Chromosomal localization of 5S rDNA in Chinese shrimp (Fenneropenaeus chinensis): a chromosome-specific marker for chromosome identification

Chinese shrimp (Fenneropenaeus chinensis) is an economically important aquaculture species in China. However, cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to isolate and analyze. In this study, fluorescence in-situ hybridization (FISH) was used to identify the chromosomes of F. chinensis. The 5S ribosomal RNA gene (rDNA) of F. chinensis was isolated, cloned and then used as a hybridization probe. The results show that the 5S rDNA was located on one pair of homologous chromosomes in F. chinensis. In addition, triploid shrimp were used to evaluate the feasibility of chromosome identification using FISH and to validate the method. It was confirmed that 5S rDNA can be used as a chromosome-specific probe for chromosome identification in F. chinensis. The successful application of FISH in F. chinensis shows that chromosome-specific probes can be developed and this finding will facilitate further research on the chromosomes of penaeid shrimps.     

Chromosomal localization of 5S rDNA in Chinese shrimp (Fenneropenaeus chinensis): a chromosome-specific marker for chromosome identification

Chinese shrimp(Fenneropenaeus chinensis)is an economically important aquaculture species in China.However,cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to isolate and analyze.In this study,fluorescence in-situ hybridization(FISH) was used to identify the chromosomes of F.chinensis.The 5S ribosomal RNA gene(rDNA)of F. chinensis was isolated,cloned and then used as a hybridization probe.The results show that the 5S rDNA was located on one pair of homologous chromosomes in F.chinensis.In addition,triploid shrimp were used to evaluate the feasibility of chromosome identification using FISH and to validate the method.It was confirmed that 5S rDNA can be used as a chromosome-specific probe for chromosome identification in F.chinensis.The successful application of FISH in F.chinensis shows that chromosome-specific probes can be developed and this finding will facilitate further research on the chromosomes of penaeid shrimps.     

Chromosomal mapping of 5S and 18S-5.8S-25SrRNA genes in Saccharina japonica(Phaeophyceae) as visualized by dual-color fluorescence in situ hybridization

It has been reported that there was a linkage of 5 S rRNA gene to 18 S-5.8 S-25 S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5 S rDNA to the 3' end of 25 S rDNA,two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica,one of species in Ochrophyta.This result supplemented the previous report that 5 S rDNA was unlinked to 25 S rDNA in this kelp.In order to simultaneously visualize this unlinkage of two genes,dual-color fluorescence in situ hybridization(FISH) technique was applied to the cytogenetics of S.japonica.Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte,respectively,metaphase nuclei hybridized simultaneously with the labeled probes of 18 S rDNA and 5 S rDNA.Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18 S-5.8 S-25 S rDNA was localized at the interstitial region of Chromosome 23,whereas 5 S rDNA resided at the sub-telomeric region of Chromosome 27.These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene,and their loci on different chromosomes indicated the physical unlinkage of 5 S rDNA to 18 S-5.8 S-25 S rDNA in this kelp.Therefore,dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S.japonica.     

Phylogenetic analyses of four species of Ulva and Monostroma grevillei using ITS, rbc L and 18S rDNA sequence data

Chlorophyta species are common in the southern and northern coastal areas of China. In recent years, frequent green tide incidents in Chinese coastal waters have raised concerns and attracted the attention of scientists. In this paper, we sequenced the 18S rDNA genes, the internal transcribed spacer (ITS) regions and the rbcL genes in seven organisms and obtained 536-566 bp long ITS sequences, 1 377-1 407 bp long rbcL sequences and 1 718-1 761 bp long partial 18S rDNA sequences. The GC base pair content was highest in the ITS regions and lowest in the rbcL genes. The sequencing results showed that the three Ulva prolifera (or U. pertusa) gene sequences from Qingdao and Nan’ao Island were identical. The ITS, 18S rDNA and rbcL genes in U. prolifera and U. pertusa from different sea areas in China were unchanged by geographic distance. U. flexuosa had the least evolutionary distance from U. californica in both the ITS regions (0.009) and the 18S rDNA (0.002). These data verified that Ulva and Enteromorpha are not separate genera.     

Nematode Diversity of Qingdao Coast Inferred from the 18S Ribosomal RNA Gene Sequence Analysis

The 18S ribosomal DNA gene （18S rDNA） sequences （approxtmately 1300 bp in length） were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode specific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin61 restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Although it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve sequences were close to Pontonema vulgate and Adoncholaimus sp., four to Daptonema procerus and two （identical） to Enoplus brews. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.     

Molecular diversity of Enteromorpha from the coast of Yantai: a dual-marker assessment

We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS （internal transcribed spacer） and 5S rDNA NTS （non-transcribed spacer） sequences. The ITS sequences showed slight nucleotide divergences between Enteromorpha linza and Enteromorpha prolifera. In contrast, multiple highly variable regions were found in the ITS region of Enteromorpha flexuosa. In general, there were more variable sites in the NTS region than in the ITS region in the three species. The variations in 5S rDNA NTS sequences indicated that the molecular diversity of Enteromorpha from the coast of Yantai is very high. However, a phylogenetic tree constructed using 5S rDNA NTS sequence data indicated that genetic differences were not directly related to geographical distribution.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

1　Introduction　PorphyraisthemainobjectforalgafarmingandplaysaveryimportantroleinChinesemarineindus tries.Recently ,therehasbeenagreatlossinPor phyracultivationduetothedegenerationoftheculti var,sothereisanincreasingdemandforgoodPor phyracultivars .Theprerequisiteofthetraditionalbreed ingandbioengineeringresearchofPorphyraistheconstructionofpurelines .Traditionally ,theclassifi cationofPorphyrawasaccordingtotheirmorphologi calcharacteristics .However ,mostmorphologicalfea turesofPorphyraar…     

Molecular phylogeny of Parapenaeopsis Alcock, 1901 (Decapoda: Penaeidae) based on Chinese materials and 16S rDNA and COI sequence

The phylogenetic relationship among Chinese Parapenaeopsis species was studied by comparing 16S rDNA and COI gene sequence. The results showed that Parapenaeopsis hardwickii (Miers, 1878) and P. cultrirostris Alcock, 1906 identified from Chinese samples were the same species. Parapenaeopsis cornuta (Kishinouye, 1900), P. incisa Liu and Wang, 1987, and P. sinica Liu and Wang, 1987 were different species. However, their systematic relationship were closer than those of other species, and they formed an advanced branch in the phylogenetic trees. Additionally, the systematic position of P. hardwickii (Miers, 1878), P. hungerfordi Alcock, 1905, and P. tenella (Bate, 1888) were not stable in the phylogenetic trees. More genetic markers such as 18S rDNA and 28S rDNA were supposed to be used to confirm the phylogenic relationship of Parapenaeopsis species.     

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.     

Culture observation and molecular phylogenetic analysis on the blooming green alga Chaetomorpha valida (Cladophorales, Chlorophyta) from China

The marine green alga Chaetomorpha valida fouls aquaculture ponds along the coastal cities of Dalian and Rongcheng, China. Unialgal cultures were observed under a microscope to determine the developmental morphological characters of C. valida. Results reveal that gametophytic filaments often produce lateral branches under laboratory culture conditions, suggesting an atypical heteromorphic life cycle of C. valida between unbranched sporophytes and branched gametophytes, which differs from typical isomorphic alternation of Chaetomorpha species. The shape of the basal attachment cell, an important taxonomic character within the genus, was found variable depending on environmental conditions. The 18S rDNA and 28S rDNA regions were used to explore the phylogenetic affinity of the taxa. Inferred trees from 18S rDNA sequences revealed a close relationship between C. valida and Chaetomorpha moniligera. These results would enrich information in general biology and morphological plasticity of C. valida and provided a basis for future identification of green tide forming algae.     

Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop (Chlamys farreri).     

Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene （18S rDNA） libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18S rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.     

Integrating a DNA barcoding project with an ecological survey: a case study on temperate intertidal polychaete communities in Qingdao, China

In this study, we integrated a DNA barcoding project with an ecological survey on intertidal polychaete communities and investigated the utility of CO1 gene sequence as a DNA barcode for the classification of the intertidal polychaetes. Using 16S rDNA as a complementary marker and combining morphological and ecological characterization, some of dominant and common polychaete species from Chinese coasts were assessed for their taxonomic status. We obtained 22 haplotype gene sequences of 13 taxa, including 10 CO1 sequences and 12 16S rDNA sequences. Based on intra- and inter-specific distances, we built phylogenetic trees using the neighbor-joining method. Our study suggested that the mitochondrial CO1 gene was a valid DNA barcoding marker for species identification in polychaetes, but other genes, such as 16S rDNA, could be used as a complementary genetic marker. For more accurate species identification and effective testing of species hypothesis, DNA barcoding should be incorporated with morphological, ecological, biogeographical, and phylogenetic information. The application of DNA barcoding and molecular identification in the ecological survey on the intertidal polychaete communities demonstrated the feasibility of integrating DNA taxonomy and ecology.     

Amplicon-Based Illumina Sequencing and Quantitative PCR Reveals Nanoplankton Diversity and Biomass in Surface Water of Qinhuangdao Coastal Area,China

Aureococcus anophagefferens caused brown tides for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To understand the population dynamics of nanoplankton during the brown tides, sequences of the V9 region of the 18 S rDNA gene, used as a marker, were analyzed by Illumina sequencing to assess nanoplankton biomass, and real-time fluorescence quantitative PCR was performed to analyze spatial variation in the 18 S rDNA copy concentrations of nanoplankton off the Qinhuangdao coast in July, 2011. The results showed that A. anophagefferens and Minutocellus polymorphus were the dominant species in the local phytoplankton community during the brown tide in July 2011. The highest 18 S rDNA copy concentrations of A. anophagefferens and M. polymorphus were detected at stations SHG and FN, respectively. The central area most strongly affected by the brown tide migrated southward from 2011 to 2013. Redundancy analysis(RDA) showed that the decreasing NOx concentration might provide suitable nutrient conditions for the A. anophagefferens outbreak. During the brown tide caused by A. anophagefferens, other phytoplankton, such as diatoms, cryptophytes, chlorophytes, dinoflagellates and other flagellates, could co-occur with it. For zooplankton, due to less selective feeding behavior, Amoebozoa was the most abundant zooplankton at station SHG, while Ciliophora was the most abundant zooplankton at other stations for its more selective feeding.     

Morphological and karyotypic variation in three wild populations of Meretrix meretrix

Three wild populations of Meretrix meretrix sampled from Dongxing, Beihai, and Shankou along the coast of Guangxi, China, were investigated with morphometry and karyometry. Six morphological indices （shell length, shell height, shell width, hinge length, total wet weight and shell weight） were measured. Differences in all morphological indices except hinge length were significant among the three populations （P 〈 0.05）. The mean values of these indices （except for the hinge length） in the Dongxing population were larger than those in the Beihai and Shankou populations, although the latter had the largest hinge length. The karyotype of the Beihai, Shankou and Dongxing samples had ten metacentric, six submetacentric, and three subtelocentric chromosome pairs. No significant difference was shown in the centromeric index values of the chromosomes in the populations （P〉0.05）. However, the order of metacentric, submetacentric and subtelocentric chromosome pairs was variable among the three populations. The results indicate a high level of inter-population variation in morphology and karyotype.     

Preliminary study on chromosomes of induced triploid in the red sea bream,Pagrosomus major

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Development of high-resolution chloroplast markers for intraspecific phylogeographic studies of Phaeocystis globosa

Phaeocystis globosa is an important harmful algal bloom causative species distributing widely in temperate and tropical coastal waters in the world.The morphological,physiological,and biochemical characte ristics are different amo ng geographic strains,which can not be distinguished with nuclear ribo somal DNA markers at present.Therefore,the genetic distance and phylogeographic relationships of nuclear 28 S rDNA D1-D2 and ITS regions,and three chloroplast intergenic spacers(petN-trnS1,trnM1-psbA,and rbcS-rpl27) were analyzed and compared among 13 strains of P.globosa isolated from the Pacific Ocean and Atlantic Ocean in this study.In addition,the nucleotide polymorphisms of 28 S rDNA D1-D2,ITS,and rbcS-rpl27 regions were evaluated in two P.globosa strains.The various levels of nucleotide polymorphism were in the nuclear 28 S rDNA D1-D2 region and ITS region,but no polymorphism was in the chloroplast rbcS-rpl27 intergenic spacer.A reasonable intraspecific phylogeographic relationship was presented by rbcS-rpl27 intergenic spacer,which had the strongest distinction to geographic strains compared to those of 28 S rDNA D1-D2 and ITS regions.In the phylogenetic tree of rbcS-rpl27 intergenic spacer,the two strains from the North Sea of the Atlantic Ocean were divided firstly from the species of P.globosa,and then formed an independent clade,while the other Atlantic strains and all of Pacific strains j oined up to build the other clade.It was implied that at least two genetically distant populations of P.globosa existed in the Atlantic coastal regions.This study provided a high-resolution chloroplast marker to analyze intraspecific phylogeographic populations of P.globosa,and preliminarily clarified the genetic relationships of the Pacific and Atlantic strains of P.globosa.     

Phylogenetic analysis of bacteria in sea ice brine sampled from the Canada Basin, Arctic Ocean

Bacterial diversity in sea ice brine samples which collected from four stations located at the Canada Basin, Arctic Ocean was analyzed by PCR-DGGE. Twenty-three 16S rDNA sequences of bacteria obtained from DGGE bands were cloned and sequenced. Phylogenetic analysis clustered these sequences within γ-proteobacteria, Cytophaga-Flexlbacter-Bacteroides （CFB） group, Firmicutes and Actinobacteria. The phylotype of Pseudoalteromonas in the γ-proteobacteria was predominant and members of the CFB group and γ-proteobacteria were highly abundant in studied sea ice brine samples.     

A NEW SPECIES OF THE GENUS NEROCILA (ISOPODA: CYMOTHOIDAE) FROM THE EAST CHINA SEA

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Molecular phylogenetic analysis of dinoflagellate Scrippsiella trochoidea isolated from the East Asian waters

Previous studies found intraspecific diversity in Scrippsiella trochoidea A. R. Loeblich III, a widely distributed calcareous cyst-producing dinoflagellate. In this study, three strains (ST-1, ST-D6 and ST-K) of S. trochoidea isolated from the East Asian waters were studied, together with other geographical strains, to resolve their phylogenetic relationships. For the three East Asian isolates, two highly diverse regions of nuclear-encoded ribosomal DNA (rDNA), the 5.8S rDNA and its flanking internal transcribed spacers 1 and 2, and the 5′ portion of the large-subunit rDNA (encompassing the “D1“ and “D2” domains) were sequenced. Homologous sequences from other geographical isolates were selected from the GenBank database and the phylogenetic relationships were inferred from the molecular data of these strains. Strains of S. trochoidea were found to cluster into three major clades (STR1, STR2 and STR3), as reported in previous studies. Two of the three strains ST-1 and ST-K, were grouped in clade STR2, the other strain, ST-D6, belonged to clade STR3. The intraspecific diversity of S. trochoidea in East Asian waters makes it necessary to carry out phylogenetic investigations in combination with data of biogeography, population dynamics, and life cycle on the ecophysiology of a region.