Isolation and characterization of polymorphic microsatellite loci in the olive flounder (Paralichthys olivaceus)

We described the isolation and characterization of 27 new microsatellite loci from olive flounder, Paralichthys olivaceus. All loci were found to be polymorphic, and had between five and 22 alleles with observed heterozygosity ranging from 0.161 to 1.0 in 31 individuals examined. These micorsatellite makers are likely to be useful for studies of genome mapping, mating systems and population genetics in this species.     

Population structure of the olive flounder (Paralichthys olivaceus) in Korea inferred from microsatellite marker analysis

The population structure of olive flounder Paralichthys olivaceus was estimated using nine polymorphic microsatellite (MS) loci in 459 individuals collected from eight populations, including five wild and three hatchery populations in Korea. Genetic variation in hatchery (mean number of alleles per locus, A = 10·2–12·1; allelic richness, A_R = 9·3–10·1; observed heterozygosity, H_O = 0·766–0·805) and wild (mean number of alleles per locus, A = 11·8–19·6; allelic richness, A_R = 10·9–16·1; observed heterozygosity, H_O = 0·820–0·888) samples did not differ significantly, suggesting a sufficient level of genetic variation in these well‐managed hatchery populations, which have not lost a substantial amount of genetic diversity. Neighbour‐joining tree and principal component analyses showed that genetic separation between eastern and pooled western and southern wild populations in Korea was probably influenced by restricted gene flow between regional populations due to the barrier effects of sea currents. The pooled western and southern populations are genetically close, perhaps because larval dispersal may depend on warm currents. One wild population (sample from Wando) was genetically divergent from the main distribution, but it was genetically close to hatchery populations, indicating that the genetic composition of the studied populations may be affected by hydrographic conditions and the release of fish stocks. The estimated genetic population structure and potential applications of MS markers may aid in the proper management of P. olivaceus populations.     

Development of 52 new polymorphic microsatellite markers for the olive flounder, Paralichthys olivaceus

We characterized 52 new microsatellite markers isolated from (GT)(n) and (CT)(n) microsatellite-enriched genomic libraries of the olive flounder (Paralichthys olivaceus). All markers were polymorphic, with eight to 30 (mean 15.1) alleles detected in 30 individuals from a single natural population. Observed heterozygosity ranged from 0.20 to 1.0. Segregation analysis within a mapping family revealed non-amplifying null alleles at six loci. These results indicate that these new microsatellite markers will be useful for population genetic, parentage, and genome mapping studies.     

A set of polymorphic trinucleotide and tetranucleotide microsatellite markers for the Japanese flounder (Paralichthys olivaceus)

We have developed the first set of trinucleotide and tetranucleotide markers for the Japanese flounder, Paralichthys olivaceus. One hundred and sixty-seven polymorphic trinucleotide and tetranucleotide microsatellites were isolated using clones derived from two libraries. Of almost 200,000 clones analysed, 0.5% presented trinucleotide or tetranucleotide repeat regions. Among the trinucleotide repeats analysed in this study, the most frequent one was (CAG)(n) and the most common tetranucleotide repeat was (GATA)(n). The position of the new markers in the genetic linkage map was determined. Markers were evenly distributed along the P. olivaceus linkage groups, without distinction between the kinds of repeats and library of origin. The markers isolated in this study contribute significantly to the genetic linkage map of the Japanese flounder.     

Five new microsatellite loci for Oliver flounder (Paralichthys olivaceus) from an expressed sequence tag (EST) library and cross‐species amplification

Five microsatellite loci have been isolated and characterized from a cDNA library of Oliver flounder, Paralichthys olivaceus. All loci were found to be polymorphic and had between four and 10 alleles. Observed and expected heterozygosities varied from 0.70 to 0.90 and from 0.52 to 0.80, respectively. Five additional fish species assessed for cross‐species amplification revealed between zero and three positive amplifications and between zero and two polymorphic loci per species.     

Application of Microsatellite Markers to Population Genetics Studies of Japanese Flounder Paralichthys olivaceus

We examined population genetic structure by means of microsatellite analysis among 7 Japanese flounder (Paralichthys olivaceus) populations collected from coastal sea areas around Japan. As was expected, all of the 11 microsatellite loci examined were variable in all populations (number of alleles per locus, 15.2–18.2; average of expected heterozygosity, 0.74–0.78). Eleven population pairs in 21 possible pairwise comparisons showed significant genetic heterogeneity associated with allele frequency distributions or fixation index (F _ST ). Modified Cavalli-Sforza chord distance (D _A ) and Nei's standard genetic distance (D _ST ) ranged from 0.051 to 0.090, and from 0.000 to 0.025, respectively. There was evidence that the populations assessed in this study were not drawn from a single panmictic population; however, it appears that Japanese flounder populations around Japan are not well-structured, as an estimate of the fixation index value among the 7 localities was very low (F _ST = 0.0025). Received April 27, 2001; accepted June 27, 2001.     

微卫星标记对牙鲆有丝分裂雌核发育家系的亲子鉴定

利用18个微卫星标记,对6个家系的26尾有丝分裂雌核发育牙鲆进行亲子鉴定,PCR扩增产物经8%非变性聚丙烯酰胺凝胶电泳检测,结果表明:1个座位在母本中表现为相同的基因型,视为单态座位,其他17个座位为多态;多态座位在亲子鉴定中的累计排除概率和累计个体识别概率分别为0.9985、0.9999;根据被测个体在17个微卫星座位的基因型,最后确认26尾子代的母本,其中7尾子代在某些座位表现出与其母本不完全匹配的基因型。利用微卫星标记可确定雌核发育后代的亲子关系,从而构建牙鲆雌核发育家系系谱,对牙鲆雌核发育的深入研究具有重要意义。     

Inheritance Characteristics of Microsatellite DNA Loci in Experimental Families of Japanese Flounder Paralichthys olivaceus

Allele segregating patterns of microsatellite DNA loci in 5 experimental families of Japanese flounder Paralichthys olivaceus and genotype frequencies for deviation from Hardy-Weinberg expectations (HWE) in natural P. olivaceus populations were studied to assess inheritability. Of the 12 microsatellite loci examined, 1 locus had a possibility of scoring errors of heterozygous individuals caused by unreproducible polymerase chain reaction amplifications of a particular allele. At the remaining 11 loci, almost all of alleles were segregated according to Mendelian transmission, and observed genotype frequencies in natural populations were consistent with HWE. The results demonstrated here would provide useful information supporting the suitability of these microsatellite loci as inheritable P. olivaceus genetic markers. Received September 25, 2000; accepted March 1, 2001     

日本牙鲆主要组织相容性复合体DAB等位基因的多态性

根据牙鲆(Paralichthys olivaceus)主要组织相容性复合体(MHC)DAB基因序列设计特异性引物,在日本牙鲆基因组中扩增了包括DAB基因完整外显子2和内含子1在内的长度为408 bp的DNA片段.对该片段克隆测序后,发现了37个MHC-DAB等位基因,各等位基因的频次以及各主型中亚型数目分布都不平衡.在第二外显子273 bp核苷酸序列中有50个位点发生变异,核苷酸多样性PI值为0.0618,在编码的氨基酸序列中存在多态变异位点30个,其中简约信息位点29个,单变异位点1个.非同义替代与同义替代的比率在抗原结合区(PBR)和非PBR编码区分别为10.0和1.62,分析表明正向选择机制可能是产生牙鲆MHC-DAB多态性的主要因素.估计的核苷酸的转换和颠换数随着遗传距离的增加而增加.各等位基因间的系统发生关系表明,19个主型分为两个群.对氨基酸组分和密码子的偏倚性以及分布频率的分析表明各同义密码子的使用频率不均衡.本研究为牙鲆MHC-DAB基因的遗传进化和分子标记辅助育种的研究提供了理论基础.     

牙鲆一株弹状病毒病原的分离与鉴定

从患病牙鲆中分离鉴定了一株弹状病毒（Paralichthys olivaceus rhabdovirus,PoRV）。用过滤除菌后的患病牙鲆组织匀浆液,接种不同的鱼类细胞,其中有7种鱼类细胞出现明显的病变在对病毒进行挑斑分离后,测定了PoRV的滴度,显示PoRV在敏感鱼类细胞（Grass Carp Ovary,GCO）中的滴度达到106.5TCID50/mL;绘制了PoRV生长曲线;经蔗糖密度梯度离心提纯PoRV,负染及宿主细胞超薄切片的电镜观察,显示PoRV大小约为60nm×200nm。测定了PoRV的理化性质,显示该病毒对有机溶剂和温度敏感,但对DNA抑制剂阿糖胞苷（Ara-c）不敏感。经SDS-PAGE电泳,对PoRV的蛋白图谱进行了分析,表明该病毒有5种主要蛋白带,其分子量大小分别约为：250kD、67kD、44kD、30kD、23kD。     

Sequences of 596 cDNA Clones (565,977 bp) of Japanese Flounder (Paralichthys olivaceus) Leukocytes Infected with Hirame Rhabdovirus

We have partially sequenced 785 sequences of 596 independent complementary DNA clones isolated from a cDNA library of Japanese flounder leukocytes infected with hirame rhabdovirus. These sequences consist of a total of 565,977 base pairs. The average size of the sequenced lengths was 721 bp. Of 596 clones, 386 (64.8%) were identified as previously reported genes by the BLASTN and BLASTX programs. About 30% of the identified clones could be recognized by only the BLASTX program. A total of 251 distinct genes were identified, and 181 of these genes are the first such genes reported from the teleostei. Approximately 27% of the identified Japanese flounder genes appear to be associated with cell division, cell structure or motility, and basic energy metabolism, 29% with gene or protein expression, and 17% with cell signaling, cell communication, and cell or organism defense. The most frequently identified expressed sequence tags of leukocytes of Japanese flounder were gelatinase b and ribosomal protein L23, which both had 1.34% prevalence. Received February 12, 1999; accepted April 13, 1999     

Comparison of genetic diversity at microsatellite loci and quantitative traits in hatchery populations of Japanese flounder Paralichthys olivaceus

Relationships of genetic diversity at microsatellite loci and quantitative traits were examined in hatchery-produced populations of Japanese flounder using a relatively straightforward experiment. Five hatchery populations produced by wild-caught and domesticated broodstocks were used to examine the effects of different levels (one to three generations) of domestication on the genetic characteristics of hatchery populations. Allelic richness at seven microsatellite loci in all hatchery populations was lower than that in a wild population. Genetic variation measured by allelic richness and heterozygosity tended to decrease with an increase in generations of domestication. In addition, the degree of genetic differentiation from a wild population increased with an increase in generations of domestication. Significant differences in three morphometric traits (dorsal and anal fin ray counts and vertebral counts) and three physiological traits (high temperature, salinity and formalin tolerance) were observed among the hatchery populations. The degree of phenotypic difference among populations was larger in morphometric traits than in physiological traits. The divergence pattern of some quantitative traits was similar to that observed at microsatellite loci, suggesting that domestication causes the decrease of genetic variation and the increase of genetic differentiation for some quantitative traits concomitantly with those for microsatellite loci. Significant positive correlation was observed between F _ST and the degree of phenotypic difference in the three morphometric traits and formalin tolerance, indicating that genetic variation at microsatellite loci predicts the degree of phenotypic divergence in some quantitative traits.     

牙鲆变态过程中的细胞凋亡

利用整体的原位TUNEL方法检测了牙鲆(Paralichthysolivaceus)变态过程中身体各器官细胞凋亡的分布及变化情况。结果如下:(1)与眼睛移动相关的脑颅骨骼的细胞凋亡右侧眼睛移动开始之后,在额骨、中筛软骨和犁骨软骨中出现细胞凋亡,并保持到眼睛移动结束;(2)中枢神经和感觉器官的细胞凋亡在眼睛移动开始之前,脊髓和脊髓鞘出现细胞凋亡,在眼睛移动开始之后,脊髓和脊髓鞘细胞凋亡停止,而在脑、眼睛和内耳出现细胞凋亡,并一直持续到眼睛移动结束;(3)与游泳、捕食和消化等功能相关的器官的细胞凋亡在眼睛移动开始后,冠状幼鳍的基部出现凋亡;在变态中后期,尾鳍基部出现细胞凋亡;下颌骨、鳃弓以及肝脏在眼睛移动开始之后,出现细胞凋亡,也一直持续到眼睛移动结束。细胞凋亡通过有序地去除多余的细胞来参与器官形态建立和重组,本研究的结果表明,在牙鲆器官功能变化过程中,细胞凋亡在与其相适应的的器官形态重塑中起着重要作用[动物学报52(2):355-361,2006]。     

Genomic Bacterial Artificial Chromosome Library of the Japanese Flounder Paralichthys olivaceus

We have constructed a genomic bacterial artificial chromosome (BAC) library from homozygous cloned Japanese flounder Paralichthys olivaceus using the pBAC-lac vector. This BAC library consists of about 49,100 clones and is deposited in 128 microtiter plates with 384 wells. The average size of inserted DNA was calculated to be 165 kb. The BAC library was determined to cover 9 times the Japanese flounder haploid genome. The Japanese flounder genomic BAC library will be useful for gene isolation as well as quantitative trait loci (QTL) analysis. Received March 1, 2000; accepted May 29, 2000.     

Cloning and characterization of phospholipase D from olive flounder (Paralichthys olivaceus)

The phospholipase D1 (PLD1) cDNA, designated PoPLD, encoding a predicted protein of 1053 amino acids in olive flounder (Paralichthys olivaceus) has been cloned. The deduced amino acid sequence shares high identity with that of PLD1s and PLD2 in human, rat and mouse. The phylogenic analysis and sequence comparison of PoPLD with other PLD isozymes were found to be closely related to the PLD1 isozyme in primary structure. The tissue expression analysis of PoPLD showed that the mRNA of PoPLD was predominantly expressed in the brain, gullet, muscle, stomach, head kidney, pyloric caeca, intestine and gill. The expression of the PoPLD gene was examined in various tissues of flounder by RT-PCR following stimulation with LPS and compared also with that of the inflammatory cytokines IL-1beta and IL-8 in various tissues of the stimulated flounder. This provides indirect evidence that PLD1 might have a relevant role in immune responses against pathogens and in inflammation. In addition, the recombinant protein of PoPLD (GFP-PoPLD), which demonstrated a phosphatidylcholine (PC)-hydrolyzing activity, was partially localized as a distinct ring-shaped form surrounding the rim of the nucleus in EPC cells. Together, our results suggest that PoPLD is similar to the mammalian PLD1 isoform, is generally widespread within olive flounder tissue, might have a relevant role in the fish immune system against pathogens and specifically may be localized in the subcellular membranes of the nuclear rim in EPC cells.     

Molecular characterization and gene expression of a CXC chemokine gene from Japanese flounder Paralichthys olivaceus

Chemokines are small, secreted cytokine peptides that have the ability to recruit a wide range of immune cells to sites of infection and disease. A novel CXC chemokine was obtained from Japanese flounder Paralichthys olivaceus. This chemokine cDNA contains an open reading frame of 333 nucleotides encoding 111 amino acid residues containing four conserved cysteine residues. The gene is composed of four exons and three introns as are those of mammalian and fish CXC chemokines. Results of homology and phylogenetic analysis revealed that the Japanese flounder CXC chemokine is closest to CXCL13 subgroup. The gene was expressed in immune-related organs, including head kidney, trunk kidney, spleen and peripheral blood leukocytes (PBLs). Japanese flounder CXC chemokine gene expression was observed at 3 and 6h after induction by LPS, but not at 3 and 6h after induction by poly I:C. These results suggest that the Japanese flounder CXC chemokine is probably associated with inflammatory as well as homeostatic functions.     

牙鲆胚胎冷冻损伤的观察

为了观察牙鲆胚胎在冷冻保存过程中的形态受损情况,将其浸入20%PM（20%丙二醇和20%甲醇1∶1的混合液）中平衡,并用程序化法和玻璃化法对其冷冻保存2h后解冻,用摄影显微镜记录其在抗冻剂里平衡时和冷冻保存后的形态。结果显示在平衡过程中胚胎卵膜出现凹陷（称为溶液损伤）,但可以恢复;在冷冻过程出现胞内冰损伤,是致命的;用程序化法冷冻保存的尾芽期胚胎卵膜和卵黄完好,胚体边缘受伤;用玻璃化法保存的尾芽期胚胎,卵膜和卵黄损伤较重,胚体损伤较轻;因此将玻璃化法和程序化法结合可以达到扬长避短的效果。     

几种因素对牙鲆胚胎玻璃化冷冻保存的影响

鱼类胚胎冷冻保存技术还远没有成熟, 为了寻找最佳的鱼类胚胎玻璃化冷冻保存条件, 我们以牙鲆(Paralichthys olivaceus) 胚胎为例, 研究了影响鱼类胚胎玻璃化冷冻保存的几个主要因子: 玻璃化液、麦管直径、胚胎阶段、平衡时间及平衡温度、洗脱浓度和洗脱时间。发现: (1) 含有多种抗冻剂的玻璃化液PMDD(2% PVP), 玻璃化稳定, 脱玻璃化率较低, 适宜进行玻璃化冷冻; (2) 尾芽期胚胎较其他时期耐受力强, 平衡40 min就足以使玻璃化液渗透完全, 时间延长, 成活率显著降低, 各个时期的胚胎对温度都比较敏感, 0°C与4°C下平衡的成活率显著高于15°C; (3) 洗脱浓度和洗脱时间对胚胎成活率影响不大; (4) 根据优化的条件, 对牙鲆两个时期的胚胎进行超低温冷冻保存实验, 共成活4次, 获得成活胚胎8粒, 其中7粒孵化出健康的鱼苗。本文为鱼类胚胎冷冻保存技术的建立提供基础资料, 并显示了牙鲆胚胎玻璃化冷冻保存是可行的。     

Phenotypic characteristics of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus)

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis , respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges–Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.