GENETIC DIFFERENTIATION WITHIN ERIOCHOIR SINENSIS(MILNE,EDWARDS) REVEALED BY ALLOZYME AND RAPD

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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)     

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

Eighteen gametophytes includingL. japonica, L. ochotensis andL. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains ofLaminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species ofLaminaria. Contribution No. 4389 from the Institute of Oceanology, Chinese Academy of Sciences. This research was supported by the State's High-tech Project of China, Marine 863 project (No. 819-03-02) and (2001AA621090).     

PHYLOGENETIC RELATIONSHIPS AMONG PUFFERFISH OF GENUS TAKIFUGU BY RAPD ANALYSIS

AmpLifications with 300 10-base primers under predetermined optimal reaction yielded 2762 reproducible amplified fragments ranging from 200 to 3000 bp. Genetic distances among five species of Takifugu and Lagocephalus spadiceus as outgreup were calculated based on the presence/absence of the amplified fragments. Topological phylogenie trees of the 5 species of Takgfugu and the outgroup were gen-erated by Parsimony and Neighbor-Joining analysis based on RAPD data set. The genetic distance between T. rubripes and T. pseudommus was almost the same as that between individuals within each species, but was much smaller than that among T. rubripes, T. pseudommus and the other species. The molecu-lar data from RAPD analysis convincingly showed that T. rubripes and T. pseudommus should be the same species.     

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 matemal 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.     

GENETIC DIVERSITY OF THE WILD AND REARED PSEUDOSCIAENA CROCEA

The genetic diversity of both wild and reared Pseudosciaena crocea (Richardson) col-lected from Guan-Jing-Yang in Ningde, China in May 1999 was investigated by random amplified poly-morphic DNA (RAPD) in the present study. The polymorphism and mean difference of the wild popula-tion as revealed by RAPD were 18.9% and 0.0960 respectively, and those of the reared stocks were rel-atively lower, with 16.7% in polymorphism and 0.0747 in mean difference. The genetic distance be-tween the two stocks was 0.0041. From the comprehensive investigation, the main reasons for the loss of genetic diversity were probably overilshing, small number of parents as broodstocks and the debatable arti-ficial ranching. Results from this study also showed that the large yellow croaker populations distributed along Fujian coastal waters including Guan-Jing-Yang still potentially wide genetic variability. It is sug-gested that genetic management and prevention should be scientifically conducted in order to maintain and improve the genetic diversity of the P. crocea population.     

Microsatellite DNA polymorphisms and the relation with body weight in sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

The relationship between microsatellite polymorphism and body weight of captive bred Chinese sea cucumber Apostichopus japonicus was investigated in two local populations in Dalian. Among ten loci discovered, nine show changes except for AJ07 loci. Seven loci were found highly polymorphic in both populations. For each locus in two populations, the average number of alleles is 6.428 6 and 6.285 7, the average observed heterozygosity at 0.225 7 and 0.245 9, the expected heterozygosity at 0.776 8 and 0.748 8, the polymorphism information content (PIC) at 0.709 2 and 0.674 6, respectively. Further analysis show significant correlation between A. japonicus body weight and occurrence markers AJ02 and AJ04. The findings of the relation may be helpful for molecular breeding, as well as the marker-assisted selection of sea cucumbers. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2006AA10A411), the Natural Science Foundation of Liaoning Province (No.20072139), the Grant of Dalian Fisheries University, the Key Laboratory Foundation of the Educational Department of Liaoning Province (No.2009S024)     

Triploidy induction with heat shocks toPenaeus chinensis and their effects on gonad development

Heat shocks effectively produced triploids inPenaeus chinensis. Fertilized eggs heat shocked (28–32°C) for 8 to 16 minutes, starting from 8 to 20 minutes after spawning, resulted in triploidy induction rates of 39%–75%. Several triploid populations were cultured to 10 cm. In a triploid population, two kinds of ovaries were observed. Histological examination showed apparent differences between these two kinds of ovaries, whereas among male shrimp, there were no such differences. Contribution No. 3465 from the Institute of Oceanology. Chinese Academy of Sciences. The study was supported by the International Foundation for Science, Asian Fisheries Society and Chinese Climbing Project PDB 6–2, and Chinese National 863 Project (No. 863-819-01-05).     

Isolation, cloning and sequencing of AFLP markers related to disease-resistance traits in Fenneropenaeus chinensis

Amplified fragment length polymorphism (AFLP) technique was used to analyze the fingerprinting of four successive generations ofFenneropenaeus chinensis to reveal their disease-resistance traits. Some loci showed quite different genetic frequencies due to artificial selection, which implied that these fragments were putative markers related to the disease-resistance trait. We developed a simple and effective method to further characterize these AFLP fragments. Specific AFLP bands were cut directly from polyacrylamide gels, re-amplified, cloned and sequenced. Eight putative genetic markers were sequenced and their sizes ranged from 63 to 209 bp. The sequences were submitted to dbGSS (database of Genome Sequence Survey); and the BLAST analysis showed low similarity to the function genes, indicating these markers were tightly linked to a disease-resistance trait but were not functional genes. This research was supported by special funds from the National Key Basic Research Program (G1999012007) and the National High-Tech Research and Development Program of China (863 Program. 2001AA620105).     

The comparison between allozyme and RAPD makers for the population genetic structure analysis of scallop Chlamys farreri

1INTRODUCTION Since the first use in human genetic research in1966,allozyme has become the most popular genetic marker for population genetics studies for almost all groups of animals and plants(Lewontin and Hubby,1966;Murphy et al.,1996).In the past seve…     

Inheritance mode of microsatellite loci and their use for kinship analysis in the Pacific oyster （Crassostrea gigas）

Five full-sib families of the Pacific oyster （Crassostrea gigas） larvae were used to study the mode of inheritance at eight microsatellite loci, and the feasibility of these markers for kinship estimate was also examined. All eight microsatellite loci were compatible with Mendelian inheritance. Neither evidence of sex-linked barriers to transmission nor evidence of major barriers to fertilization between gametes from the parents was shown. Three of the eight loci showed the presence of null alleles in four families, demonstrating the need to conduct comprehensive species-specific inheritance studies for microsatellite loci used in population genetic studies. Although the null allele heterozygotes were considered as homozygotes in the calculation of genetic distance, offspring from five full-sib families were unambiguously discriminated in the neighbor-joining dendrogram. This result indicates that the microsatellite markers may be capable of discriminating between related and unrelated oyster larvae in the absence of pedigree information, and is applicable to the investigation of the effective number of parents contributing to the hatchery population of the Pacific oyster.     

Genetic variation of natural and cultured stocks of Paralichthys olivaceus by allozyme and RAPD

Population genetics of the left-eyed flounder, Paralichthys olivaceus, including natural and cultured stocks distributed in the coastal waters near Qingdao of eastern maritime China, was analyzed in allozyme and RAPD. The results showed that among total 29 gene loci of 15 isozymes, 9 and 7 were po- lymorphic in natural and cultured stocks, respectively. The status of genetic diversity in P. olivaceus is low in terms of polymorphic loci in chi-square test and genetic departure index of Hardy-Weinberg equi- librium. More alleles in IDHP, CAT, GDH and Ldh-C allozymes were found in the fish, which could be used as markers in assortive breeding and distinguishing stock, population or species evolution. Total 88 and 86 RAPD bands ranging from 200 to 2 500 bp were recognized individually in average of 7.8–8.0 bands per primer. The genetic diversity in cultured stock is lower than that in natural ones showing an ob- viously decreasing genetic divergence. Therefore, effective countermeasures must be taken to protect ge- netic resources of marine cultured fishes. The 2 markers have their own pros and cons. Combining the 2 markers to investigate the genetic variation of populations is suggested. The results provide basic data of this flounder and they are useful for studying genetic improvement and genetic resources of the fish.     

Isolation and pharmacological activities of bromophenols from Rhodomela confervoides

Four bromophenols were isolated from the extract of marine red alga,Rhodomela confervoides by column chromatography and HPLC methods. By means of spectroscopic methods inclding IR, MS, 1D, and 2D-NMR techniques, their structures were elucidated as (1) 3-bromo-4,5-dihydroxy benzoic acid methyl ester; (2) bis (2,3-dibromo-4,5-dihydroxybenzyl) ether; (3) 3,4-dibromo-5-(methoxymethyl)-1,2-benzenediol and (4) 3-bromo-4,5-dihydroxy-benzaldehyde. Compound 1 was first isolated from the algae in nature, and 1, 4 were found to have selective cytotoxic activities against KB, Bel 7402 and A549 cells, 2 showed powerful antibacterial activities againstStaphylococcus aurens andPseudomonas aeruginosa. Supported by National “863” Program (No. 2004AA625030, 2001AA620503), Quingdao marine sciences project (No. 04-2-NN-26) and Key Knowledge Innovative Project of the Chinese Academy of Sciences (KZCX3-SW-215).     

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China （LCDV-cn） genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene. Supported by High Technology Research and Development Program of China (863 Program, No. 2006AA100309)     

紫红笛鲷随机扩增多态性DNA及遗传多样性分析

采用RAPD技术分析了湛江近海紫红笛鲷 (LutjanusargentimaculatusForsk l)的遗传多样性。从 12 0个随机引物中筛选出了 14个引物。 14个引物共检测到 173个位点 ,其中多态位点比例 (P)为 6 8.79%,遗传距离 (D)为 0 .2 2 2 8,遗传多样性指数 (H)为 0 .190 4。结果表明 ,目前湛江近海的紫红笛鲷自然群体的遗传多样性仍然维持在良好水平 ,捕捞尚未对其造成明显影响。     

Genetic study of Kelp"901"strain

Based on DNA extraction and optimization of random amplified reaction (RAPD) to the gametophytes and sporophytes of Kelp “901” strain, genetic study on variation was conducted to its parents and offsprings of F₆, F₇, F₈, and F₉ generation. RAPD results have shown that among 30 selected primers for gametophytes, 297 loci ranging from 200 to 3 000 bp were obtained in the average of 9.9 loci for each primer. This indicated a high polymorphic rate with RAPD detection. UPGMA (unweighted pair-group method arithmetic average) analysis showed that each male and female gametophyte of a generation could be clustered into one pair separately. The genetic distances of the Kelp 901 generation were 0.3212–0.4767, and the maximum was between F₇ and F₈ (0.4767). Identity analysis showed that F₆ generation was more close to the female parent (0.6593), and F₇ generation was more close to the male parent (0.578 8). To the sporophytes study in 24 selected primers for RAPD amplification, 191 loci ranging from 230–2800 bp were obtained, in the average to each primer of 8.0 loci. The heterozygosity to six populations were male parent (0.2239), female parent (0.1072), F₆ (0.2164), F₇(0.2286), F₈(0.2296) and F₉(0.3172). The nearest genetic distance was 0.083 5(F₈, F₉). Total heterozygosity (H_T) of F₆, F₇, F₈ and F₉ generations was 0.3186, the average heterozygosity (H_S) for F₆, F₇, F₈ and F₉ generations was 0.2480, and deduced coefficient of population differentiation (G_st) was 22.2%. Six sequence characterized amplified regions (SCAR) were preliminary screened through RAPD analysis. It needed to be verified in detail as they are significant for molecular marker assistance in breeding and selectingLaminaria.     

Competition Among Dinoflagellate Alexandrium Tam—arense, Raphidophyte Heterosigma Carterae and Diatom Skeletonema Costatum under Combinations ofTwo Temperatures and Five Salinities

Competition among HAB (Harmful Algal Bloom) species Dinoflagellate Alexandrium tamarense, Raphidophyte Heterosigma carterae, and Diatom Skeletonema costatum was studied in the laboratory. Experiments with these three major HAB species under combinations of different salinities (10, 18, 25, 30, 35) and temperatures (19℃, 25℃) were carried out. The results showed that S. costatum successfully competed with the other two species at salinities of 18, 25,30, and 35 at temperatures of 19℃ and 25℃. However, H. carterae showed its advantage at low salinity of 10 and became the single dominant species at salinity 10 and 25℃. A. tamarense could not compete successfully with the other two species especially at low salinities. However, it could remain at low density in the presence of higher densities of other algae.     

近江牡蛎两个野生种群的遗传多样性分析

采用RAPD分子标记和rDNA-ITS1序列分析了近江牡蛎Crassostrea rivularis湛江官渡和阳江程村野生种群的遗传多样性。12个RAPD引物共扩增出8838条片段,157个位点,平均每个引物可产生13个位点,片段长度在200～2200bp之间。湛江种群和阳江种群的多态位点比例分别为89.62%和89.57%,遗传多样性指数分别为0.4170和0.4334。种群间平均遗传距离为0.0327,平均遗传相似性为0.9681,平均遗传分化系数为0.0437。得到近江牡蛎18S、5.8S部分序列和ITS1全部序列,其中ITS1序列片段长度为478～485bp,共有11个变异位点,两个为转换(A/G),其他为插入/缺失(A/-、T/-)。湛江和阳江种群各获得8个单倍型,其中有一个单倍型为两个种群共享。两个种群的CG碱基平均含量较高,分别为58.29%和58.41%。种群间的遗传分化系数0.0254。结果说明,近江牡蛎湛江种群和阳江种群间具有较高的遗传多样性和较低的遗传分化。     

Studies on extraction procedure and antioxidative activity of phlorotannins fromSargassum kjellmanianum

Known only in phaeophyceae, phlorotannins (brown algal polyphenols) are natural products with potential uses in pharmacology. This study yielded an extraction procedure to obtain high purity, high molecular weight phlorotannins fromSargassum kjellmanianum and revealed the characteristics of their infrared and flourescence spectra. The antioxidative activity of phlorotannins, which was about 2.6 times as strong as that of 0.02% BHT (tert-butyl-4-hydroxytoluene), showed potential for preventing oil rancidification. Contribution No. 2918 from the Institute of Oceanology, Chinese Academy of Sciences. The project was supported by The State Ninth-five Key Program of China. (No. 96-916-04-01).