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.     

Characterization of a cDNA encoding a homolog of actin-related protein 4 from a marine red alga Porphyra yezoensis

ABSTRACT:   A cDNA ( PyARP4 ) containing an open reading frame for a protein of 573 amino acids was identified in the marine red alga Porphyra yezoensis . The conceptual PyARP4 protein exhibits significant similarity to actin-related protein (ARP) 4 in the terrestrial plant Arabidopsis . Comparison of the deduced amino acid sequence showed moderate sequence identity (30%) to a conventional actin in P. yezoensis , as seen in comparisons between ARP and conventional actins of other organisms. A putative bipartite nuclear localization signal and an actin motif were found within the PyARP4 amino acid sequence. In a phylogenetic analysis, the PyARP4 was found to cluster with the ARP4 of other organisms. The expression level of PyARP4 did not change significantly among four developmental stages of life cycle and was lower than that of a conventional actin. This cDNA therefore may serve as a useful internal standard in gene expression analyses of differentially expressed genes in P. yezoensis .     

Genetic comparison between torafugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and its closely related species karasu <Emphasis Type="Italic">Takifugu chinensis</Emphasis>

Abstract:   Sequence analyses of mitochondrial (mt) and nuclear genes were performed for genetic comparison between two Takifugu pufferfish species: torafugu T. rubripes and karasu T.  chinensis . With a sequence coverage of 20% in mtDNA, 640, 308, 344, 522 and 697 bp encoding mt 16S ribosomal RNA (rRNA), adenosine triphosphatase 6 ( ATPase 6 ), nicotinamide adenine dinucleotide dehydrogenase subunit 4 ( ND4 ), ND5 and cytochrome b (cyt b ), respectively, among 24 wild torafugu, 24 wild karasu and six hybrid-like samples, 15% of the torafugu identified by external color patterns showed nucleotide sequences consistent with karasu. Meanwhile, sequences of 60% karasu were consistent with those registered for torafugu (AJ421455). As for the hybrid-like samples, two possessed karasu-specific sequences in some base positions while torafugu-specific sequences in others. The remaining hybrid-like samples possessed torafugu-specific sequences. On the other hand, the mt control region did not show such type of consistency. Analysis of nuclear melanocortin receptor genes ( MC1R , MC4R ) among 54 samples showed 99–100% inter- and intraspecific sequence identity. Partial nuclear 18S  rRNA, complete internal transcribed spacer 1 ( ITS1 ), partial 5.8S  rRNA and ITS2 genes showed similar levels of identity, indicating a very low level of variation in their respective gene fragments between the two Takifugu species.     

Natural hybridization between <Emphasis Type="Italic">Pinctada fucata</Emphasis> and <Emphasis Type="Italic">Pinctada maculata</Emphasis> inferred from internal transcribed spacer regions of nuclear ribosomal RNA genes

ABSTRACT:   Genetic evidence of the occurrence of natural hybridization between female Pinctada fucata and male Pinctada maculata among wild pearl oysters ( n  = 20) collected for use as the mother shell for private pearl farming in the Oshima Strait at Amami-o-shima, Kagoshima Prefecture, Japan, were obtained. A polymerase chain reaction-based species identification method for Pinctada was developed using polymorphisms in the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA (rRNA) gene. This method enabled the amplification of the ITS regions using a primer set specific for P. maculata and P. fucata . However, 10 of 20 individuals morphologically identified as P. fucata had sequences specific to both P. maculata and P. fucata in the ITS region. These putative hybrids showed sequences of a maternally inherited mitochondrial 16S rRNA gene, identical to that of P. fucata . Shells of the putative hybrids were difficult to discriminate from those of P. fucata exhibiting similar taxonomic traits. Moreover, the hybrids exhibited slower growth than P. fucata but faster growth than P. maculata .     

Complete nucleotide sequence and variation of mitochondrial DNA from 10 individuals of walleye pollock, Theragra chalcogramma

ABSTRACT:   The complete mitochondrial genome sequence of 10 walleye pollocks, Theragra chalcogramma , from the Japan Sea and Bering Sea was determined. The 16 568–16 571 bp genome contains the same 37 mitochondrial structural genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in all other vertebrates analyzed, in an organization identical to that of other bony fish. The major non-coding region had several conserved sequence features. Nucleotide variations of ND1, ND5, and control region were high, and these regions appear to be good candidates for high-resolution markers in population studies.     

黄姑鱼异质雌核发育子代的遗传分析

为了解黄姑鱼（Nibea albiflora）异质雌核发育子代的基因纯合情况,利用微卫星标记（SSR）和扩增片段长度多态性标记（AFLP）对黄姑鱼异质雌核发育家系进行遗传鉴定和分析。结果显示：（1）雌核发育家系在4个SSR位点和5对AFLP引物组合扩增出的位点均未发现父本特异性等位条带,表明雌核发育体比率为100%。（2）用于遗传分析的7个SSR位点在雌核发育家系和正常交配家系中均未见完全纯合的情况,雌核发育家系7个SSR位点的平均纯合度为0.382,是正常交配家系（0.161）的2.37倍。雌核发育家系各个体的纯合位点数为0~6个,纯合位点所占比例为0~85.7%。（3）5对AFLP引物共扩增出182条清晰的扩增条带,其中有21条父本特异性条带和16条母本特异性条带。16条母本特异性条带中有7个条带在雌核发育家系中显著偏分离（P<0.05）。雌核发育家系和正常交配家系多态性条带比例分别为14.7%和20.3%。（4）雌核发育家系与母本的遗传相似度高于与正常交配家系的遗传相似度,正常交配家系同父本和母本的遗传距离大致相同。研究结果表明,黄姑鱼异质雌核发育二倍体家系的遗传纯合度显著高于正常交配家系,人工诱导雌核发育是促进基因纯合的一个有效途径,它不仅可以加速有利基因的纯合固定,还可以加速有害基因的淘汰,从而有效提高育种效率。     

Identification of currently cultivated Porphyra species by PCR-RFLP analysis

ABSTRACT:   Porphyra yezoensis and P. tenera are the representative species of the marine crop Porphyra (nori) in Japan. Since the two species are extremely similar to each other in morphology, nori breeders have tentatively classified many strains of cultivated Porphyra into the two species without strict species identification. In order to facilitate rapid and reliable identification of the many strains of Porphyra currently cultivated in various Japanese regions, 24 conchocelis strains were examined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis using the plastid RuBisCo spacer and the nuclear internal transcribed spacer regions. From the results, all the strains were identified as P. yezoensis f.  narawaensis , and P. tenera was not detected. Hence, it was confirmed that most of the Porphyra strains currently cultivated in Japan are P. yezoensis f.  narawaensis , and that intensive selective breeding has led to a reduced genetic diversity in the stock used for nori cultivation.     

Genetic information of three pure lines of Porphyra yezoensis (Bangiales, Rhodophyta) obtained by AFLP analysis

Porphyra (Bangiales, Rhodophyta), which includes several valuable marine crops, has recently received great interest as a model plant for fundamental and applied studies in marine sciences. Amplified fragment length polymorphisms (AFLPs) are a robust and efficient means for genetic mapping, linkage analysis of genetic characters for breeding and population studies in land plant genomes. To examine whether AFLPs are applicable as genetic markers in the present study, we detected AFLP markers with three pure lines in order to promote genetic analysis in Porphyra yezoensis . The following five sets of AFLP primer pairs (E-AA, M-CAA) (E-AA, M-CAC) (E-AA, M-CAG) (E-AA, M-CAT) (E-AA, M-CTA) were tested with template DNAs from three pure lines and they showed a total of 227 bands. This suggests that AFLP markers are promising tools for genetic analysis in Porphyra .     

Effect of a seaweed mixture on serum lipid level and platelet aggregation in rats

ABSTRACT:   To assess the effect of a seaweed mixture on lipid levels in serum as well as platelet aggregation in rats, Eisenia bicyclis ('Arame'), Hizikia fusiformis ('Hijiki') and Undaria pinnatifida sporophylls ('Mekabu'), all brown seaweeds, and Porphyra yezoensis ('Susabinori'), a red seaweed, were powdered and mixed in a ratio of 45:30:20:5 (w/w). When rats were fed a cholesterol-rich diet containing this mixture of seaweeds (9–10% w/w) for 28 days, serum total cholesterol, LDL-cholesterol, free cholesterol, and triglyceride levels declined significantly to 49.7%, 48.1%, 49.0% and 74.8%, respectively, of those of the control. Serum HDL-cholesterol, however, was unchanged. Though activated partial thromboplatin time, prothrombin time, antithrombin III activity, and fibrinogen levels in plasma were unchanged, the maximal ADP- and collagen-induced platelet aggregation decreased significantly to 89.0% and 85.5% control levels, respectively. These results indicate that this mixture of E. bicyclis , H. fusiformis , U. pinnatifida sporophylls, and P. yezoensis , is useful for the prevention of hyperlipidemia and thrombosis in rats.     

Complete mitochondrial DNA sequence of the Japanese flying fish <Emphasis Type="Italic">Cypselurus hiraii</Emphasis>

ABSTRACT:   In this study, to develop a technique that enables authentication of processed seafood, the complete nucleotide sequence of the mitochondrial genome for the Japanese flying fish Cypselurus hiraii was determined. Three segments spanning the entire genome were amplified using polymerase chain reaction, and products were subsequently used as templates for direct sequencing with 60 primers. The genome (16 528 base pairs) was found to contain the same 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as those found in other vertebrate mitochondrial genomes, with the gene order being identical to that typical of vertebrates. A major non-coding region between the tRNA^Pro and tRNA^Phe genes (868 base pairs) appears to be the control (D-loop) region, as it has several conserved blocks characteristic of control regions.     

The use of green fluorescent protein as a marker for monitoring a probiotic Bacillus S11 in the black tiger shrimp Penaeus monodon

To monitor the probiotic Bacillus S11 (BS11) in vivo , wild-type cells were transformed with the green fluorescent protein (GFP)-expressing plasmid, pAD44-12, carrying the gfp mut3a gene under the constitutive Bacillus cereus UW85 promoter, and its non-GFP control plasmid pAD. Transformants with pAD44-12 (BS11-GFP), not pAD (BS11-pAD), expressed detectable but not too high levels of green fluorescence. Chloramphenicol resistance (BS11-GFP, BS11-pAD) and GFP fluorescence (BS11-GFP) as markers suggested that plasmid retention was 78–79% for both BS11-GFP and BS11-pAD cells after approximately 50 generations of growth without antibiotic selection. When mixed into shrimp feed at a final concentration ∼10⁵ CFU g⁻¹, the inclusion of viable transformed bacteria in fed shrimps was observed. After feeding shrimp three times daily in 400-L cement tanks for 9 weeks, no significant differences in the average shrimp weight, the number of BS11 in either the culture water or in the shrimp's gut were seen between shrimp fed BS11-GFP and BS11-pAD or BS11, suggesting that expression of the gfp mut3a gene has no detectable effect on BS11 properties and shrimp growth. Histological examination of sections of shrimp's intestines following feeding with BS11-GFP demonstrated that BS11-GFP in shrimp feed survived and adhered onto the shrimp intestines' surface. BS11-GFP thus has good potential as a non-invasive marker tag for short-term experiments.     

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.     

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 .     

Utilization of tissue habitats by Myxobolus wulii Landsberg & Lom, 1991 in different carp hosts and disease resistance in allogynogenetic gibel carp: redescription of M. wulii from China and Japan

Myxobolus wulii (= Myxosoma magna ) was first described from the gills of goldfish, Carassius auratus auratus, in China. Subsequently, a myxosporean infecting the hepatopancreas of allogynogenetic gibel carp, C. auratus gibelio , was designated as a different species, Myxobolus guanqiaoensis , although the morphological features were almost identical to those of M. wulii . In Japan, an unidentified Myxobolus sp. was found in the gills and hepatopancreas of goldfish. Morphological and molecular analyses in the present study identified these myxosporeans as M. wulii , which was thus shown to use different habitats in the host fish. Phylogenetic analyses of small subunit ribosomal RNA gene sequences showed that M. wulii is closely related to two gill-infecting Myxobolus species, M. ampullicapsulatus and M. longisporus . Fish infected with M. wulii in the hepatopancreas exhibit swollen abdomens and chronic mortality. Hepatopancreas tissues are virtually destroyed and replaced with plasmodia of M. wulii . A remarkable difference in susceptibility to M. wulii between two clones of allogynogenetic gibel carp was observed, suggesting that resistance to the myxosporean infection was established in a clone of fish bred by allogynogenesis.     

三疣梭子蟹Sox基因HMG盒的克隆分析

利用能扩增人类SRY基因HMG盒的一对简并引物,分别对三疣梭子蟹雌雄个体基因组DNA进行扩增,均得到216 bp和约400 bp的两条带,不存在性别差异,通过PCR-SSCP分析,未发现雌蟹或雄蟹所特有的Sox基因。对216 bp的带进行克隆测序得到6个Sox基因,分别命名为PTSox21、PTSox12、PTSox11a、PTSox11b、PTSox11c和PTSox4。其中,PTSox21、PTSox12和PTSox4氨基酸序列与人类Sox21、Sox12和Sox4基因的同源性分别为90%、66%和63%,PTSox11a、PTSox11b和PTSox11c氨基酸序列均与人类Sox11基因有63%的同源性。此外,PTSox11a和PTSox11b的氨基酸序列之间的同源性达到了98.6%,只在第44位氨基酸残基不同。与其他物种Sox基因氨基酸序列的同源性比较发现,PTSox21与饰纹姬蛙Sox2a有92%的同源性,而PTSox12、PTSox11a、PTSox11b和PTSox11c均与意大利蜜蜂的Sox1有73%的同源性,PTSox4与意大利蜜蜂的Sox1有72%的同源性。     

Complete mitochondrial genome of the sea cucumber Stichopus sp. and its application in the identification of this species

The complete sequence of mitochondrial DNA (mtDNA) from Stichopus sp. (Echinodermata: Holothuroidea: Stichopodidae: Stichopus) was acquired using conventional PCR and long PCR followed by cloning and sequencing. The mtDNA is a circular molecule of 16 257 bp in length, containing the set of 37 genes, including 13 protein‐coding genes (PCGs), 22 tRNA genes and two ribosomal RNA genes. The plus strand consists of 30.9% A, 23.7% C, 16.0% G and 29.3% T bases (AT skew = 0.027; GC skew = ?0.194). All 13 PCGs encode a total of 3782 amino acids and all 22 tRNA genes were predicted to be capable of folding into a clover‐leaf secondary structure. Intergenetic regions in the mitochondrial genome of Stichopus sp. contain 903 bp in total, with the largest continuous region (674 bp, AT% = 58.6) between tRNA‐Thr and tRNA‐Pro. Analysis of phylogenetic relationship of family Stichopodidae and genetic distances (species among the family Stichopodidae and species within Stichopus monotuberculatus group) based on the partial cox1 sequence demonstrates that Stichopus sp. from the South China Sea is a member of S. monotuberculatus complex.     

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 .     

Occurrence of postmortem myoliquefactive kudoosis in Atlantic mackerel, Scomber scombrus L., from the North Sea

Members of the myxosporean genus Kudoa occur in various marine teleosts worldwide. Several species are of concern to the fishery and aquaculture industries as they may produce unsightly cysts in the fish host's musculature or are associated with postmortem myoliquefaction of the fish muscle, commonly referred to as 'soft flesh'. This study describes the occurrence and effects on a host of a Kudoa species in Atlantic mackerel, Scomber scombrus , from the northern North Sea. Generalized postmortem myoliquefaction associated with Kudoa sp. occurred in 0.8% of the examined fish, i.e. 11 of 1339 mackerel developed 'soft flesh'. There was a significant difference in the prevalence of myoliquefaction between medium sized (400–600 g) and large mackerel (>600 g). The prevalence reached 8.9% in the latter host size group. No subclinical infections of Kudoa sp. were detected when examining fresh muscle ( n  = 103) and blood ( n  = 165) samples for spores using light microscopy. Affected mackerel developed generalized myoliquefaction after 38–56 h post-catch. No inflammatory host response was associated with the presence of plasmodia within single body muscle fibres of 'soft flesh' affected fish. Based on comparison of myxospore dimensions and analysis of the nuclear small subunit (SSU) ribosomal DNA, the present Kudoa species is assigned to Kudoa thyrsites . However, due to the species' apparently very wide geographical distribution and host range, its varying effect on different fish host species, together with the still unknown life cycle of Kudoa spp., the taxonomic status of K. thyrsites appears not to be fully resolved.     

Intraspecific diversity of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Harvey) Suringar (Laminariales,Phaeophyta) from Japan,China and Korea,based on the <Emphasis Type="Italic">cox1</Emphasis> gene and ITS2 sequences

As a trial to develop a method of authenticating the place of origin of circulated Undaria pinnatifida products, we investigated their intraspecific genetic diversity using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) and the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA (rDNA) sequence. Four dried U. pinnatifida products labeled with their origins (one from Japan, one from China and two from Korea), natural plants collected from three locations (two from Japan and one from China), and cultivated plants collected from two locations (one from Japan and one from China) were used in the present study. The amplified fragments of cox1 were 664 bp in length, and the aligned sequences were highly homologous. Among the nine sequences, no insertions or deletions were found and six substitution positions were detected, and they were classified into five haplotypes. In contrast, multiple highly variable regions were found in ITS2, and some of them carried a restriction site for Mbo II. Polymerase chain reaction-restriction fragment length polymorphism analysis showed different restricted profiles among the tested samples. The availability of molecular markers for authenticating food products of U. pinnatifida is discussed.     

海胆纲线粒体基因组特征及基因差异位点分析

研究了海胆纲6个线粒体基因组均编码后生动物标准的37个基因。6个海胆线粒体基因组的基因排列完全相同。海胆纲和球海胆属线粒体基因组13个蛋白质编码基因和2个核糖体RNA基因的差异位点分析表明,差异位点数最多的基因为nad5基因,其次为nad4基因和cox1基因。因此,在海胆纲和球海胆群体遗传学的研究中,nad5、nad4和cox1基因是理想的分子标记,用于分析海胆内部不同群体之间的遗传多样性。