Haijin Liu

Production and verification of heterozygous clones in Japanese flounder, Paralichthys olivaceus by microsatellite marker

Mitotic gynogenetic diploids Japanese flounder Paralichthys olivaceus were produced by activating eggs with ultra violet (UV) irradiated sperm of red sea bream ( Pagrus major ), followed by hydrostatic pressure treatment to block the first mitotic division. By crossing two mitotic gynogenetic diploid females with two males, two heterozygous clones of Japanese flounder were produced. Microsatellite marker was used to confirm the genetic status of maternal parents and their progenies. 20 polymorphic microsatellite markers were chosen, covering 16 out of the total 24 linkage groups. The four maternal parents used in this experiment were completely heterozygous, while four mitotic gynogenetic diploids were homozygous for each marker. The genotypes of heterozygous clone progenies were identical and the combination of parental alleles, demonstrates the successful development of cloning. Key words: Japanese flounder, microsatellite, clone, gynogenesis

Gonadal Transcriptome Analysis in Sterile Double Haploid Japanese Flounder

Sterility is a serious problem that can affect all bionts. In teleosts, double haploids (DHs) induced by mitogynogenesis are often sterile. This sterility severely restricts the further application of DHs for production of clones, genetic analysis, and breeding. However, sterile DH individuals are good source materials for investigation of the molecular mechanisms of gonad development, especially for studies into the role of genes that are indispensable for fish reproduction. Here, we used the Illumina sequencing platform to analyze the transcriptome of sterile female DH Japanese flounder in order to identify major genes that cause sterility and to provide a molecular basis for an intensive study of gonadal development in teleosts. Through sequencing, assembly, and annotation, we obtained 52,474 contigs and found that 60.7% of these shared homologies with existing sequences. A total of 1225 differentially expressed unigenes were found, including 492 upregulated and 733 downregulated genes. Gene Ontology and KEGG analyses showed that genes showing significant upregulation, such as CYP11A1, CYP11B2, CYP17, CYP21, HSD3β, bcl2l1, and PRLR, principally correlated with sterol metabolic process, steroid biosynthetic process, and the Jak-stat signaling pathway. The significantly downregulated genes were primarily associated with immune response, antigen processing and presentation, cytokine–cytokine receptor interaction, and protein digestion and absorption. Using a co-expression network analysis, we conducted a comprehensive comparison of gene expression in the gonads of fertile and sterile female DH Japanese flounder. Identification of genes showing significantly different expression will provide further insights into DH reproductive dysfunction and oocyte maturation processes in teleosts.

Constructing a genetic linkage map and mapping quantitative trait loci for skeletal traits in Japanese flounder

A genetic linkage map of Japanese flounder was constructed using 165 doubled haploids (DHs) derived from a single female. A total of 574 genomic microsatellites (type II SSRs) and expressed sequence tag (EST)-derived markers (EST-SSRs) were mapped to 24 linkage groups. The length of linkage map was estimated as 1270.9 centiMorgans (cM), with an average distance between markers of 2.2 cM. The EST-SSRs were used together with type II SSR markers to construct the Japanese flounder genetic linkage map which will facilitate identify quantitative trait locus (QTL) controlling important economic traits in Japanese flounder. Thus, twelve skeletal traits at 2 years of age were measured for all DHs. Forty-one QTLs were detected on 14 linkage groups and totally account for a small proportion of phenotypic variation (4.5 to 17.3%). Most of QTLs detected distribute on linkage groups 5 (9 QTLs), 8 (9 QTLs), 9 (5 QTLs) and 20 (4 QTLs), in which, some QTLs perform the pleiotropy.

Production and verification of a 2 nd generation clonal group of Japanese flounder, Paralichthys olivaceus

Clonal fishes are useful tools in biology and aquaculture studies due to their isogenicity. In Japanese flounder (Paralichthys olivaceus), a group of homozygous clones was created by inducing meiogynogenesis in eggs from a mitogynogenetic homozygous diploid. As the clones reached sexual maturity, meiogynogenesis was again induced in order to produce a 2nd generation clonal group of Japanese flounder. After 3 months, there were 611 healthy, surviving individuals. Twenty-four microsatellite markers, that covered all the linkage groups of Japanese flounder, were used to identify the homozygosity of the 2nd generation clones; no heterozygous locus was detected. This indicates that the production of a 2nd generation clonal group of Japanese flounder was successful. Restriction-site DNA associated sequencing at the genomic level also confirmed the homozygosity and clonality of the 2nd generation clonal group. Furthermore, these 2nd generation clones had a small coefficient of variation for body shape indices at 210 days of age and showed a high degree of similarity in body characteristics among individuals. The successful production of 2nd generation clones has laid the foundation for the large-scale production of clonal Japanese flounder.

Choice of microsatellite markers for identifying homozygosity of mitotic gynogenetic diploids in Japanese flounder Paralichthys olivaceus.

A set of 72 microsatellite markers distributed evenly among 24 linkage groups were selected from the published genetic linkage maps of Japanese flounder Paralichthys olivaceus. In two normal diploid full-sib families, the test for Mendelian inheritance showed that genotypic segregation deviations were not significant at all analysed loci. To estimate microsatellite-centromere map distances, four meiotic gynogenetic diploid lines were produced by the activation of eggs using UV irradiated sperm of red seabream Pagrus major and cold-shock treatment to block the extrusion of the second polar body. Under the assumption of complete interference, 21 markers were located in the centromeric region, 39 in the telomeric region and the rest in the intermediate region of linkage groups. A total of 192 mitotic gynogenetic diploids from one spawn were identified by these markers. Genotype analysis showed that the number of homozygous individuals decreased as microsatellite-centromere map distance increased on each linkage group.

The complete mitochondrial genome sequence of Takifugu flavidus (Tetraodontiformes: Tetrodontidae)

Abstract The complete mitochondrial genome of Takifugu flavidus (Tetraodontiformes: Tetrodontidae) was obtained in this study. The mitogenome is 16,449 bp in size and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 non-coding regions: origin of light-strand replication (OL) and control region (D-loop). The overall nucleotide composition of the heavy strand was 29.88% A, 25.81% T, 15.28% G and 29.03% C, with a slight AT bias of 55.69%. Except for ND6 gene and eight tRNA genes, other genes are encoded on the heavy strand. The mitochondrial genome data of T. flavidus should contribute to phylogenetic analysis and studies on genetic structure, as well as molecular phylogeny and species identification of Tetrodontidae.

A species-specific primer pair for distinguishing between Paramisgurnus dabryanus and Misgurnus anguillicaudatus based on mitochondrial DNA polymorphisms

Abstract Paramisgurnus dabryanus and Misgurnus anguillicaudatus (family Cobitidae) are loaches with high morphological similarity. In this study, we designed primers to distinguish between Paramisgurnus dabryanus and Misgurnus anguillicaudatus based on the length differences in the mitochondrial COXII to tRNALys gene region. Samples of P. dabryanus and M. anguillicaudatus from different geographical locations were collected and amplified to verify primer specificity. The results of electrophoresis revealed the successful amplification of all P. dabryanus and M. anguillicaudatus DNA samples, which had distinct, specific-specific sizes (214 bp for P. dabryanus and 285 bp for M. anguillicaudatus). In conclusion, the new primers provide fast, reliable, and accurate identification between P. dabryanus and M. anguillicaudatus.

Cytological studies on induced mitogynogenesis in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The effect of hydrostatic pressure treatment on the induction of mitogynogenesis in the eggs of Japanese flounder Paralichthys olivaceus (Temminck et Schlegel) by using heterospecific sperm were studied. Before treatment, the eggs were at metaphase of the first mitosis. The spindle was disassembled by the treatment and then resembled in its pretreatment position, and the chromosomes were rearranged, i.e., the first mitosis was not blocked. During the second mitotic cycle, only a monopolar spindle was assembled in each blastomere and the chromosomes doubled, but cell cleavage was blocked. In the third cycle, mitosis proceeded normally with a bipolar spindle in each blastomere. Flow cytometric analysis of ploidy demonstrated that mitogynogenetic larvae were all diploid. The ultraviolet light-irradiated sperm of the red sea bream (Pagrus major) was condensed, formed a dense chromatin body, and randomly entered one blastomere.

Analysis of genetic diversity in Bohai natural population of Paralichthys olivaceus

To analyze the genetic diversity of natural population and select the specific microsatellite markers for identifying effectively genetic characteristics of population in Japanese flounder( Paralichthys olivaceus)in this study,seventy-four w ild individuals captured from the Bohai Sea w ere used to form the experimental population. A set of 72 microsatellite markers located on different regions in linkage group w ere chosen to carry out the genetic analysis. In these markers,17 w ere located in the centromeric region,36 in the distant region from the centromere and 19 in the intermediate region of linkage groups. Analysis show ed that the number of alleles( A) ranged from 6. 400 to 7. 389,number of effective alleles( N e) ranged from 4. 469 to5. 129 and Shannons information index( I) ranged from 1. 565 to 1. 683 in different regions of linkage group. The observed heterozygosity( H o),unbiased expected heterozygosity( H e) and polymorphism information content( PIC) w as from 0. 568 to 0. 593,from 0. 738 to 0. 753 and from 0. 707 to 0. 746,respectively. The Hardy-Weinberg departure value( d) w as low er than- 0. 200,w hile,genetic distance( G D)among individuals in population w as above than 0. 620. Each genetic parameter demonstrated that there w as richer genetic diversity in experimental population and relatively farther genetic distance among individuals.This population w as suitable for conducting selective breeding as founder population. Significant difference did not exist in the genetic parameters obtained by microsatellite markers located on different regions in linkage group,w hich verified that there w as no necessary correlation betw een the positions of marker located and the degree of genetic diversity of markers. How ever,these polymorphic markers could be used as marker candidates to analyze the genetic structure in population of Japanese flounder.

Identifying genetic characteristics of different diploids in Japanese flounder based on M-C mapping

To analyze the effects of microsatellite markers located in different positions in linkage group on identifying genetic characteristics of different diploids in Japanese flounder in this study,the mature spawners selected from Japanese flounder selection and breeding flounder were used to produce the normal diploids(ND),meiotic gynogenetic diploids(MGD-1),successive two generation meiotic gynogenetic diploids(MGD-2)and inbred diploid(MGD1H)by crossing mature female from MGD-1 with induced sex reversed male.A set of 72 microsatellite markers distributed evenly in 24 linkage groups were selected from the published genetic linkage maps of Japanese flounder.To estimate microsatellite-centromere map distances,four MGD-1 lines were produced.Under the assumption of complete interference,17 markers were located in the centromeric region,36 in the distant region from the centromere and 19 in the intermediate region of linkage groups.The genetic characteristics of four types of diploid in Japanese flounder were identified by these markers.Analysis showed that the number of allele(A)and polymorphism information content(PIC)have limited variation range,in which the A and PIC of ND were the highest while those of MGD1H were the lowest.In four types of diploid,the observed heterozygosity decreased and homozygosity increased as microsatellite-centromere map distance increased.The highest and lowest percentages of homozygous individuals were found in centromeric region from 8.8% to 29.1% and in telomeric region from 2.4% to 23.2%,respectively.The variation ranges of MGD-1and MGD-2 were significantly higher than that of the other diploids.The results demonstrated that choice of microsatellite markers located in different positions in linkage group has great impact on identifying genetic characteristics of different diploids in Japanese flounder.