Jun-ya Aoki

Quantitative Trait Loci (QTL) Associated with Resistance to a Monogenean Parasite (Benedenia seriolae) in Yellowtail (Seriola quinqueradiata) through Genome Wide Analysis

Benedenia infections caused by the monogenean fluke ectoparasite Benedenia seriolae seriously impact marine finfish aquaculture. Genetic variation has been inferred to play a significant role in determining the susceptibility to this parasitic disease. To evaluate the genetic basis of Benedenia disease resistance in yellowtail (Seriola quinqueradiata), a genome-wide and chromosome-wide linkage analyses were initiated using F1 yellowtail families (n = 90 per family) based on a high-density linkage map with 860 microsatellite and 142 single nucleotide polymorphism (SNP) markers. Two major quantitative trait loci (QTL) regions on linkage groups Squ2 (BDR-1) and Squ20 (BDR-2) were identified. These QTL regions explained 32.9–35.5% of the phenotypic variance. On the other hand, we investigated the relationship between QTL for susceptibility to B. seriolae and QTL for fish body size. The QTL related to growth was found on another linkage group (Squ7). As a result, this is the first genetic evidence that contributes to detailing phenotypic resistance to Benedenia disease, and the results will help resolve the mechanism of resistance to this important parasitic infection of yellowtail.

Second generation physical and linkage maps of yellowtail (Seriola quinqueradiata) and comparison of synteny with four model fish

BackgroundPhysical and linkage maps are important aids for the assembly of genome sequences, comparative analyses of synteny, and to search for candidate genes by quantitative trait locus analysis. Yellowtail, Seriola quinqueradiata, is an economically important species in Japanese aquaculture, and genetic information will be useful for DNA-assisted breeding. We report the construction of a second generation radiation hybrid map, its synteny analysis, and a second generation linkage map containing SNPs (single nucleotide polymorphisms) in yellowtail.ResultsApproximately 1.4 million reads were obtained from transcriptome sequence analysis derived from 11 tissues of one individual. To identify SNPs, cDNA libraries were generated from a pool of 500 whole juveniles, and the gills and kidneys of 100 adults. 9,356 putative SNPs were detected in 6,025 contigs, with a minor allele frequency ≥25%. The linkage and radiation hybrid maps were constructed based on these contig sequences. 2,081 markers, including 601 SNPs markers, were mapped onto the linkage map, and 1,532 markers were mapped in the radiation hybrid map.ConclusionsThe second generation linkage and physical maps were constructed using 6,025 contigs having SNP markers. These maps will aid the de novo assembly of sequencing reads, linkage studies and the identification of candidate genes related to important traits. The comparison of marker contigs in the radiation hybrid map indicated that yellowtail is evolutionarily closer to medaka than to green-spotted pufferfish, three-spined stickleback or zebrafish. The synteny analysis may aid studies of chromosomal evolution in yellowtail compared with model fish.

Functional Analysis of Promoter Region from Eel Cytochrome P450 1A1 Gene in Transgenic Medaka

Abstract: Transcription of the CYP1A1 genes in mammals and fish is stimulated by polyaromatic hydrocarbons. DNA sequencing analysis revealed that CYP1A1 gene in eel (Anguilla japonica) contains two kinds of putative cis-acting regulatory elements, XRE (xenobiotic-responsive element) and ERE (estrogen-responsive element). XRE is known as the enhancer that is responsible for the inducibility of the genes of CYP1A1 and some other drug-metabolizing enzymes. In the eel CYP1A1 gene, XRE motifs are distributed as follows: five times in the region from −2136 to −1125 bp, XRE(−6) to (−2); once in the proximal basal promoter region, XRE(−1); and once in the first intron, XRE(+1). The region between XRE(−2) and XRE(−1) contains three ERE motifs. To investigate the function of the cis-acting regulatory elements in the eel CYP1A1 gene, recombinant plasmids prepared with its 5′ upstream sequence and the structural gene for luciferase were microinjected into fertilized eggs of medaka at the one-cell stage. Hatched fry were treated with 3-methylcholanthrene, and the transcription efficiency was assayed using competitive polymerase chain reaction analysis. Deletion of the region containing the five XREs, XRE(−6) to XRE(−2), and the point mutation of XRE(−1) reduced the inducible expressions by 75% and 56%, respectively, showing apparent dependency of the drug induction on the XREs. Constitutive expression, however, was not significantly affected by deletion or disruption of the XREs. When the region between XRE(−2) and XRE(−1) containing no XREs but three ERE motifs was internally deleted, the inducible expression and the constitutive expression were reduced by 88% and 75%, respectively. Replacement of this region with a partial fragment of eel CYP1A1 complementary DNA, with slight alteration of the distance between the five XREs and XRE(−1), reduced the inducible expression and the constitutive expression by 91% and 60%, respectively. These results strongly suggest that not only XRE but also other regulatory elements, possibly ERE, play an important role in induced and constitutive expressions of the eel CYP1A1 gene.

Isolation and Sequence Analysis of the Eel Cytochrome P450 1A1 Gene

Abstract: The putative CYP1A1 gene was isolated from a genomic library of eel (Anguilla japonica) chromosomal DNA. Sequence analysis of 8184 bp showed that the clone obtained was the structural gene of CYP1A1, which consisted of seven exons and six introns in a region approximately 5800 bp in length, as well as a 5′ upstream region of about 2300 bp. The initiation codon was found not in the first exon, but in the second exon, as was reported for the CYP1A genes of mammals and fish. The GC box, CAAT box, and TATA box were located proximately to the first exon, at 110, 62, and 38 bp upstream of the transcriptional start site, respectively. We found six core sequences of xenobiotic-responsive elements (XREs) in the 5′ upstream region, and also one in the first intron. All the XRE core sequences in the 5′ upstream region were unique in their localization in the sense strand. The XRE core sequence in the first intron was in the antisense strand.

Identification of Sex-Linked SNPs and Sex-Determining Regions in the Yellowtail Genome

Unlike the conservation of sex-determining (SD) modes seen in most mammals and birds, teleost fishes exhibit a wide variety of SD systems and genes. Hence, the study of SD genes and sex chromosome turnover in fish is one of the most interesting topics in evolutionary biology. To increase resolution of the SD gene evolutionary trajectory in fish, identification of the SD gene in more fish species is necessary. In this study, we focused on the yellowtail, a species widely cultivated in Japan. It is a member of family Carangidae in which no heteromorphic sex chromosome has been observed, and no SD gene has been identified to date. By performing linkage analysis and BAC walking, we identified a genomic region and SNPs with complete linkage to yellowtail sex. Comparative genome analysis revealed the yellowtail SD region ancestral chromosome structure as medaka-fugu. Two inversions occurred in the yellowtail linage after it diverged from the yellowtail-medaka ancestor. An association study using wild yellowtails and the SNPs developed from BAC ends identified two SNPs that can reasonably distinguish the sexes. Therefore, these will be useful genetic markers for yellowtail breeding. Based on a comparative study, it was suggested that a PDZ domain containing the GIPC protein might be involved in yellowtail sex determination. The homomorphic sex chromosomes widely observed in the Carangidae suggest that this family could be a suitable marine fish model to investigate the early stages of sex chromosome evolution, for which our results provide a good starting point.

Construction of a radiation hybrid panel and the first yellowtail ( Seriola quinqueradiata ) radiation hybrid map using a nanofluidic dynamic array

BackgroundYellowtail (Seriola quinqueradiata) are an economically important species in Japan. However, there are currently no methods for captive breeding and early rearing for yellowtail. Thus, the commercial cultivation of this species is reliant upon the capture of wild immature fish. Given this, there is a need to develop captive breeding techniques to reduce pressure on wild stocks and facilitate the sustainable development of yellowtail aquaculture. We constructed a whole genome radiation hybrid (RH) panel for yellowtail gene mapping and developed a framework physical map using a nanofluidic dynamic array to use SNPs (single nucleotide polymorphisms) in ESTs (expressed sequence tags) for the DNA-assisted breeding of yellowtail.ResultsClonal RH cell lines were obtained after ionizing radiation; specifically, 78, 64, 129, 55, 42, and 53 clones were isolated after treatment with 3,000, 4,000, 5,000, 6,000, 8,000, or 10,000 rads, respectively. A total of 421 hybrid cell lines were obtained by fusion with mouse B78 cells. Ninety-four microsatellite markers used in the genetic linkage map were genotyped using the 421 hybrid cell lines. Based upon marker retention and genome coverage, we selected 93 hybrid cell lines to form an RH panel. Importantly, we performed the first genotyping of yellowtail markers in an RH panel using a nanofluidic dynamic array (Fluidigm, CA, USA). Then, 580 markers containing ESTs and SNPs were mapped in the first yellowtail RH map.ConclusionsWe successfully developed a yellowtail RH panel to facilitate the localization of markers. Using this, a framework RH map was constructed with 580 markers. This high-density physical map will serve as a useful tool for the identification of genes related to important breeding traits using genetic structural information, such as conserved synteny. Moreover, in a comparison of 30 sequences in the RH group 1 (SQ1), yellowtail appeared to be evolutionarily closer to medaka and the green-spotted pufferfish than to zebrafish. We suggest that synteny analysis may be potentially useful as a tool to investigate chromosomal evolution by comparison with model fish.

An Eel Cytochrome P450 1A Gene Having No XRE Sequences in Its 5′ Upstream Region of 1600 bp

Abstract: The gene family cytochrome P450 (CYP) 1A is ubiquitous among animals, and CYP1A1 genes have been identified in teleost and mammals. We isolated another CYP1A gene, which is inducibly expressed by exposure to 3-methylcholanthrene, from the genomic library of the eel (Anguilla japonica). The genomic clone obtained, approximately 17,500 bp in length, contained the structural gene of the CYP1A and a 5′ upstream region of about 1600 bp. Sequence analysis of the 5′ upstream region and the first and second exons revealed that the initiation codon was in the second exon, as in the CYP1A genes reported for mammals and teleosts. CAAT and TATA boxes were found 51 and 29 bp upstream from the transcriptional start site, respectively. Unlike the CYP1A1 genes of eel and other animals, we found no xenobiotic responsive element (XRE) core sequences in the 5′ upstream region studied (1631 bp) or in the first intron, whereas three peculiar regions, each composed of multiple repeats of the trinucleotide TAA, were found between 824 and 1356 bp upstream from the transcriptional start site. Absence of XRE core sequences and presence of the multiple repeats in the 5′ upstream region sequenced may suggest that the expression mechanism of the eel CYP1A gene is somewhat different from the mechanisms of other reported CYP1A genes.

Constructing a 'Chromonome' of Yellowtail (Seriola quinqueradiata) for Comparative Analysis of Chromosomal Rearrangements

To investigate chromosome evolution in fish species, we newly mapped 181 markers that allowed us to construct a yellowtail (Seriola quinqueradiata) radiation hybrid (RH) physical map with 1,713 DNA markers, which was far denser than a previous map, and we anchored the de novo assembled sequences onto the RH physical map. Finally, we mapped a total of 13,977 expressed sequence tags (ESTs) on a genome sequence assembly aligned with the physical map. Using the high-density physical map and anchored genome sequences, we accurately compared the yellowtail genome structure with the genome structures of five model fishes to identify characteristics of the yellowtail genome. Between yellowtail and Japanese medaka (Oryzias latipes), almost all regions of the chromosomes were conserved and some blocks comprising several markers were translocated. Using the genome information of the spotted gar (Lepisosteus oculatus) as a reference, we further documented syntenic relationships and chromosomal rearrangements that occurred during evolution in four other acanthopterygian species (Japanese medaka, zebrafish, spotted green pufferfish and three-spined stickleback). The evolutionary chromosome translocation frequency was 1.5-2-times higher in yellowtail than in medaka, pufferfish, and stickleback.

Identification of Sex-associated SNPs of Greater Amberjack (Seriola dumerili)

The sex determination systems of fish are highly diverse compared with those of mammals. Thus, performing investigations using nonmodel fish species helps to understand the highly diverse sex determination systems of fish. Because greater amberjack (Seriola dumerili) is one of the most important edible fish globally and knowledge of its sex determination system is economically important in the field of aquaculture, we are interested in the mechanisms of sex determination of Seriola species. In this study, we identified sex-associated SNPs of greater amberjack using SNP information of 10 males and 10 females by an association test. We determined that the sex-associated SNPs were on chromosome 12 and mainly covered with two scaffolds (about 7.1 Mbp). Genotypes of sex-associated SNPs indicated that females are the heterogametic sex (ZZ/ZW). Furthermore, we compared the genomic structure of greater amberjack with those of Japanese amberjack (Seriola quinqueradiata), California yellowtail (Seriola dorsalis), and medaka (Oryzias latipes). Whole-genome alignments and synteny analysis indicated that the sex determination system of greater amberjack is markedly different from that of medaka and implied that the sex determination system is conserved in the Seriola species.