Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus

<p>Abstract</p> <p>Background</p> <p>Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (<it>Marsupenaeus japonicus</it>), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome.</p> <p>Results</p> <p>Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of <it>Daphnia </it>and <it>Drosophila </it>suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences.</p> <p>Conclusions</p> <p>Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our result suggested that it is difficult to analyze the shrimp genome following normal analytical methodology. Hence, it is necessary to avoid repetitive sequence (such as segmental duplications) when studying the other unique structures in the shrimp genome.</p

Marker-assisted breeding for viral disease resistance in Japanese flounder (Paralichthys olivaceus)

DNA marker technologies can be used for genetic improvement through selection of favorable traits such as disease resistance. These traits are generally modeled as being controlled by many genes of small additive effects, known as quantitative trait loci (QTL). Construction of a genetic linkage map based on DNA markers at a large number of sites in the fish genome is necessary to identify QTL controlling traits of disease resistance. By identifying markers associated with high per- formance QTL in different strains or species, it may be possible to improve the performance of such traits in other strains through intro- gression of the desired QTL. One of the goals of selective breeding programs is to integrate genetic marker information from pedigreed brood stock into successful management and culture. Such an approach, termed marker- assisted selection (MAS) and/or marker- assisted gene introgression (MAI), is expect- ed to increase genetic response by affecting efficiency and accuracy of selection

Table1_Y-specific amh allele, amhy, is the master sex-determining gene in Japanese flounder Paralichthys olivaceus.DOCX

Japanese flounder (Paralichthys olivaceus) is an important marine fish species of both fisheries and aquaculture in Northeast Asia. The commercial interest for all-female progenies due to several sex-related traits has prompted basic research on the mechanisms of sex determination in this species. By conducting a linkage analysis of the sex-determining locus, we initially identified 12 microsatellite markers linked to sex in 11 scaffolds, whose localization was restricted to a specific region of linkage group 9. Sequence analysis of this region identified 181 genes based on the UniProt database annotations. Among them, the amh gene was considered a potential candidate for sex determination because this gene is known to have taken over the role of sex determination in many teleosts. An in-depth sequence analysis of both the coding and non-coding regions of amh in XX and XY individuals detected nine SNPs linked with maleness. However, because these substitutions were synonymous, the upstream and downstream regions of amh were also investigated and a male-specific variant with deletions in the promoter region was detected. This truncated Y-specific amh variant was named amhy, and the amh shared by both sexes was named amhx. The association analysis using both females and males of the genotypic sex inferred by the presence/absence of amhy found complete association with phenotypic sex and genotype. Gene expression analysis in larvae derived from a single-pair progeny by quantitative real-time PCR detected amhy transcripts in the larval trunks between 20 and 100 days after hatching only in XY larvae. Localization of amhy by in situ hybridization was detected in presumptive Sertoli cells of XY gonads. Expression of amhx was almost undetectable in both XX and XY genotypes. Loss of Amh function by CRISPR-Cas9 induced male-to-female sex reversal, indicating that this gene was necessary for the masculinization of XY individuals. In conclusion, the complete linkage of amhy with males, its early expression in XY gonads before testicular differentiation, and the induction of sex reversal by loss-of-function mutation support the view that amhy is the sex-determining gene in this species.</p

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

<div><p>Benedenia infections caused by the monogenean fluke ectoparasite <i>Benedenia seriolae</i> 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 (<i>Seriola quinqueradiata</i>), 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 (<i>BDR-1</i>) and Squ20 (<i>BDR-2</i>) 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 <i>B. seriolae</i> 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.</p></div

Localization of significant markers for Benedenia disease resistance in linkage group Squ2F and Squ20F with family A.

<p>Squ(linkage group)F; marker distance in female map. (A) Squ2F, (B) Squ20F. Map positions and LOD scores are based on a simple interval mapping QTL analysis using the software MapQTL 5. Marker absolute map distances are given in (cM). 95% confidence probability LOD support interval was indicated as Gray bold line. Horizontal lines across each plot indicate LOD siginificance threshold, <i>P_g</i>; genome-wide significance threshold.</p

Significant markers for Benedenia disease resistance multiple-QTL model mapping in linkage group Squ2F and Squ20F with family A.

<p>Squ(linkage group)F; marker distance in female map. (A) Squ2F, (B) Squ20F. This figure was described using by MapQTL 5.</p

Multiple QTL model mapping results of the significant markers for Benedenia disease resistance in linkage group 2 and 20 in family A.

<p>Locus; marker name, LOD; Lod scores, % Var; percent of variance explained, Effect; estimated effect. Squ(linkage group)F; F is dam allele in female linkage group. Values in bold are LOD max in peak of each QTL marker position and each value.</p