Shijun Xiao

Gene map of large yellow croaker ( Larimichthys crocea ) provides insights into teleost genome evolution and conserved regions associated with growth

The genetic map of a species is essential for its whole genome assembly and can be applied to the mapping of important traits. In this study, we performed RNA-seq for a family of large yellow croakers (Larimichthys crocea) and constructed a high-density genetic map. In this map, 24 linkage groups comprised 3,448 polymorphic SNP markers. Approximately 72.4% (2,495) of the markers were located in protein-coding regions. Comparison of the croaker genome with those of five model fish species revealed that the croaker genome structure was closer to that of the medaka than to the remaining four genomes. Because the medaka genome preserves the teleost ancestral karyotype, this result indicated that the croaker genome might also maintain the teleost ancestral genome structure. The analysis also revealed different genome rearrangements across teleosts. QTL mapping and association analysis consistently identified growth-related QTL regions and associated genes. Orthologs of the associated genes in other species were demonstrated to regulate development, indicating that these genes might regulate development and growth in croaker. This gene map will enable us to construct the croaker genome for comparative studies and to provide an important resource for selective breeding of croaker.

Comparative analysis of the GBLUP, emBayesB, and GWAS algorithms to predict genetic values in large yellow croaker (Larimichthys crocea).

BackgroundThe advances of sequencing technology accelerate the development of theory of molecular quantitative genetics such as QTL mapping, genome-wide association study and genomic selection. This paper was designed to study genomic selection in large yellow croaker breeding. The aims of this study were: (i) to estimate heritability values of traits in large yellow croaker; (ii) to assess feasibility of genomic selection in the traits of growth rate and meat quality; (iii) to compare predictive accuracies affected by different algorithms and training sizes, and to find what training sizes could reach ideal accuracies; (iv) to compare results of GWAS with genomic prediction, and to assess feasibility of pre-selection of significant SNPs in genomic selection. 500 individuals were tested in the trait of body weight and body length, while 176 were tested in the percentage of n-3 highly unsaturated fatty acids (n-3HUFA) in muscle. GBLUP and emBayesB were used to perform genomic prediction.ResultsGenotyping-By-Sequencing method was used to construct the libraries for the NGS sequencing and find ~30,000 SNPs. Heritability estimates were 0.604, 0.586 and 0.438 for trait of body weight, body length and n-3HUFA, respectively. The predictive abilities estimated by GBLUP showed higher than that by emBayesB in traits of body weight and body length. However, the result was just the opposite in n-3HUFA. According to fit the curve of predictive accuracy, we estimated that at least 1000 individuals in training set could reach an accuracy of 0.8 in body weight and body length. GBLUP, emBayesB and GWAS could not always find significant SNPs associated with phenotypes consistently. Significant SNPs were selected by emBayesB could obtain the largest proportions to explain total additive genetic variances.ConclusionsThis research showed that genomic selection was feasible in large yellow croaker breeding. We suggest doing a test before deciding to use which algorithm in specific trait in genomic prediction. We estimated required training sizes to reach ideal predictive accuracies and assessed feasibility of pre-selection of SNPs successfully. Because of high mortality rate of fish and high cost in genomic sequencing, genomic selection may be more suitable for applying on some traits which cannot be measured on candidates directly.

Identification of Immune-Related Genes and Development of SSR/SNP Markers from the Spleen Transcriptome of Schizothorax prenanti.

Schizothorax prenanti (S. prenanti) is mainly distributed in the upstream regions of the Yangtze River and its tributaries in China. This species is indigenous and commercially important. However, in recent years, wild populations and aquacultures have faced the serious challenges of germplasm variation loss and an increased susceptibility to a range of pathogens. Currently, the genetics and immune mechanisms of S. prenanti are unknown, partly due to a lack of genome and transcriptome information. Here, we sought to identify genes related to immune functions and to identify molecular markers to study the function of these genes and for trait mapping. To this end, the transcriptome from spleen tissues of S. prenanti was analyzed and sequenced. Using paired-end reads from the Illumina Hiseq2500 platform, 48,517 transcripts were isolated from the spleen transcriptome. These transcripts could be clustered into 37,785 unigenes with an N50 length of 2,539 bp. The majority of the unigenes (35,653, 94.4%) were successfully annotated using non-redundant nucleotide sequence analysis (nt), and the non-redundant protein (nr), Swiss-Prot, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. KEGG pathway assignment identified more than 500 immune-related genes. Furthermore, 7,545 putative simple sequence repeats (SSRs), 857,535 single nucleotide polymorphisms (SNPs), and 53,481 insertion/deletion (InDels) were detected from the transcriptome. This is the first reported high-throughput transcriptome analysis of S. prenanti, and it provides valuable genetic resources for the investigation of immune mechanisms, conservation of germplasm, and molecular marker-assisted breeding of S. prenanti.

Genomic Selection Using Extreme Phenotypes and Pre-Selection of SNPs in Large Yellow Croaker (Larimichthys crocea)

Genomic selection (GS) is an effective method to improve predictive accuracies of genetic values. However, high cost in genotyping will limit the application of this technology in some species. Therefore, it is necessary to find some methods to reduce the genotyping costs in genomic selection. Large yellow croaker is one of the most commercially important marine fish species in southeast China and Eastern Asia. In this study, genotyping-by-sequencing was used to construct the libraries for the NGS sequencing and find 29,748 SNPs in the genome. Two traits, eviscerated weight (EW) and the ratio between eviscerated weight and whole body weight (REW), were chosen to study. Two strategies to reduce the costs were proposed as follows: selecting extreme phenotypes (EP) for genotyping in reference population or pre-selecting SNPs to construct low-density marker panels in candidates. Three methods of pre-selection of SNPs, i.e., pre-selecting SNPs by absolute effects (SE), by single marker analysis (SMA), and by fixed intervals of sequence number (EL), were studied. The results showed that using EP was a feasible method to save the genotyping costs in reference population. Heritability did not seem to have obvious influences on the predictive abilities estimated by EP. Using SMA was the most feasible method to save the genotyping costs in candidates. In addition, the combination of EP and SMA in genomic selection also showed good results, especially for trait of REW. We also described how to apply the new methods in genomic selection and compared the genotyping costs before and after using the new methods. Our study may not only offer a reference for aquatic genomic breeding but also offer a reference for genomic prediction in other species including livestock and plants, etc.

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.

Molecular cloning and functional characterization of a QM protein in large yellow croaker (Larimichthys crocea)

Since it was proposed to be a tumor suppressor in 1991, QM protein has attracted intensive and wide attention in plants, animals and fungi research fields. Up to date, however, the function of QM protein in fish immunity remains unknown. In this investigation, a QM gene (named as LycQM gene) was cloned from large yellow croaker (Larimichthys crocea), and LycQM protein was expressed in Escherichia coli and purified. The LycQM gene was ubiquitously transcribed in multi-tissues, including spleen, muscle, heart, liver, intestine, blood and head kidney. By quantitative real-time RT-PCR analysis, we found the highest and the lowest expression level of LycQM gene in head kidney and in heart, respectively. Time course analysis showed that LycQM expression was obviously up-regulated in blood and head kidney after immunization with polyinosinic polycytidynic acid (poly I:C), formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus and bacterial lipopolysaccharides (LPS). Moreover, as demonstrated by RNAi assays, LycQM protein could regulate the activity of phenoloxidase, a key enzyme in the proPO activation system of immunity. These results suggested that LycQM protein might play an important role in the immune response against microorganisms in large yellow croaker.

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.

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.