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.     

Molecular cytogenetic of the Amoy croaker,A rgyrosomus amoyensis(Teleostei,Sciaenidae)

The family Sciaenidae is remarkable for its species richness and economic importance. However, the cytogenetic data available in this fish group are still limited, especially those obtained using fl uorescence in situ hybridization(FISH). In the present study, the chromosome characteristics of a sciaenid species, Argyrosomus amoyensis, were examined with several cytogenetic methods, including dual-FISH with 18 S and 5 S rDNA probes, and a self-genomic in situ hybridization procedure(Self-GISH). The karyotype of A. amoyensis comprised 2 n=48 acrocentric chromosomes. A single pair of nucleolar organizer regions(NORs) was located at the proximal position of chromosome 1, which was positive for silver nitrate impregnation(AgNO_3) staining and denaturation-propidium iodide(DPI) staining but negative for Giemsa staining and 4',6-diamidino-2-phenylindole(DAPI) staining, and was confi rmed by FISH with 18 S rDNA probes. The 5 S rDNA sites were located at the centromeric region of chromosome 3. Telomeric FISH signals were detected at all chromosome ends with dif ferent intensities, but internal telomeric sequences(ITSs) were not found. Self-GISH resulted in strong signals distributed at the centromeric regions of all chromosomes. C-banding revealed not only centromeric heterochromatin, but also heterochromatin that located on NORs, in interstitial and distal telomeric regions of specifi c chromosomes. These results suggest that the karyotype of Amoy croaker was relatively conserved and primitive. By comparison with the reported cytogenetic data of other sciaenids, it can be deduced that although the karyotypic macrostructure and chromosomal localization of 18 S rDNA are conserved, the distribution of 5 S rDNA varies dynamically among sciaenid species. Thus, the 5 S rDNA sites may have different evolutionary dynamics in relation to other chromosomal regions, and have the potential to be ef fective cytotaxonomic markers in Sciaenidae.     

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.     

Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.     

3种柔鱼线粒体基因与核核糖体基因片段序列比较分析

为准确检测柔鱼（Ommαstrephesbartram川、茎柔鱼（ Dosidicus gigas）与阿根廷滑柔鱼（ IIIex argentinus ） 的种间遗传差异,对线粒体16SrRNA、细胞色素b（Cytb）与编码核糖体大亚基的基因（28SrDNA）片段序列进 行测定。经比对获得同源片段序列的长度分别为444、430、464坤,其中16SrRNA与28SrDNA基因片段上分别存在3处和47处碱基插入/缺失。核昔酸组成分析表明;3种柔鱼在3个基因片段上的核音酸组成差异不显著, 在线粒体2个基因片段上的A＋T含量（16SrRNA;69.90%、72.01%、74.66%; Cytb; 63.61%、68.91%、71.65% ） 均明显高于C＋C含量（16SrRNA;30.10%、27.99%、25.34%; Cytb; 36.39%、31.09%、28.35% ）,而在28SrDNA 基因片段上的A＋T含量（37.16%、36.74%、38.29% ）明显低于C＋C含量（62.84%、63.26%、61.71 %）0 3种柔鱼 在28SrDNA基因片段上检测到的核昔酸替代率最低,为6.68%,而蛋白质编码基因Cytb核昔酸替代率最高,为 20.93%,核营酸替代均发生在密码子第3位点上,而且未引起氨基酸替代。基于邻接法、最大简约法与最大似然 法重建的系统树显示,柔鱼与茎柔鱼的亲缘关系较近。根据C严b基因片段序列分析,柔鱼与茎柔鱼和阿根廷滑柔鱼的分歧时间分别为653-790万a和765 - 925万a,种间分化事件发生在中新世至上新世间。     

Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.     

Identification of Microplankton in South China Sea with Image-Matching Individual PCR

In this study, marine microplankton were identified by combining standard light microscopy with Sanger 18S rRNA gene sequencing. The image-matching individual PCR technique was applied to identify the image collectable unicellular microplankton to genera. Instead of pure strain culture and morphological identification, microplankton individual cells were isolated and fixed with glutaraldehyde, frozen and stored for months. Finally, they were imaged under a microscope and molecularly identified via phylogenetic analysis of their 18S ribosomal RNA gene(18S rDNA). Microplankton cells were collected at 30 locations in South China Sea, and were assigned to 21 known and 4 unidentified genera(2 uncultured fungi and 2 uncultured stramenopiles) with phylogenetic analysis in parallel to the morphological identification.     

Molecular survey of sulphate-reducing bacteria in the deep-sea sediments of the west Pacific Warm Pool

The sulfate-reducing bacteria (SRB) community in the deep-sea sediments of the west Pacific Warm Pool (WP) was surveyed by molecular phylogenetic analyses using primers targeting the 16S rRNA gene fragments of SRB. Specific 16S rRNA gene libraries from five sediment layers (1-cm, 3-cm, 6-cm, 10-cm and 12-cm layer) of the 12-cm core of WP-0 were constructed. The clones in the five libraries were differentiated by restriction fragment length polymorphism (RFLP) and representative clones were selected to sequence. It was found that the clones fell into four groups, which were closest related to Desulfotomaculum, Desul- facinum, Desulfomonile and Desulfanuticus. Desulfacinum-like clones were only detected in the upper layers of the sediment core, whereas Desulfomonile-like clones were only present in the deeper layers. Fluorescence in situ hybridization (FISH) was further carried out to visualize and count the SRB and bacteria in the five sediment layers. It was found that SRB constituted only a small proportion of the bacteria community (0.34%-1.95%), it had the highest content in the 3-cm layer (1.95%) and had a depth- related decreasing tendency along the 12- cm core.     

Isolation and identification of bacteria associated with the surfaces of several algal species

We conducted this study to assess the diversity of bacteria associated with the surfaces of algae based on 16S rDNA sequence analyses.Twelve strains of bacteria were obtained from the surfaces of the following four species of algae:Gracilaria textorii,Ulva pertusa,Laminaria japonica,and Polysiphonia urceolata.The isolated strains of bacteria can be divided into two groups:Halomonas and Vibrio,in physiology,biochemical characteristics and 16S rDNA sequence analyses.The phylogenetic tree constructed based on ...     

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).     

Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii

Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill) stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively.     

Archaeal diversity and abundance within different layers of summer sea-ice and seawater from Prydz Bay,Antarctica

Fluorescence in-situ hybridization （FISH） and 16S rRNA gene clone library analyses were used to determine the abundance and diversity of archaea in Prydz Bay, Antarctica. Correlation analysis was also performed to assess links between physicochemical parameters and archaeal abundance and diversity within the sea-ice. Samples of sea-ice and seawater were collected during the 26th Chinese National Antarctic Research Expedition. The results of FISH showed that archaea were relatively abundant within the top layer of the sea-ice, and correlation analysis suggested that the concentration of 4NH＋ might be one of the main factors underlying this distribution pattern. However, using 16S rRNA gene libraries, archaea were not detected in the top and middle layers of the sea-ice. All archaeal clones obtained from the bottom layer of the sea-ice were grouped into the Marine Group I Crenarchaeota while the archaeal clones from seawater were assigned to Marine Group I Crenarchaeota, Marine Group II Euryarchaeota, and Marine Group III Euryarchaeota. Overall, the ifndings of this study showed that the diversity of archaea in the sea-ice in Prydz Bay was low.     

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.     

16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea,Echinodermata)

Cultured Apostichopusjaponicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopusjaponicus individuals.     

Structure analysis of the complete mitochondrial genome in cultivation variety ��Rongfu��

In this report, complete mitochondrial genome sequences of Laminaria cultivation variety ‘Rongfu’ were obtained. The results showed the length of circular molecule of mtDNA was 37 638 bp (64.7% A+T), encoding three rRNAs (23S, 16S and 5S), 25 tRNAs, 35 known mitochondrial proteins and 3 ORFs. Sequence alignment indicated its mtDNA genome was very similar to that of Laminaria japonica. Phylogenetic trees inferred from concatenated 30 mitochondrial genes showed that ‘Rongfu’, Laminaria japonica, Laminaria longipedalis, Laminaria diabolica, Laminaria religiosa and Laminaria ochotensis clustered together. In addition, compared with mitochondrial genome of L. japonica, ‘Rongfu’ mtDNA lacked a non-coding region of 19 nucleotides, which was located between rRNA small subunit gene 3 (rps3) and rRNA small subunit gene 9 (rps9). Seven cultivation varieties of China were divided into two groups based on this non-coding region which was absent in ‘Rongfu’, ‘Fujian’ and ‘Sanhai’ while present in ‘Ailunwan’, ‘Dongfang No.2’, ‘Dongfang No.3’ and ‘Zaohoucheng’. So this variation can be used in germplasm identification of cultivation variety.     

Sequence analysis of the ITS region and 5.8S rDNA of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The sequences of the ITS(internal transcribed spacer) and 5.8S rDNA of three cultivated strains of Porphyra haitanensis thalli(NB,PT and ST) were amplified,sequenced and analyzed.In addition,the phylogenic relationships of the sequences identified in this study with those of other Porphyra retrieved from GenBank were evaluated.The results are as follows:the sequences of the ITS and 5.8S rDNA were essentially identical among the three strains.The sequences of ITS1 were 331 bp to 334 bp,while those of the 5.8...     

钦州湾牡蛎线粒体16S rRNA和COⅠ基因片段的序列变异分析

利用线粒体16S rRNA基因和线粒体细胞色素氧化酶亚基Ⅰ(cytochrome oxidaseⅠ,COⅠ)基因片段初步研究钦州湾牡蛎(Ostrea)的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16S rRNA基因部分长度为413bp,COⅠ基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A+T比例:16S rRNA基因59.8%;COⅠ基因60.5%。对位排序比较表明,16S rRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COⅠ片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎(Crassostrea hongkongensis)16S rRNA和COⅠ序列与白肉牡蛎的遗传距离均为0.000,有明巨牡蛎(Crassostrea ariakensis)16S rRNA和COⅠ序列和红肉牡蛎(red meat ostrea)的遗传距离分别为0.000和0.011,白肉牡蛎16S rRNA和COⅠ序列和红肉牡蛎之间的遗传距离分别为0.035和0.146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16S rRNA和COⅠ基因在种间存在明显的多态性,证实了16S rRNA和COⅠ基因序列适用于牡蛎的系统学分析。     

Primary species recognitionand phylogeny of Chondrus （Gigartinales, Rhodophyta） using 18S rDNA sequence data

The nuclear-encoded small subunit ribosomal RNA gene (18S rDNA) of 16 isolates of Chondrus from 8 countries were sequenced. A total of 1796 nucleotides were obtained and aligned with the phylogenetic analysis conducted. The results suggest that the entity from Dalian, China, regarded as C. sp1 is C. pinnulatus. The C. sp2 previously depicted as C. yendoi or Mazzaella japonica may belong to genus Chondrus. So, 4 Chondrus species, i.e. C. ocellatus, C. nipponicus, C. armatus, and C. pinnulatus are distributed in China. However, the entity from Connemara, Ireland, named C. crispus, is not a Chondrus species but that of Mastocarpus stellatus, although it is morphologically similar to C. crispus. Phylogenetic analysis based on complete 18S rDNA sequence data shows that genus Chondrus includes 3 main lineages: the Northern Pacific lineage, containing C. ocellatus, C. yendoi, and C. nipponicus; C. armatus, and C. pinnulatus form the sub-North Pacific lineage; and the Northern Atlantic Ocean lineage, comprising samples of C. crispus from Canada, Portugal, Ireland, Germany and France. The phylogenetic relationships indicate that genus Chondrus might have a North Pacific ancestral origin, radiated to North Atlantic area, and then formed the species C. crispus.     

钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析

利用线粒体16SrRNA基因和线粒体细胞色素氧化酶亚基Ⅰ（cvtocllrome oxidase Ⅰ,COI）基因片段初步研究钦州湾牡蛎（Ostxea）的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16SrRNA基因部分长度为413bp,COI基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A＋T比例：16SrRNA基因598％;COI基因605％。对位排序比较表明,16SrRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COI片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎（Crassostrea hongkongensis）16SrRNA和COI序列与白肉牡蛎的遗传距离均为0000,有明巨牡蛎（Crassostrea ariakensis）16SrRNA和COI序列和红肉牡蛎（redmeatostxea）的遗传距离分别为0000和0011,白肉牡蛎16SrRNA和COI序列和红肉牡蛎之间的遗传距离分别为0035和0146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16SrRNA和COI基因在种间存在明显的多态性,证实了16SrRNA和COI基因序列适用于牡蛎的系统学分析。