Gen Hua Yue

A high-resolution linkage map for comparative genome analysis and QTL fine mapping in Asian seabass, Lates calcarifer

BackgroundHigh density linkage maps are essential for comparative analysis of synteny, fine mapping of quantitative trait loci (QTL), searching for candidate genes and facilitating genome sequence assembly. However, in most foodfish species, marker density is still low. We previously reported a first generation linkage map with 240 DNA markers and its application to preliminarily map QTL for growth traits in Asian seabass (Lates calcarifer). Here, we report a high-resolution linkage map with 790 microsatellites and SNPs, comparative analysis of synteny, fine-mapping of QTL and the identification of potential candidate genes for growth traits.ResultsA second generation linkage map of Asian seabass was developed with 790 microsatellite and SNP markers. The map spanned a genetic length of 2411.5 cM, with an average intermarker distance of 3.4 cM or 1.1 Mb. This high density map allowed for comparison of the map with Tetraodon nigroviridis genome, which revealed 16 synteny regions between the two species. Moreover, by employing this map we refined QTL to regions of 1.4 and 0.2 cM (or 400 and 50 kb) in linkage groups 2 and 3 in a population containing 380 progeny; potential candidate genes for growth traits in QTL regions were further identified using comparative genome analysis, whose effects on growth traits were investigated. Interestingly, a QTL cluster at Lca371 underlying growth traits of Asian seabass showed similarity to the cathepsin D gene of human, which is related to cancer and Alzheimers disease.ConclusionsWe constructed a high resolution linkage map, carried out comparative mapping, refined the positions of QTL, identified candidate genes for growth traits and analyzed their effects on growth. Our study developed a framework that will be indispensable for further identification of genes and analysis of molecular variation within the refined QTL to enhance understanding of the molecular basis of growth and speed up genetic improvement of growth performance, and it also provides critical resource for future genome sequence assembly and comparative genomics studies on the evolution of fish genomes.

A Microsatellite Linkage Map of Barramundi, Lates calcarifer

Barramundi (Lates calcarifer) is an important farmed marine food fish species. Its compact genome (∼700 Mb) is among the smallest genomes of food fish species. We established a first-generation genetic linkage map of Barramundi with a mapping panel containing three parents (two males and one female) and 93 progeny. A total of 240 microsatellite markers were mapped into 24 linkage groups. Among these markers, 10 were located in ESTs and known genes. The total lengths of the female and male maps were 873.8 and 414.5 cM with an average marker spacing of 6.20 and 4.70 cM, respectively. Comparing the flanking sequences of the 240 Barramundi microsatellites with the assembled whole-genome sequences of Tetraodon nigrovidiris revealed 55 homologous sequences located in 19 of the 21 chromosomes of T. nigrovidiris. The map will not only enable the mapping of quantitative trait loci, but also provide new resources for understanding the evolution of fish genomes.

Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers

The whole broodstock of two Hungarian common carp farms—80 and 196 individuals—was analyzed by using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. Ten polymorphic RAPD markers and four microsatellites were selected to genotype both of the stocks. As expected, microsatellite analysis revealed more detailed information on genetic diversities than RAPD assay. Results obtained with both types of DNA markers showed lack of major differences between the genetic structure of the two stocks: heterozygosity values and allele frequencies were very similar. Dendrograms created from both sets of data did not show grouping of individuals according to stocks. Genotypes from the two stocks were also compared to those from a limited number of samples collected from other hatcheries and two rivers. Allele frequencies in the groups were similar, with the exception of wild carps. An interesting observation was that three private microsatellite alleles were found in the eight wild carp individuals, compared to the seven detected in the rest of the samples tested (372 individuals).

The intestinal microbiome of fish under starvation

BackgroundStarvation not only affects the nutritional and health status of the animals, but also the microbial composition in the host’s intestine. Next-generation sequencing provides a unique opportunity to explore gut microbial communities and their interactions with hosts. However, studies on gut microbiomes have been conducted predominantly in humans and land animals. Not much is known on gut microbiomes of aquatic animals and their changes under changing environmental conditions. To address this shortcoming, we determined the microbial gene catalogue, and investigated changes in the microbial composition and host-microbe interactions in the intestine of Asian seabass in response to starvation.ResultsWe found 33 phyla, 66 classes, 130 orders and 278 families in the intestinal microbiome. Proteobacteria (48.8%), Firmicutes (15.3%) and Bacteroidetes (8.2%) were the three most abundant bacteria taxa. Comparative analyses of the microbiome revealed shifts in bacteria communities, with dramatic enrichment of Bacteroidetes, but significant depletion of Betaproteobacteria in starved intestines. In addition, significant differences in clusters of orthologous groups (COG) functional categories and orthologous groups were observed. Genes related to antibiotic activity in the microbiome were significantly enriched in response to starvation, and host genes related to the immune response were generally up-regulated.ConclusionsThis study provides the first insights into the fish intestinal microbiome and its changes under starvation. Further detailed study on interactions between intestinal microbiomes and hosts under dynamic conditions will shed new light on how the hosts and microbes respond to the changing environment.

A consensus linkage map of the grass carp (Ctenopharyngodon idella) based on microsatellites and SNPs

BackgroundGrass carp (Ctenopharyngodon idella) belongs to the family Cyprinidae which includes more than 2000 fish species. It is one of the most important freshwater food fish species in world aquaculture. A linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution. The aim of this study is to construct a first generation genetic map of grass carp using microsatellites and SNPs to generate a new resource for mapping QTL for economically important traits and to conduct a comparative mapping analysis to shed new insights into the evolution of fish genomes.ResultsWe constructed a first generation linkage map of grass carp with a mapping panel containing two F1 families including 192 progenies. Sixteen SNPs in genes and 263 microsatellite markers were mapped to twenty-four linkage groups (LGs). The number of LGs was corresponding to the haploid chromosome number of grass carp. The sex-specific map was 1149.4 and 888.8 cM long in females and males respectively whereas the sex-averaged map spanned 1176.1 cM. The average resolution of the map was 4.2 cM/locus. BLAST searches of sequences of mapped markers of grass carp against the whole genome sequence of zebrafish revealed substantial macrosynteny relationship and extensive colinearity of markers between grass carp and zebrafish.ConclusionsThe linkage map of grass carp presented here is the first linkage map of a food fish species based on co-dominant markers in the family Cyprinidae. This map provides a valuable resource for mapping phenotypic variations and serves as a reference to approach comparative genomics and understand the evolution of fish genomes and could be complementary to grass carp genome sequencing project.

Rapid isolation of DNA from fresh and preserved fish scales for polymerase chain reaction.

Abstract: We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence.

A genome scan for quantitative trait loci affecting growth-related traits in an F1 family of Asian seabass ( Lates calcarifer )

BackgroundBody weight and length are economically important traits in foodfish species influenced by quantitative trait loci (QTL) and environmental factors. It is usually difficult to dissect the genetic and environmental effects. Asian seabass (Lates calcarifer) is an important marine foodfish species with a compact genome (~700 Mb). The recent construction of a first generation linkage map of Asian seabass with 240 microsatellites provides a good opportunity to determine the number and position of QTL, and the magnitude of QTL effects with a genome scan.ResultsWe conducted a genome scan for QTL affecting body weight, standard length and condition factors in an F1 family containing 380 full-sib individuals from a breeding stock by using 97 microsatellites evenly covering 24 chromosomes. Interval mapping and multiple QTL model mapping detected five significant and 27 suggestive QTL on ten linkage groups (LGs). Among the five significant QTL detected, three (qBW2-a, qTL2-a and qSL2-a) controlling body weight, total and standard length respectively, were mapped on the same region near Lca287 on LG2, and explained 28.8, 58.9 and 59.7% of the phenotypic variance. The other two QTL affecting body weight, qBW2-b and qBW3, were located on LG2 and 3, and accounted for 6.4 and 8.8% of the phenotypic variance. Suggestive QTL associated with condition factors are located on six different LGs.ConclusionThis study presents the first example of QTL detection for growth-related traits in an F1 family of a marine foodfish species. The results presented here will enable further fine-mapping of these QTL for marker-assisted selection of the Asian seabass, eventually identifying individual genes responsible for growth-related traits.

A first generation microsatellite- and SNP-based linkage map of Jatropha.

Jatropha curcas is a potential plant species for biodiesel production. However, its seed yield is too low for profitable production of biodiesel. To improve the productivity, genetic improvement through breeding is essential. A linkage map is an important component in molecular breeding. We established a first-generation linkage map using a mapping panel containing two backcross populations with 93 progeny. We mapped 506 markers (216 microsatellites and 290 SNPs from ESTs) onto 11 linkage groups. The total length of the map was 1440.9 cM with an average marker space of 2.8 cM. Blasting of 222 Jatropha ESTs containing polymorphic SSR or SNP markers against EST-databases revealed that 91.0%, 86.5% and 79.2% of Jatropha ESTs were homologous to counterparts in castor bean, poplar and Arabidopsis respectively. Mapping 192 orthologous markers to the assembled whole genome sequence of Arabidopsis thaliana identified 38 syntenic blocks and revealed that small linkage blocks were well conserved, but often shuffled. The first generation linkage map and the data of comparative mapping could lay a solid foundation for QTL mapping of agronomic traits, marker-assisted breeding and cloning genes responsible for phenotypic variation.

Mutation rate and pattern of microsatellites in common carp (Cyprinus carpio L.).

Microsatellites are popular molecular markers in genetic and evolutionary studies. Their mutational dynamics have been extensively studied in humans and fruit flies, but few data were available in fish. By genotyping 55 individuals of a F1 pedigree, we investigated the mutation rates and patterns of 49 microsatellites in one of the most important fresh water fish species, the common carp (Cyprinus carpio L.). The overall mutation rate of the 49 loci was 5.56×10−4/locus/generation (95% confidence interval 1.52×10−4 and 1.63×10−3). The change of allele size was between +2 to −5 repeat units, assuming that the mutation allele arose from the parental allele most similar in size to the mutant.

Analysis of Stress-Responsive Transcriptome in the Intestine of Asian Seabass (Lates calcarifer) using RNA-Seq

Identification of differentially expressed genes (DEGs) and regulated pathways in response to stressors using a whole-genome approach is critical to understanding the mechanisms underlying stress responses. We challenged Asian seabass with lipopolysaccharide (LPS), Vibrio harveyi, high salinity and fasting, and sequenced six cDNA libraries of intestine samples using Roche 454 RNA-seq. Over 1 million reads (average size: 516 bp) were obtained. The de novo assembly obtained 83 911 unisequences with an average length of 747 bp. In total, 62.3% of the unisequences were annotated. We observed overall similar expression profiles among different challenges, while a number of DEGs and regulated pathways were identified under specific challenges. More than 1000 DEGs and over 200 regulated pathways for each stressor were identified. Thirty-seven genes were differentially expressed in response to all challenges. Our data suggest that there is a global coordination and fine-tuning of gene regulation during different challenges. In addition, we detected dramatic immune responses in intestines under different stressors. This study is the first step towards the comprehensive understanding of the mechanisms underlying stress responses and supplies significant transcriptome resources for studying biological questions in non-model fish species.