Species identification of <Emphasis Type="Italic">Pinctada imbricata</Emphasis> using intergenic spacer of nuclear ribosomal RNA genes and mitochondrial 16S ribosomal RNA gene regions

ABSTRACT:   For pearl production, pearl oyster seeds from foreign pearl oysters as well as hybrids between native and such foreign pearl oysters are produced in Japanese hatcheries. However, it is very difficult to identify these pearl oysters and hybrids based on morphological measurements. Thus, a molecular identification method for distinguishing Atlantic pearl oysters Pinctada imbricata from the Indian-Pacific pearl oyster group including P. martensii and P. fucata , was developed. The polymerase chain reaction (PCR) products of the partial intergenic spacer (IGS) of nuclear ribosomal RNA (rRNA) genes exhibited length polymorphism between P. imbricata (590 bp) and the other two species (427 bp). The restriction fragment length polymorphism analysis of the PCR products (PCR-RFLP) cleaved with Mse  I observed in the IGS of nuclear rRNA genes also gave different profiles between P. imbricata and the other two species. The difference in PCR-RFLP using Alu  I was also detected in the mitochondrial 16S rRNA gene regions between P. imbricata and the other two species. Thus, the method developed enables the distinction of P. imbricata from P. martensii and P. fucata .     

Ecology,physiology and genetics of a phycodnavirus infecting the noxious bloom-forming raphidophyte <Emphasis Type="Italic">Heterosigma akashiwo</Emphasis>

Abstract:   Heterosigma akashiwo virus (HaV) is a large icosahedral virus (∼0.2 μm) harboring a double-stranded DNA (dsDNA) genome (∼294 kbp). The virus is the only member of the genus Raphidovirus in the family Phycodnaviridae. Since its first discovery, a number of ecologic, physiologic and genetic studies about HaV have been conducted; especially, the relationship between H. akashiwo and HaV in nature was studied and viral infection is now regarded as a significant factor influencing the dynamics and termination of H. akashiwo blooms. HaV infection has considerable impacts on H. akashiwo populations in both aspects of fluctuation in biomass (quantity) and changes in clonal composition (quality). Partial sequencing of the HaV genome revealed that a number of genes showed considerable similarity to those of other protist-infecting viruses; still, the phylogenetic position of HaV suggested a number of enigmas in host–virus coevolution. Here are summarized the ecology, physiology and genetics of HaV especially from the viewpoint of the host–virus relationship.     

小球藻psbA基因的克隆与序列分析

psbA基因编码光合系统II反应中心的D1蛋白,是叶绿体基因组中一个重要的光调控基因。对小球藻属4个种的6株小球藻psbA基因进行了PCR扩增和测序,并结合GenBank上已有的2条小球藻psbA基因序列,使用MEGA3.1软件对psbA序列进行了遗传距离值的计算和系统发育树的构建。结果显示8株小球藻psbA基因的遗传距离值为0.012~0.167。除了原始小球藻F-2和蛋白核小球藻820分别与蛋白核小球藻F-5、F-9及与普通小球藻Cvq关系极近之外,其他6株小球藻基本上按照种的划分进行聚类,该结果与用小球藻的ITS和rbcL序列分析的结果一致。     

Mitochondrial DNA variation and population structure of the Japanese mitten crab Eriocheir japonica

ABSTRACT:   The Japanese mitten crab Eriocheir japonica is a common grapsid species found throughout freshwater and estuarine regions in Japan. In order to obtain information on the genetic variation and population structure of this species, a polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis was conducted on the cytochrome oxidase subunit I (COI) of mitochondrial DNA, on 666 individuals from 19 sample sites covering the three main geographic regions of Japan (Main Islands, Okinawa, and Ogasawara). Genetic analysis using seven restriction enzymes produced an array of 61 composite haplotypes. Three regional groups corresponding to the three geographic regions were clearly identified by cluster and molecular variance model ( amova ) analyses. Each of the three groups showed dominant haplotypes that were almost completely absent in populations from the other geographic areas. Comparison with published information for other species indicates that the degree of genetic divergence between these three main groups is equivalent to the genetic distance between congeneric species. Thus, the population structure of the Japanese mitten crab, as inferred from mtDNA analysis, is formed by genetically distinct groups that closely reflect their geographic distribution in the Japanese archipelago as well as restricted gene flow.     

Species identification method for <Emphasis Type="Italic">Scombrops boops</Emphasis> and <Emphasis Type="Italic">Scombrops gilberti</Emphasis> based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

ABSTRACT:   Gnomefish Scombrops boops and Scombrops gilberti are commercially important fishes in Japan, but these species are often confused in the markets because of their morphological similarity. To identify these two species, we performed nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis on 16S ribosomal RNA (rRNA) gene and the control region in mitochondrial DNA. Five and 12 nucleotide substitutions were observed between species in the 777-bp 16S rRNA gene and 471-bp control region, respectively. Diagnostic restriction sites for discriminating between S. boops and S. gilberti were found in the 16S rRNA gene, but not in the control region. Polymerase chain reaction (PCR)–RFLP analysis using two enzymes, Eco NI and Mva I, clearly discriminated between S. boops and S. gilberti identified by meristic characters. The PCR–RFLP analysis identified most of the 168 Scombrops young caught in the coastal waters of the Izu and Miura peninsulas as S. boops , suggesting that S. gilberti juveniles are rare in this area.     

湖北地区克氏原螯虾白斑综合征病毒变异区ORF14/15、ORF23/24基因序列比较分析

试验扩增、克隆了在湖北地区采集的19份克氏原螯虾(Procambarus clarkii)白斑综合征病毒(White spot syndrome virus,WSSV)阳性样品的变异区ORF14/15和ORF23/24基因,通过测序比较分析了湖北各WSSV毒株与Gen Bank公布的标准毒株间在变异区ORF14/15及ORF23/24基因的差异性。结果显示,19份WSSV阳性样品中有部分样品在变异区扩增出ORF14/15、ORF23/24基因片段,变异区基因序列分析发现,与Gen Bank已公布的标准毒株相比,存在大片段缺失。在变异区ORF14/15,有3个毒株扩增出1 442 bp的片段,4个毒株扩增出630 bp的片段,基于变异区ORF14/15构建的系统进化树显示,这些毒株归属两个不同的分支。在变异区ORF23/24,有2个毒株扩增出大小为2 096 bp的片段,进化分析发现这2个毒株在变异区ORF23/24的遗传距离较近。     

Genetic diversity and population history of the migratory catfishes Pangasianodon hypophthalmus and Pangasius bocourti in the Cambodian Mekong River

ABSTRACT:   Polymerase chain reaction (PCR) restriction fragment length polymorphism analysis of mitochondrial DNA was applied to the genetic structure and evolutionary history of the more ancestral Pangasianodon hypophthalmus ( n  = 82), and the recently speciated catfish Pangasius bocourti ( n  = 90) from the Cambodian Mekong River. Both pangasiids were characterized by a lack of genetic population structure that may result from high levels of contemporary gene flow. Genetic diversity was lower in P. hypophthalmus than in P. bocourti . However, a different evolutionary history was inferred for both species based on genealogical and demographic analyses (mismatch analysis, Tajima's D- and Fu's F _S-tests). The genetic profile of the more ancestral P. hypophthalmus shows indications of a recent population bottleneck, whereas the recently speciated P. bocourti shows signatures of historical population expansion. This study stresses the importance of preserving the migration routes in the Cambodian Mekong basin in order to maintain the genetic diversity and long-term integrity of both species.     

基于matK、rbcL和ITS序列的5种大叶藻系统发育研究

运用PCR直接测序法,对采自爱尔兰、日本、韩国和中国的大叶藻、矮大叶藻、丛生大叶藻和红须根虾形藻及GenBank中的宽叶大叶藻5种大叶藻叶绿体的matK、rbcL和核糖体ITS的部分序列进行测定,并比较分析了5种大叶藻3个目的片段的核苷酸序列,结果发现胞嘧啶(C)在3个目的片段上的含量均较低。ITS片段检测到172处核苷酸替换,表现出丰富的遗传多态性。matK基因片段上有52处核苷酸替换,rbcL基因片段上有20处核苷酸替换,且大部分替换来自于第三密码子的同义替换,种间在氨基酸水平上产生了一定的分化。基于ITS、matK和rbcL 3个目的片段,利用邻接法、最大简约法、最大似然法和贝叶斯法构建的系统发育树结果基本一致,明显分为3大支。矮大叶藻与大叶藻属间3种大叶藻的核苷酸最小差异值为19.33%,在分子数据上达到了属的水平。基于matK和rbcL基因片段拼接序列的分析结果表明:大叶藻与丛生大叶藻的分化时间在上新世(2～2.7百万年前),与宽叶大叶藻的分化时间在上新世(4～5.3百万年),与矮大叶藻的分化时间在渐新世(27～36百万年前),与红须根虾形藻的分化时间在始新世(33～44百万年前)。4个地区大叶藻的...     

Simple differentiation of two closely related species Porphyra tenera and Porphyra yezoensis (Bangiophyceae, Rhodophyta) based on length polymorphism of actin-related protein 4 gene (ARP4)

ABSTRACT:   Genetic polymorphisms were investigated to develop a simple and rapid method to differentiate between the two closely related species, Porphyra tenera and Porphyra yezoensis . Polymerase chain reaction (PCR) using the specific primer pair of the ARP4 gene gave length polymorphic single fragments of genomic DNAs from five strains of P. tenera (Japan T-8, JTW; Korea KTY1, KTY2, KTY3) and seven strains of P. yezoensis (Japan TU-1, TU-2, TUH-25, JHU, JA-1; Korea KGJ, KPH). All strains of P. yezoensis had introns 60 bp longer than that of P. tenera . Multiple cleaved amplified polymorphic sequence (CAPS) markers were also developed to differentiate P. tenera and P. yezoensis . This is the first report of length polymorphisms that can be used to differentiate between the two species using only PCR amplification with agarose gel electrophoresis. It is expected that the length polymorphism and plentiful CAPS profiles obtained in this study will be useful in the assessment of genetic diversity within P. tenera and P. yezoensis as well as in breeding science that requires the collection of various strains of the two species.     

Complete nuclear ribosomal DNA sequence analysis of Pacific abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Pacific abalone Haliotis discus hannai (Haliotidae, Gastropoda) is an economically important shellfish species in northern China. The complete nuclear ribosomal DNA (nrDNA) of Pacific abalone was amplified, sequenced, and analyzed. The length of the nrDNA was determined to be around 10.7 kb, and to contain, in order, small subunit ribosomal RNA (nrSSU) genes (1871 bp), internal transcribed spacer (ITS, 759–762 bp), large subunit ribosomal RNA (nrLSU) gene (3411 bp), and an intergenic spacer (IGS, 4624–4654 bp). The SSU and LSU regions were almost identical in different individuals, and show little variation from those of other abalone species. The two different variations of the ITS2 region were presented, and this phenomenon also existed in other species. A phylogeny tree was constructed, based on ITS region sequence datasets, to determine the evolutionary relationships of abalones. Abalones have two major subclades, mainly distributed in the North Pacific, Europe and Australia. The IGS region of the nrDNA was sequenced and analyzed for the first time. Several repeat fragments were present upstream of the sequence, and were significantly different between individuals (93.86% sequence identity). The complete nrDNA sequence will be useful for the classification, identification, phylogeny, germplasm management, and breeding of this shellfish.     

Changes in intracellular polyamine concentration during growth of Heterosigma akashiwo (Raphidophyceae)

ABSTRACT:   Of the free, conjugated and bound forms of polyamines, the free form of spermidine was the most abundant polyamine in Heterosigma akashiwo throughout the growth period except for the lag phase. Free spermidine content increased remarkably during the exponential growth phase and increased as the growth rate increased. The maximum growth yield of H. akashiwo was reduced by the addition of methylglyoxal bis-guanylhydrazone (MGBG) and the reduced growth yield could be counteracted by the addition of spermidine to the medium. It is concluded that spermidine plays a significant role in the growth of H. akashiwo . These results are similar to those obtained in Chattonella antiqua that belongs to same taxonomic Class as H. akashiwo .     

Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae

A study was conducted to evaluate the probiotic properties of endogenous rainbow trout microbiota against pathogenic Lactococcus garvieae. A total of 335 bacterial strains were isolated from rainbow trout and screened for antagonistic activity against L. garvieae using an agar spot assay. Antagonistic strains were grouped by PCR amplification of repetitive bacterial DNA elements (rep‐PCR) and identified by 16S rRNA gene sequence analysis. The results revealed that the antagonistic strains belonged to the genera Lactobacillus, Lactococcus and Leuconostoc. Further probiotic characteristics, such as specific growth rate, doubling time, resistance to biological barriers, antibiotic resistance, hydrophobicity and production of antimicrobial substances, were also studied. These strains were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The antagonistic efficacy was maintained after sterile filtration and was sensitive to proteinase K, indicating that proteinaceous extracellular inhibitory compounds were at least partially responsible for pathogen antagonism. Based on these results, these strains should be further studied to explore their probiotic effects in challenge experiments in vivo. This study shows clear evidence that the indigenous trout‐associated microbiota may provide a defensive barrier against L. garvieae.     

Complete mitochondrial DNA sequence,gene organization and genetic variation of control regions in <Emphasis Type="Italic">Parargyrops edita</Emphasis>

ABSTRACT:   The complete nucleotide sequence of the mitochondrial genome of Parargyrops edita was determined by gene amplification using long polymerase chain reaction and direct sequencing techniques. The genome with 16 640 bp contained 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes). The content and order of genes was identical to those in typical teleosts. The major non-coding region of 964 bp in length with several conserved sequence features were identified as the control region (CR). Both COI and ND4 began with a GTG start codon, which is unusual in other fishes. The genetic variation of the CR from 27 wild samples was evaluated. A total of 536 bp consensus sequence of CR was aligned and 26 unique haplotypes were identified. The haplotype diversity (h) was estimated to be 0.997 (standard deviation [SD] 0.011) and nucleotide diversity ( π ) was 0.014 (SD 0.008) for the sample collected from coastal waters of Guangdong Province. It showed a very high level of genetic variation in the sample and also suggested that mtDNA CR sequence analysis can be use to evaluate the genetic diversity and genetic structure of P. edita .     

两种常见外来入侵赤潮藻的PCR鉴定

米氏凯伦藻与环状异帽藻为日本海域流入我国的两种外来入侵赤潮藻。利用核糖体ITS区分别设计出针对该两种藻的特异性PCR引物。通过prmier5.0软件设计多对引物,经PCR扩增,琼脂糖凝胶电泳检测、以筛选目标藻的特异性引物,并以链状亚历山大藻、立玛原甲藻、牟氏角毛藻、赤潮异弯藻作为阴性对照,做进一步PCR验证。筛选到米氏凯伦藻最佳引物Ki1F3/Ki1B3和环状异帽藻最佳引物YiF3a/YiB3a。两对特异性引物成功鉴定了两种外来入侵藻,而对其它藻种则是阴性反应,可为赤潮的预测预报提供分子鉴定基础。     

Identification and molecular typing of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> isolated from pond-cultured tilapia in China

Bacteria strains with strong virulence were isolated from pond-cultured tilapia in China. They were identified as Streptococcus agalactiae by biochemical assays, and confirmed by 16S ribosomal RNA (rRNA) and group B Streptococcus (GBS)-specific gene cfb analyses. Multiplex polymerase chain reaction (PCR) assay of the alpha C protein (ACP) gene and capsular polysaccharide antigen (cps) gene was employed to identify their molecular serotype (MS). Amplification of the ACP gene produced a 400-bp C alpha protein gene (bca) fragment, suggesting that these isolates belong to MS Ia, Ib or II; amplification of cps produced a 790-bp amplicon, indicating that they belong to MS Ia/III-3. An additional PCR based on nucleotide difference in the cps H–I region of MS Ia and III further suggested that the isolates belong to serotype MS Ia. Moreover, multi-locus sequence typing (MLST) indicated that these strains were of sequence type 7 (ST-7). These results showed that isolates from different regions of China shared the same MS and ST. However, none of the isolated ST-7 GBS corresponded to the capsular serotype, suggesting that these fish GBS possessed specific molecular characteristics not present in human or other animals. Data from this study will facilitate the understanding of epidemiology and nosogenesis of tilapia GBS and the establishment of effective disease prevention methods.     

Development of a novel PCR assay based on the 16S-23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR‐based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S–23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false‐positive results with non‐L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.     

Genetic diversity and divergence among clam <Emphasis Type="Italic">Cyclina sinensis</Emphasis> populations assessed using amplified fragment length polymorphism

ABSTRACT:   The genetic diversity and population structure of Cyclina sinensis were assessed using amplified fragment length polymorphism (AFLP). Three hundred and fifty-four AFLP loci were analyzed in 160 individuals collected from Lvshun, Lianyungang, Yueqing and Dongxing of coastal China. High levels of genetic diversity were detected, where the percentage of polymorphic loci and average expected heterozygosity ranged 88.4–98.9% and 0.304–0.365, respectively. Analysis of molecular variance showed that variation among populations (24.4%) was highly significant ( P  < 0.001). Accordingly, the global fixation index ( θ _B) averaged over loci was high (0.205). The large genetic differences among populations indicate restricted gene flow, congruent with limited dispersal capability of C. sinensis . The unweighted pair group method with arithmetic mean (UPGMA) tree topology constructed on the basis of Nei's genetic distances between populations showed a clear separation of the northern two populations from the southern two populations, suggesting that gene flow between the northern and southern regions is extremely low. This finding is additionally supported by the separate clustering of the four populations in the results of principal coordinate analysis. The useful information obtained in this study will offer insights to fine-tune conservation and fishery management measures in the future.     

Rapid identification of eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">Anguilla anguilla</Emphasis> by polymerase chain reaction with single nucleotide polymorphism-based specific probes

ABSTRACT:   Standard molecular techniques, such as sequencing and restriction fragment length polymorphism analysis after polymerase chain reaction (PCR) amplification are relatively complicated, and species identification can take a long time when using such techniques. We established a quick method, using PCR with species-specific TaqMan Minor Groove Binder (MGB) probes based on single nucleotide polymorphism (SNP) to distinguish the two eel species Anguilla japonica and Anguilla anguilla . This method can be used in processed products. Partial sequences of the mitochondrial 16S rRNA gene were compared between A. japonica and A. anguilla to design a primer pair common to both A. japonica and A. anguilla and probes specific to A. japonica and A. anguilla . Different fluorescence intensities were produced in two PCR microtubes each containing A. japonica - and A. anguilla -specific probes for one target sample. We observed the fluorescence intensity of PCR products in microtubes under ultraviolet transillumination, with similar results to those obtained by real-time PCR. Therefore, SNP-based PCR is a powerful tool for identifying materials of processed foods from either A. japonica or A. anguilla .