were identified. Additionally, a total of 6 391 simple sequence repeats (SSRs) were identified from the transcriptome, among which 4 498 SSRs had enough flanking regions to design primers for polymerase chain reactions (PCR). To access the polymorphism of these markers, 30 primer pairs were randomly selected for PCR amplification and validation in 30 individuals, and 12 primer pairs (40.0%) exhibited obvious length polymorphisms. This work applied RNA-Seq to assemble and analyze a live transcriptome in L. crocea. With gene annotation and sequence information, genes related to growth and immunity were identified and massive SSR markers were developed, providing valuable genetic resources for future gene functional analysis and selective breeding of L. crocea.

SNP discovery in large yellow croaker (Larimichthys crocea) using Roche 454 pyrosequencing sequencing platform

Large yellow croaker, one of the most important economic marine fish in China, is mainly distributed in coastal regions of East Asia. Its wild stocks have been collapsed due to overfishing and habitat degradation. However, genetic analysis of the species for population conservation and molecular-aided selection has been largely hindered by insufficient molecular markers. In this study, we used Roche 454 pyrosequencing to develop single nucleotide polymorphism (SNP) markers in large yellow croaker transcriptome. From 4993 transcripts with functional annotation, 5343 putative SNP were detected. Among them, 26 SNPs were randomly selected and 15 loci exhibited significant polymorphisms in genotyping by Sequenom MassARRAY. To our knowledge, this is the first report on the SNP marker development in large yellow croaker based on 454 pyrosequencing RNA sequencing. These SNP could serve as useful marker resources in genetic analysis for population conservation and economic performance improvement of large yellow croaker.

MicroRNAs identification and bioinformatics analysis in large yellow croaker Larimichthys crocea using an integrated comparative and ab initio approach

MicroRNAs (miRNAs) are a group of small, endogenous, single-stranded non-coding RNAs that post-transcriptionally regulate gene expression levels. Previous studies have revealed that miRNAs play key roles in multiple biological processes, such as growth and development in both animals and plants. Computational identification is an efficient method for miRNA prediction in organisms with a reference genome before high-throughput miRNA sequencing experiments. In this study, we employed an integrated strategy combining the homology-based and ab initio approaches to predict miRNAs from the genome and transcriptome of large yellow croaker, one of the most commercially important marine fish in China and East Asia. A total of 418 miRNA molecules, including 287 miRNAs by the homology-based method and 131 miRNAs by the ab initio approach, were identified for large yellow croaker. Additionally, 16 053 target genes were predicted and annotated for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Meanwhile, we analysed single nucleotide polymorphisms (SNPs) around large yellow croaker miRNA and found that the miRNA seed regions were significantly less prone to mutations, indicating that the miRNA sequences were under strict natural selection based on their essential regulation functions in living cells. Twenty-two SNPs were identified in large yellow croaker miRNA seed regions, which dramatically influenced the miRNA-gene regulation networks. This is the first reported miRNA detection from both the genome and transcriptome using the integrated strategy for large yellow croaker species, and the miRNA and SNP analyses in this work provide important resources and a reference for subsequent miRNA functional investigations in large yellow croaker.

Transcriptome Profiling of the Abdominal Skin of Larimichthys crocea in Light Stress

Large yellow croaker (Larimichthys crocea), one of the most important marine fish species in China, can change its abdominal skin color when it is shifted from light to dark or from dark to light, providing us an opportunity of investigating the molecular responding mechanism of teleost in light stress. The gene expression profile of fish under light stress is rarely documented. In this research, the transcriptome profiles of the abdominal skin of L. crocea exposed to light or dark for 0 h, 0.5 h and 2 h were produced by next-generation sequencing (NGS). The cluster results demonstrated that stress period, rather than light intensity (e.g., light or dark), is the major influencing factor. Differently expressed genes (DEGs) were identified between 0 h and 0.5 h groups, between 0 h and 2 h groups, between 0.5 h light and 0.5 h dark, and between 2 h light and 2 h dark, respectively. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that the genes relating to immunity, energy metabolism, and cytoskeletal protein binding were significantly enriched. The detailed analysis of transcriptome profiles also revealed regular gene expression trends, indicating that the elaborate gene regulation networks underlined the molecular responses of the fish to light stress. This transcriptome analysis suggested that systematic and complicated regulatory cascades were functionally activated in response to external stress, and coloration change caused by light stress was mainly attributed to the change in the density of chromatophores for L. crocea. This study also provided valuable information for skin coloration or light stress research on other marine fish species.