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.     

Analysis of new microsatellite markers developed from reported sequences of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

The expressed sequence tags (ESTs) of Japanese flounder, Paralichthys olivaceus, were selected from GenBank to identify simple sequence repeats (SSRs) or microsatellites. A bioinformatic analysis of 11111 ESTs identified 751 SSR-containing ESTs, including 440 dinucleotide, 254 trinucleotide, 53 tetranucleotide, 95 pentanucleotide and 40 hexanucleotide microsatellites respectively. The CA/TG and GA/TC repeats were the most abundant microsatellites. AT-rich types were predominant among trinucleotide and tetranucleotide microsatellites. PCR primers were designed to amplify 10 identified microsatellites loci. The PCR results from eight pairs of primers showed polymorphisms in wild populations. In 30 wild individuals, the mean observed and expected heterozygosities of these 8 polymorphic SSRs were 0.71 and 0.83 respectively and the average PIC value was 0.8. These microsatellite markers should prove to be a useful addition to the microsatellite markers that are now available for this species.     

南海海域4种笛鲷的微卫星DNA分析

采用聚丙烯酰胺凝胶电泳技术,从20对西大西洋笛鲷(Lutjanus campechanus)的微卫星引物中筛选适用于红鳍笛鲷(L.erythopterus)、勒氏笛鲷(L.russellii)、紫红笛鲷(L.argentimaculatus)和约氏笛鲷(L.johnii)基因组分析的微卫星引物,分析4种笛鲷的遗传多样性及系统发生。经反应条件的优化,从20对引物中筛选出17对可稳定扩增出特异片段的引物。通过测序证实扩增产物含微卫星位点后,以该17对引物对4个种的群体进行遗传多样性分析。其中16对可在约氏笛鲷、勒氏笛鲷、紫红笛鲷基因组中扩增出重复性好的特异性条带,分别有65%、65%、60%呈现出种内多态;15对可在红鳍笛鲷中扩增出重复性好的特异性条带,并且全部呈现出种内多态。四种笛鲷中,红鳍笛鲷的多态性最高,高度多态基因座占检测座位的66.67%;勒氏笛鲷的多态性最低,低度多态基因座占43.75%。获得3个种间特异性分子标记。用DS和DA两种方法计算4种笛鲷的遗传距离,构建了系统发生树。     

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.     

利用RAPD和ISSR引物对三系杂交水稻及其父母本的鉴定

为了鉴定杂交种子与父母本的亲缘关系,用20条随机引物和21条ISSR引物在供试的一组三系杂交水稻F1、父母本及对照进行扩增。20条RAPD引物共扩增出78个条带,其中28条为多态性条带;21条ISSR引物共扩增出72个条带,其中多态性条带为20条。将这些多态性条带进行聚类分析,F1与父、母亲缘关系较近归为一类,随后它们才与对照归为一类。结果表明RAPD和ISSR引物的PCR扩增均能有效地鉴定三系杂交水稻的杂种F1与组合中父、母本的遗传关系。与RAPD相比ISSR引物在三系杂交水稻及其父母本的鉴定中具有稳定性更好的优点。     

Application of RAPD in genetic diversity study onGracilaria lemaneiformis III. Phase and sex related markers

Random amplification of polymorphic DNA (RAPD) was conducted by using 10 random primers (P-1 to P-10) inGracilaria lemaneiformis. Phase and sex specific bands were amplified by primers P-2, P-6, P-7 and P-8: for P-2 a 1.4 kb band was found in female gametophytes and tetrasporophytes, for P-6, a 0.6 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.76 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.72 kb band appeared in female gametophytes and tetrasporophytes; for P-8, a 0.73 kb band only appeared in male gametophytes.

     

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)     

奥利亚罗非鱼微卫星位点的筛选

从GenBank上选取40对尼罗罗非鱼(Oreochromis niloticus)的微卫星引物,分别在奥利亚罗非鱼(Oreo-chromis aureus)(83系)、红色奥利亚罗非鱼、奥利亚罗非鱼(02系)和尼罗罗非鱼的基因组上进行扩增。40对引物中有37对(92.5%)可进行有效扩增,32对引物(80%)可检测到个体间等位基因的多态性。其中引物UNH168与奥利亚罗非鱼的性别相关,在雌性个体中可扩增出二条大小不同的特异带(分别为135和171 bp),在雄性个体中则只有一条(171 bp)。将特异条带回收、克隆并测序,结果显示雌雄个体中171 bp条带的序列完全相同,包括103bp的侧翼序列和34个CA重复,在雌性个体中获得的135 bp条带则只有16个CA重复。引物UNH846、UNH860和UNH995可鉴别奥利亚罗非鱼和尼罗罗非鱼,UNH890可鉴别出红色奥利亚罗非鱼。可见,大部分的尼罗罗非鱼微卫星位点存在于奥利亚罗非鱼中。     

长江下游鳊鱼遗传多样性的RAPD分析

应用RAPD技术对长江下游鳊鱼群体的遗传多样性进行分析,从40个随机引物中筛选出28个引物对鳊鱼24个个体的基因组DNA进行扩增,共检测到178个位点,分子片段在0.2～2.0kb之间,其中多态位点83个,占46.63%;个体间最大的遗传距离为0.1326,最小的遗传距离为0.0476,平均遗传距离为0.0885;群体的Nei基因多样性指数为0.2593,Shannon多样性指值为0.0986。与其他鱼类的遗传多样性研究结果相比,长江下游鳊鱼群体的遗传多样性处于中等水平。     

基于近缘EST-SSR引物的企鹅珍珠贝养殖和野生群体的遗传标记

利用近源物种马氏珠母贝的EST-SSR引物对企鹅珍珠贝DNA进行扩增,获得7对可扩增出目标条带的近缘分子标记,分析野生群体(YS)和养殖群体(YZ)企鹅珍珠贝的遗传多样性。结果显示,7个位点共获得26个等位基因;野生和养殖群体的等位基因数分别为3~5和3~4,平均期望杂合度(He)分别为0.623 5、0.496 8;平均观测杂合度(Ho)分别为0.431 8、0.302 8;HNUPM047位点的多态信息含量较低,为0.446 7,其他位点均在0.5以上,说明该遗传标记可提供比较丰富的遗传信息。表明养殖群体遗传多样性水平低于野生群体。     

东、黄海鲐鱼群体遗传差异的RAPD分析

利用随机扩增多态DNA(RAPD)技术分别分析了五岛西部日本鲐、东海西部日本鲐和澳洲鲐三群体的样本,共产生226个分子标记,其中143个为多态性标记,多态百分率达到63.3%,每条引物产生的平均标记数为7.29。五岛西部日本鲐、东海西部日本鲐和澳洲鲐群体内遗传相似度分别为0.949、0.877和0.936,而群体间遗传距离依次为:五岛西部日本鲐-东海西部日本鲐(0.2357)<东海西部日本鲐-澳洲鲐(0.3030)<五岛西部日本鲐-澳洲鲐(0.3528),五岛西部日本鲐与东海西部日本鲐个体在UPGMA系统树独立聚类为两个类群,初步支持东、黄海的日本鲐存在东海西部和五岛西部的两个种群的观点。Shannon多样性指数表明,其遗传多样性大小依次为东海西部日本鲐、澳洲鲐、五岛西部日本鲐。     

Inheritance of 15 microsatellites in the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>: segregation and null allele identification for linkage analysis

Microsatellites were screened in a backcross family of the Pacific oyster, Crassostrea gigas. Fifteen microsatellite loci were distinguishable and polymorphic with 6 types of allele-combinations. Null alleles were detected in 46.7% of loci, accounting for 11.7% of the total alleles. Four loci did not segregate in Mendelian Ratios. Three linkage groups were identified among 7 of the 15 segregating loci. Fluorescence-based automated capillary electrophoresis (ABI 310 Genetic Analyzer) that used to detect the microsatellite loci, has been proved a fast, precise, and reliable method in microsatellite genotyping. Supported by the National Natural Science Foundation of China (NO.40730845, 39825121)     

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 analysis with ISSR PCR on green algae <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR （ISSR PCR）. Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity （h^＊） and Shannon index of diversity （I^＊） in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones （0.190 3 and 0.274 8）, which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.     

Inheritance Pattern of Microsatellite Loci and Their Use for Kinship Analysis m the Japanese Scallop Patinopecten yessoensis

The inheritance mode of seven microsatellite markers was investigated in Patinopecten yessoensis larvae from four con-trolled crosses, and the feasibility of using these markers for kinship estimation was also examined. All the seven microsatellite loci were compatible with Mendelian inheritance. Neither sex-linked barriers to transmission nor major barriers to fertilization between gametes from the parents were evident. Two of the seven loci showed the presence of null alleles in two families, suggesting the need to conduct comprehensive species-specific inheritance studies for microsatellite loci used in population genetic studies. However, even if the null allele heterozygotes were considered as homozygotes in the calculation of genetic distance, offspring from four fami-lies were all unambiguously discriminated in the neighbor-joining dendrogram. This result indicates that the microsatellite markers used may be capable of discriminating between related and unrelated scallop larvae in the absence of pedigree information, and of investigating the effective number of parents contributing to the hatchery population of the Japanese scallop.     

Inheritance pattern of microsatellite loci and their use for kinship analysis in the Japanese scallop Patinopecten yessoensis

The inheritance mode of seven microsatellite markers was investigated in Patinopecten yessoensis larvae from four controlled crosses, and the feasibility of using these markers for kinship estimation was also examined. All the seven microsatellite loci were compatible with Mendelian inheritance. Neither sex-linked barriers to transmission nor major barriers to fertilization between gametes from the parents were evident. Two of the seven loci showed the presence of null alleles in two families, suggesting the need to conduct comprehensive species-specific inheritance studies for microsatellite loci used in population genetic studies. However, even if the null allele heterozygotes were considered as homozygotes in the calculation of genetic distance, offspring from four families were all unambiguously discriminated in the neighbor-joining dendrogram. This result indicates that the microsatellite markers used may be capable of discriminating between related and unrelated scallop larvae in the absence of pedigree information, and of investigating the effective number of parents contributing to the hatchery population of the Japanese scallop.     

Assessing the Germplasm of Laminaria （Phaeophyceae） with Random Amplified Polymorphic DNA （RAPD） Method

Eighteen gametophytes including L.japonica ,L.ochotensis and L.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 gamtophytes of the same cell lines could be grouped in pairs respectively,It indicated that RAPD analysis could be used not only to distin-gusih different strains of Laminariz ,but also to distinguish male and female gametophyte within the same cell lines,There is ambiguous systematic relationship if judged mereely by the present data It seems that the use of RAPD marker is limited to elucidstion of the phylogenetic relationship among the species of Laminaria.     

Inheritance Pattern of Microsatellite Loci and Their Use for Kinship Analysis in the Japanese Scallop Patinopecten yessoensis

The inheritance mode of seven microsatellite markers was investigated in Patinopecten yessoensis larvae from four con-trolled crosses,and the feasibility of using these markers for kinship estimation was also examined. All the seven microsatellite loci were compatible with Mendelian inheritance. Neither sex-linked barriers to transmission nor major barriers to fertilization between gametes from the parents were evident. Two of the seven loci showed the presence of null alleles in two families,suggesting the...     

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。