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1.
1 Introduction Microsatellite DNAs, also called simple sequence re-peats (SSRs), are stretches of DNA, consisting of tan-demly repeating mono-, di-, tri-, tetra- or penta-nucleotide units. They are evenly distributed throughout the ge-nomes of most eukaryotic species (Tohn et al., 2000). Their high information content about the divergence of genome, which is directly related to the number of alleles at each locus, and the ease of PCR assays make them ex-cellent molecular markers (Powell …  相似文献   

2.
The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species.  相似文献   

3.
The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species.  相似文献   

4.
1 Introduction Microsatellites or simple sequence repeats (SSRs) are tandemly repeated motifs of one to six bases found in all prokaryotic and eukaryotic genomes analysed to date (Zane et al., 2002). Due to their hyper-variable and co-dominant nature, relatively high abundance and random distribution in the genome, microsatellites are among the most efficient class of molecular markers. Such repeats display high polymorphism because of variation in repeat length and can be rapidly analysed t…  相似文献   

5.
A complementary DNA (cDNA) library was constructed from half-smooth tongue sole spleen. A long-read expressed sequence tag (EST) database was generated, containing 3100 cDNA clones, of which 220 clones were fully sequenced. A total of 1060 non-redundant simple sequence repeats (SSRs) were obtained from the cDNA library. An average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric repeats (51.4%) are the most abundant class of SSR in the dataset. The dimeric, trimeric, tetrameric and hexameric repeats are represented in decreasing proportions of 27.2%, 16.0%, 2.8% and 1.9%, respectively. The frequency of pentameric repeats was observed the least (only 0.7%). Most of the monomeric and dimeric repeats are distributed in 3′ and 5′ un-translation region. If translation regions are considered merely, trimeric repeats are the highest, accounting for 57% of the total microsatellites.  相似文献   

6.
1　Introduction　Generallyknownasacodominantgeneticmarker ,microsatellitehasbeenwidelyusedinstudiesonpopu lationgenetics,high resolutiongenotyping ,genemap ping ,evolution ,linkageanalysis ,conservationbiology ,behaviouralecology ,relationsbetweenparasite…  相似文献   

7.
Turbot(Scophthalmus maximus) is a flatfish species commercially important for aquaculture.In this study,we generated a microsatellite-enriched genomic DNA library for Scophthalmus maximus,and then isolated and characterized 45 microsatellite loci by genotyping 30 individuals.The observed number of alleles ranged from 2 to 19 with an average of 6.24,while the effective number of alleles ranged from 1.30 to 11.11 with an average of 3.66.The expected heterozygosities varied from 0.235 to 0.925 4 and polymorphic information content ranged from 0.204 4 to 0.903 3,with an average of 0.622.Twelve loci deviated significantly from Hardy-Weinberg equilibrium,and no significant linkage disequilibrium was observed between any pair of loci after Bonferroni correction.In cross-species amplification,five flatfish species(Paralichthys lethostigma,Verasper moseri,platichthys stellatus,Hippoglossoides dubius and Cynoglossus semilaevis) showed at least one polymorphic locus.These polymorphic microsatellite loci should prove useful for population analysis of turbot and other related species.  相似文献   

8.
A muscle cDNA library of Chinese shrimp (Fenneropenaeus chinensis) was constructed with the SMART™ cDNA Library Construction Kit. The titer of optimal primary library was 7.7×105 pfu mL−1 and that of the amplified library was 3.0×109 pfu mL−1. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin I gene. This library provided a useful resource for the functional genomic research of F. chinensis.  相似文献   

9.
The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COΙ. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COΙ so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.  相似文献   

10.
Microsatellite markers have become one kind of the most important molecular tools used in various researches. A large number of microsatellite markers are required for the whole genome survey in the fields of molecular ecology, quantitative genetics and genomics. Therefore, it is extremely necessary to select several versatile, low-cost, efficient and time- and labor-saving methods to develop a large panel of microsatellite markers. In this study, we used Zhikong scallop (Chlamys farreri) as the target species to compare the efficiency of the five methods derived from three strategies for microsatellite marker development. The results showed that the strategy of constructing small insert genomic DNA library resulted in poor efficiency, while the microsatellite-enriched strategy highly improved the isolation efficiency. Although the mining public database strategy is time- and cost-saving, it is difficult to obtain a large number of microsatellite markers, mainly due to the limited sequence data of non-model species deposited in public databases. Based on the results in this study, we recommend two methods, microsatellite-enriched library construction method and FIASCO-colony hybridization method, for large-scale microsatellite marker development. Both methods were derived from the mi-crosatellite-enriched strategy. The experimental results obtained from Zhikong scallop also provide the reference for microsatellite marker development in other species with large genomes.  相似文献   

11.
A complementary DNA (cDNA) library was constructed from half-smooth tongue sole spleen. A long-read expressed sequence tag (EST) database was generated, containing 3100 cDNA clones, of which 220 clones were fully sequenced. A total of 1060 non-redundant simple sequence repeats (SSRs) were obtained from the cDNA library. An average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric repeats (51.4%) are the most abundant class of SSR in the dataset. The dimeric, trimeric, tetrameric and hexameric re- peats are represented in decreasing proportions of 27.2%, 16.0%, 2.8% and 1.9%, respectively. The frequency of pentameric repeats was observed the least (only 0.7%). Most of the monomeric and dimeric repeats are distributed in 3' and 5' un-translation region. If translation regions are considered merely, trimeric repeats are the highest, accounting for 57% of the total microsatellites.  相似文献   

12.
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).  相似文献   

13.
To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the library was 20 kb. This library was screened for antibiotic activity using paper disc assaying. Two clones displayed the antibacterial activity against Micrococcus tetragenus. The metabolites of these two clones were analyzed through HPLC. The result showed that their metabolites were quite different from those of the host E. coli DH5α and the host containing vector pHZ132. This study may present a new approach to exploring bioactive metabolites of sponge symbionts.  相似文献   

14.
The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.  相似文献   

15.
1　Introduction　Photosyntheticdinoflagellatesareimportantprima ryproducersandcausesoftoxic‘redtide’ .Theyarefoundinmostaquaticenvironmentsandformamajorpartofthemodernplankton .Dinoflagellatesmaybeimportantindicatorsofenvironmentalstatus (Dale ,1996 ) .…  相似文献   

16.
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.  相似文献   

17.
The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene （rDNA） fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.  相似文献   

18.
Two Large-insert genomic bacterial artificial chromosome （BAC） libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.  相似文献   

19.
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20.
A muscle cDNA library of Chinese shrimp (Fenneropenaeus chinensis) was constructed with the SMARTTM cDNA Li- brary Construction Kit. The titer of optimal primary library was 7.7×105 pfu mL-1 and that of the amplified library was 3.0×109 pfu mL-1. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin Ⅰ gene. This library provided a useful resource for the functional genomic research ofE chinensis.  相似文献   

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