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     

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。     

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.     

Isolation and characterization of new microsatellite markers from the Japanese flounder (Paralichthys olivaceus)

The isolation and characterization of eight polymorphic microsatellite loci from a Japanese flounder partial genomic library are reported. The eight markers isolated in this study were highly polymorphic and their positions on the linkage genome map of the Japanese flounder were determined. Therefore, they are useful for ecological studies of wild populations. These markers are more effective than other markers with no information of chromosomal locations.     

Analysis of Microsatellite DNA Polymorphisms in Rockfish Sebastes thompsoni and Application to Population Genetics Studies

Population differentiation and relationships among 6 natural rockfish populations collected from northern coastal seas around Japan were assayed using microsatellite DNA loci. Seven loci examined were polymorphic in all populations. The number of alleles per locus ranged from 6.7 to 9.3, and the average of observed and expected heterozygosity ranged from 0.63 to 0.68, and from 0.66 to 0.69, respectively. The observed genotype frequencies at each locus were almost in agreement with Hardy-Weinberg expectations with two exceptions (P < .05). The allele frequencies of 16 population pairs were significantly different (P < .05). Genetic distance (D _A ) between 6 populations ranged from 0.03 to 0.08. According to a neighbor-joining tree generated from the D _A values, the 6 populations fell into 3 clusters. These clusters were correlated with the geographical positions of each population; larval dispersions due to water current were also found to have an effect on these results. Received May 2, 2000; accepted July 17, 2000.     

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     

微卫星评价牙鲆雌核发育二倍体纯合性

采用8个微卫星座位分别对牙鲆减数雌核发育二倍体家系和卵裂雌核发育二倍体家系的纯合性进行检验。卵裂雌核发育二倍体在所有检测座位全部纯合。减数雌核发育二倍体在部分座位发生纯合,但未发现在所有座位全部纯合的个体,在Poli9TUF、Poli9-8TUF、Poli11TUF、Poli13TUF、Poli23TUF、Poli30TUF、Poli123TUF和Poli130TUF座位,杂合子比例分别为1·0000、1·0000、0·1944、0·9459、0·8611、1·0000、0·7778和0·8000,平均杂合子比例为0·8224。由此表明,牙鲆除了Poli11TUF外,在其余7个座位均具有很高的重组率。研究结果显示,牙鲆卵裂雌核发育二倍体一代即可形成纯合子;而减数雌核发育二倍体由于具有较高的重组率,使其与母本的遗传同质性较高。     

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.     

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.     

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.     

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]。     

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.     

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.     

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.     

海水中镉对牙鲆的生物有效性研究

采用暴露实验方法,研究了海水中镉在牙鲆内脏、肌肉、鳃组织内蓄积及在清洁海水中排放规律,以及海水中总有机碳（TOC）浓度对镉吸收的影响.结果表明,镉暴露浓度为0.5 mg·L^-1时,牙鲆各组织镉蓄积量随暴露时间增加而增大,第10天达吸收平衡,Cd蓄积量顺序为内脏（147.73 mg·kg^-1）>鳃（15.55 mg·kg^-1）>肌肉（4.578 mg·kg^-1）,表明内脏是重金属镉蓄积的主要部位.染毒13 d后,将牙鲆移入清洁海水中,结果表明,随排放时间增加,各组织镉蓄积量明显下降,排放至第15天时各组织镉排出率为内脏团（80.66%）>鳃（73.66%）>肌肉（56.84%）,表明受镉污染牙鲆经清洁海水排放后,有望达到食用标准.海水中TOC浓度对镉蓄积量有明显影响.随着TOC浓度升高,各组织内镉蓄积量明显下降,表明海水TOC能降低镉的生物有效性.     

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。     

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.     

牙鲆出血病病原的初步研究

从患出血病的牙鲆 (Paralichthysolivaceus)的肝、脾、肾组织中分离到 3个菌株。经人工感染证实PHK致病性强 ,为病原菌 ,经鉴定为沙鱼弧菌 (Vibriocarchariae)。药敏试验结果显示 ,该菌株对红霉素、庆大霉素、先锋噻肟、菌必治、链霉素、氟嗪酸、多粘菌素B、头孢西丁、环丙沙星、头孢克肟、先锋必敏感 ;对萘啶酸、头孢噻吩、氯霉素、四环素、先锋Ⅴ、痢特灵、青霉素G、先锋孟多、氨苄青霉素、强力霉素等不敏感。