Ze Yuan Zhu

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.

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.

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.

Construction of a BAC library and mapping BAC clones to the linkage map of Barramundi, Lates calcarifer

BackgroundBarramundi (Lates calcarifer) is an important farmed marine food fish species. Its first generation linkage map has been applied to map QTL for growth traits. To identify genes located in QTL responsible for specific traits, genomic large insert libraries are of crucial importance. We reported herein a bacterial artificial chromosome (BAC) library and the mapping of BAC clones to the linkage map.ResultsThis BAC library consisted of 49,152 clones with an average insert size of 98 kb, representing 6.9-fold haploid genome coverage. Screening the library with 24 microsatellites and 15 ESTs/genes demonstrated that the library had good genome coverage. In addition, 62 novel microsatellites each isolated from 62 BAC clones were mapped onto the first generation linkage map. A total of 86 BAC clones were anchored on the linkage map with at least one BAC clone on each linkage group.ConclusionWe have constructed the first BAC library for L. calcarifer and mapped 86 BAC clones to the first generation linkage map. This BAC library and the improved linkage map with 302 DNA markers not only supply an indispensable tool to the integration of physical and linkage maps, the fine mapping of QTL and map based cloning genes located in QTL of commercial importance, but also contribute to comparative genomic studies and eventually whole genome sequencing.

A standard panel of microsatellites for Asian seabass (Lates calcarifer)

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 +/- 0.71 (range: 6-19), and the expected heterozygosity averaged 0.76 +/- 0.02 (range: 0.63-1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM-T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3-5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 x 10 and 20 x 20) demonstrated a high power of these markers for revealing parent-sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.

A simple and efficient method for isolating polymorphic microsatellites from cDNA

BackgroundMicrosatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish.ResultsThe method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern.ConclusionNormalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution.

Eleven polymorphic microsatellites isolated from red swamp crayfish, Procambarus clarkii

Eleven microsatellites were isolated from a genomic DNA library enriched for CA‐ and GA‐repeats, and were characterized in 48 individuals of red swamp crayfish Procambarus clarkii. All 11 microsatellites were polymorphic with an average of allele number of 6.6 per locus. Genotypic distributions of these 11 markers were found to be in conformance with the expectations of Hardy–Weinberg equilibrium. All 11 markers were unlinked. These markers are being used to study the invasion routine, genetic diversity and population structure of the species P. clarkii.

Novel polymorphic microsatellites for studying genetic diversity of red Asian arowanas

Asian arowana (Scleropages formosus) is a highly endangered fish species listed in Appendix 1 of CITES since 1980. Fourteen novel polymorphic microsatellites were isolated from a CA-enriched partial genomic DNA library, and were used for studying genetic diversity of 41 red arowanas from the wild population. Surprisingly the average allele number of the 14 polymorphic microsatellites was as high as 12.4/locus. The average observed heterozygosity was 0.78 ranging from 0.51 to 0.95, and the gene diversity was quite high (0.78). All these data suggest that high level of genetic diversity existed in the red Asian arowana population.

The complete nucleotide sequence of the mitochondrial genome of Tetraodon nigroviridis

The fresh water pufferfish Tetraodon nigroviridis is a model organism for studying evolution of genome and gene functions, but its mitochondrial genome (mtDNA) sequence is still not available. We determined the complete nucleotide sequence of its mtDNA using shotgun sequencing. The T. nigroviridis mtDNA was 16,462 bp, and contained 13 protein coding genes, 22 tRNAs, 2 rRNAs and a major non-coding region. The gene order was identical to the common type of vertebrate mtDNA, whereas the G+C content in the sense strand was 46.9%, much higher than most other fish species. One hundred and three SNPs were detected in the control region of the mtDNA of 35 individuals, a majority of SNPs were detected in the 5′ end of the control region. A phylogenetic study including 21 fish species was performed on concatenated amino acid sequences of 12 protein coding genes, and revealed that the T. nigroviridis was clustered with Fugu rubripes into a group. The complete mtDNA sequence and SNPs in its control region will be useful in studying fish evolution, in differentiating different Tetraodon species and in analyzing genetic diversity within T. nigroviridis.