Genetic diversity in two Japanese flounder populations from China seas inferred using microsatellite markers and COI sequences

Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea (Qingdao population, QD) and East China Sea (Zhoushan population, ZS) using 10 polymorphic microsatellite loci and cytochrome c oxidase subunit I (COI) sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data (F_st =0.048 7, P<0.001) and mitochondrial DNA data (F_st =0.128, P<0.001) both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.     

黄姑鱼(Nibea albiflora)雌核发育的人工诱导及鉴定

采用紫外线(UV)灭活的同源精子通过冷休克抑制第二极体排放诱导黄姑鱼(Nibea albiflora)的雌核发育。结果表明,黄姑鱼精子最适的灭活剂量为420mJ/cm2,紫外照射剂量与黄姑鱼精子和卵子授精后的受精率及孵化率之间表现出明显的Hertwig效应。多次试验筛选的黄姑鱼雌核发育的最适条件为授精后2min于3℃处理8min。雌核发育仔鱼经形态学和流式细胞仪鉴定为二倍体。进一步利用10对微卫星分析了遗传物质在亲本和子代中的传递情况,结果表明雌核发育后代的等位基因完全来自于母本,没有父本基因参与;雌核发育后代在5个微卫星位点具有较高的重组率,使其与母本保持了高度同质性。     

白消安及其与高温联合处理对黄姑鱼(Nibea albiflora)性腺发育的影响

黄姑鱼（Nibea albiflora）的性成熟周期短,养殖技术成熟,是合适的生殖细胞移植受体。不育受体的制备在鱼类生殖细胞移植中非常关键。为了优化不育黄姑鱼的制备方法,采用白消安和白消安-高温（30℃）对黄姑鱼进行处理,设置5个组：对照组（Con组）、白消安10 mg/kg组（Bu10组）、白消安40 mg/kg组（Bu40组）、白消安10 mg/kg高温组（Bu10H组）、白消安40 mg/kg高温组（Bu40H组）,白消安以不同剂量进行2次的腹腔注射,间隔两周。采用组织学、qPCR以及原位杂交等方法分析白消安及其与高温联合处理对黄姑鱼性腺及其生殖细胞和体细胞发育的影响。研究结果表明白消安处理对黄姑鱼生殖细胞的影响具有明显的性别差异：雄鱼在处理28 d后其性腺重和GSI较对照组显著下降,进一步组织学观察发现雄鱼精巢在处理14 d时已经出现空腔,qPCR分析发现雄鱼vasa基因表达在处理期间显著降低,且在处理第48 d仍处于下调状态。然而,白消安及其与高温联合处理并未对雌鱼生殖细胞产生明显的影响。研究还发现高温处理组出现较高的死亡率,但雄鱼生殖细胞的消除效果并未显著增强。因此,白消安处理能够消除黄姑鱼雄鱼性腺内的生殖细胞,以40 mg/kg白消安2次（间隔两周）注射后消除效果最好,能够作为生殖细胞移植受体鱼。     

半滑舌鳎和塞内加尔鳎养殖群体遗传变异的RAPD分析

采用RAPD方法对半滑舌鳎和塞内加尔鳎的养殖群体进行遗传变异分析。从78条随机引物中筛选出20条用于群体遗传学分析,2个群体共扩增出260条DNA片段,其中半滑舌鳎187条,塞内加尔鳎180条,片段大小为200-2 000 bp。半滑舌鳎和塞内加尔鳎养殖群体的多态片段比例分别为63.10%和60.56%,Nei的多样性指数和Shannon的信息指数分别为0.266 4和0.237 5,0.385 2和0.349 8。与其它鲆鲽鱼类养殖群体相比,半滑舌鳎和塞内加尔鳎养殖群体的遗传多样性水平略低;2种鱼群体间的遗传相似性系数和遗传距离分别为0.458 3和0.723 1,它们的遗传变异主要来自群体内。     

黄姑鱼(Nibea albiflora Richardson)早期生长发育研究

为研究黄姑鱼胚胎发育特征和胚后发育的仔、稚、幼鱼发育阶段的形态学,2010-06-08在实验室利用显微镜和显微镜数码摄像显示系统对黄姑鱼胚胎发育过程进行连续观察拍摄,描述并记录其发育过程和时间。结果表明,黄姑鱼卵为圆球形的端黄卵,盘状分裂,中央有一个油球,卵径为(0.89±0.028)mm,油球直径为0.25mm;在水温为24℃、S为27条件下经22.5h完成孵化,胚胎发育可分为24期。胚后发育阶段,1～3日龄为卵黄囊期仔鱼;4日龄仔鱼开口,卵黄囊完全消失,此时仔鱼处于混合营养阶段,进入前弯曲期;6日龄仔鱼油球完全消失,进入外源性营养阶段;16日龄仔鱼尾鳍担鳍骨形成,脊椎末端上曲,进入弯曲期;21日龄仔鱼尾下骨后缘与尾索垂直,鳍条数不断增加,同时背鳍、臀鳍和腹鳍原基出现,进入后弯曲期;26日龄鱼苗全身被菊花状黑色素细胞,尾柄处开始被鳞,进入稚鱼期;31日龄以后,鱼苗全身被鳞,进入幼鱼期。在水温为25～27℃,S为26～28的条件下,仔、稚、幼鱼全长与日龄具有显著的正相关性。研究表明,黄姑鱼进入稚鱼期之后,其生长速率明显增大,为减少自残,应及时分池和投足饵料。     

低温胁迫对黄姑鱼(Nibea albiflora)抗氧化酶、Na~+/-K~+-ATP酶及Hsp70蛋白含量的影响

为探讨低温处理对黄姑鱼幼鱼生理机能的影响,以18℃为对照组,设置8℃、10℃和14℃3个低温胁迫组,测定不同温度急性胁迫对黄姑鱼幼鱼(Nibea albiflora)超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、Na~+/-K~+-ATP酶活力以及热休克蛋白70(Hsp70)蛋白含量的影响。研究结果表明,低温胁迫组(8℃、10℃和14℃)的SOD和CAT酶活力随着胁迫温度的降低及其胁迫时间的延长呈先升高后降低的趋势,在72 h时恢复到对照组水平。肌肉中的Na~+/-K~+-ATP酶活力则随处理时间的延长呈现先降低后升高的趋势,10℃和14℃组能够在72 h时恢复到对照组水平,而8℃组则在72 h仍显著低于对照组。低温胁迫下黄姑鱼肌肉的Hsp70含量随着处理时间的延长呈先升高后降低趋势,10℃和14℃组能够在72 h时恢复到对照组水平,而8℃组则在72 h仍显著高于对照组。由此说明,SOD、CAT、Na~+/-K~+-ATP以及Hsp70蛋白参与了黄姑鱼低温胁迫应答过程,可以作为其低温胁迫应答的标志物。     

星斑川鲽(Platichthys stellatus)养殖群体的RAPD分析

利用RAPD技术对星斑川鲽养殖群体的遗传多样性进行了研究。将实验用星斑川鲽养殖群体的活鱼苗解剖,取其肌肉,高盐法抽提获得总DNA,质量鉴定和浓度测定后用随机引物进行PCR扩增,从20个随机引物中筛选出18个用于群体遗传学分析,共扩增出133条带,谱带大小为100~2 000 bp,大多数谱带分布在250~1 000bp。多态谱带数为83,每个引物扩增谱带数为1~14条,平均为7条。多态谱带比例为62.41%,Shannon的信息指数和Nei的多样性指数分别为0.21和0.316 6。     

小黄鱼(♀)×大黄鱼(♂)杂交子代遗传特征分析

通过对小黄鱼(♀)×大黄鱼(♂)杂交子代及其双亲染色体核型和线粒体COI基因序列的比较分析,旨在对其杂交遗传特性进行研究。其中染色体核型分析表明:小黄鱼的核型为2n=6sm+42t,NF=54;大黄鱼的核型为2n=2sm+22st+24t,NF=72;杂交子代为2n=8sm+12st+28t,NF=68。杂交子代和亲本在染色体数目上一致,但在组型上存在差异,可能是由于杂交过程中双亲染色体组发生了融合和重配。同时,对杂交子代及其亲本672bp的COI基因同源序列进行分析,表明杂交子代与其母本小黄鱼的序列几乎一致,利用Kitumra-2-Parameter-Distance双参数模型分析显示杂交子代与小黄鱼的遗传距离为0.011,该数值接近其种内平均遗传距离。基于COI基因片段序列构建的系统树显示,杂交子代与小黄鱼聚成一支。杂交子代在线粒体COI基因上表现出严格的母系遗传特征。     

牙鲆sox9基因的克隆与表达研究

通过Genome walking方法克隆得到了牙鲆(Paralichthys olivaceus)sox9的基因组全长序列,对其在成鱼各组织的差异表达进行了RT-PCR分析.结果显示:牙鲆sox9基因全长3573 bp,包含3个外显子和2个内含子,编码的蛋白全长478 aa,含有高度保守的HMG结构域;sox9基因在性腺的表达具有两性差异,精巢中的表达明显强于卵巢中的表达,属1个性别相关基因,但其在肝脏、心脏、鳃、脑、眼和胃等多种组织中也有不同程度的表达.据此进一步分析和讨论等sox9基因在牙鲆性腺发育中的作用.     

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus： karyotype, allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish. Supported by National Natural Science Foundation of China (No. 30571445), National High-Technology Research and Development Program (863 Program, No. 2006AA10A404), and Project from the Ministry of Science and Technology of China (No. 2006DKA30470-017)