Multiple primer PCR for the identification of anisakid nematodes from Taiwan Strait

There were six major larval anisakid species found in commercial marine fishes caught in the Minnan fishing ground in the Taiwan Strait: Anisakis physeteris, Anisakis pegreffii, Raphidascaris trichiuri, Contracaecum aduncum, Contracaecum muraenesoxi, Contracaecum sp. For rapid identification of the parasite species above, a single and a multiple primer PCR (multiplex PCR) method, using specific primers based on aligned sequences of the internal transcribed spacer ITS-1, 5.8S, and ITS-2 of nuclear ribosomal DNA, were jointly used for the rapid identification of these anisakid larvae. The primers yielded distinct PCR products for each of the anisakid nematodes, providing rapid and accurate tools for identifying anisakid nematodes with distinct geographical distribution.     

Analysis of nucleotide sequence polymorphism of internal transcribed spacers of ribosomal genes in diploid <Emphasis Type="Italic">Aegilops</Emphasis> (L.) species

The nucleotide sequence of the fragment of the internal transcribed spacer (ITS) of rDNA comprising the full-length ITS1, the gene encoding 5.8S rRNA, and part of the ITS2 sequence was determined in 22 samples of five diploid Aegilops species. The full alignment length of compared sequences was 524 bp. Species-specific substitutions were found in the ITS nucleotide sequence of rDNA of different Aegilops species. Intraspecific differences in ITS structure in diploid Aegilops species were detected for the first time. Polymorphism of the ITS nucleotide sequence within the same sample was revealed, which might be due either to differences between the genomes of individual plants comprising the sample or to the presence of several types of ribosomal genes in the genome of one plant. In general, both interspecific and intraspecific variability of the ITS nucleotide sequences of rDNA is extremely low. In total, 26 variable sites, twelve of which were informative, were identified.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 193–197.Original Russian Text Copyright © 2005 by Goryunova, Chikida, Gori, Kochieva.     

Sequence differences in the internal transcribed spacer ribosomal DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma)

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5–7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.     

Phylogeny of the genus<Emphasis Type="Italic">Goodyera</Emphasis> (Orchidaceae; Cranichideae) in Korea based on nuclear ribosomal DNA ITS region sequences

We sequenced the nuclear ribosomal internal transcribed spacer (ITS) regions to determined the phylogenetic relationships of the severalGoodyera species in Korea and to measure the extent of their differentiation region. ITS 1 was 238 to 239 bp long while ITS 2 was 258 to 259 bp. The 5.8S coding region was 156 bp long. Sequence divergences among species, calculated by Kimura’s two- parameter method, ranged from 0.0 to 5.4%. The most parsimonious tree, with a consistency index of 0.935 and a retention index of 0.937, was produced with 337 steps. Our ITS sequence results demonstrate the monophyly of KoreanGoodyera and support previous morphological, geographical, and RAPD data analyses.     

Characterisation of Ascaris from human and pig hosts by nuclear ribosomal DNA sequences.

The sequences of the nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S rRNA gene and the second internal transcribed spacer were determined for Ascaris samples from pigs and humans from different geographical regions. The sequences of the 5.8S gene and the second internal transcribed spacer were the same for all samples examined, whereas all Ascaris samples from humans had six (1.3%) nucleotide differences in the first internal transcribed spacer compared with those from pigs. These differences provided some support for the existence of separate species of Ascaris or population variation within this genus. Using a nucleotide difference within a site for the restriction enzyme HaeIII, a PCR-linked restriction fragment length polymorphism method was established which allowed the delineation of the Ascaris samples from pigs and humans used herein. Exploiting the sequence differences in the first internal transcribed spacer, a PCR-based single-strand conformation polymorphism method was established for future analysis of the genetic structure of pig and human Ascaris populations in sympatric and allopatric zones.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species

The organization of 5S ribosomal RNA genes (rDNA) was examined for threeJapanese Laminaria species, L. japonica, L.religiosa and L. ochotensis. The linkage of 5SrDNA with the 18S-5.8S-25S rDNAs unit known in the brown algaScytosiphon lomentaria could not be detected inLaminaria. Instead, the tandem repeats of 5S rDNA were notassociated with the 18S-5.8S-25S rDNAs unit. The nucleotide sequence of 5S rDNAwas completely identical among these three species and its length was 118bp. However, a difference of nucleotide arrangement was detectedinthe spacer region of the tandemly repeated 5S rDNAs. Several nucleotideinsertion / deletions and substitutions were confirmed between differentindividuals of L. japonica, which were collected from notonly disjunct localities, but also the same locality. The lengths of the spacerregion of L. japonica, L. religiosaand L. ochotensis were 247–252 bp, 232bp and 252 bp, respectively.     

Nucleotide sequence of the 17S–25S spacer region from rice rDNA

Summary The nucleotide sequence of a spacer region between rice 17S and 25S rRNA genes (rDNAs) has been determined. The coding regions for the mature 17S, 5.8S and 25S rRNAs were identified by sequencing terminal regions of these rRNAs. The first internal transcribed spacer (ITS1), between 17S and 5.8S rDNAs, is 194–195 bp long. The second internal transcribed spacer (ITS2), between 5.8S and 25S rDNAs, is 233 bp long. Both spacers are very rich in G+C, 72.7% for ITS1 and 77.3% for ITS2. The 5.8S rDNA is 163–164 bp long and similar in primary and secondary structures to other eukaryotic 5.8S rDNAs. The 5.8S rDNA is capable of interacting with the 5′ terminal region of 25S rDNA.     

Molecular evidence for cryptic species within Cylicostephanus minutus (Nematoda: Strongylidae)

The nucleotide sequences of the first and second internal transcribed spacers of nuclear ribosomal DNA were determined for adults of Cylicostephanus minutus from different geographical origins. The lengths of first and second internal transcribed spacer sequences ranged from 370 to 372bp and 215 to 216bp, respectively. Pairwise sequence comparisons revealed that some individuals of C. minutus had identical first and second internal transcribed spacer sequences, whereas others differed by 3.0% and 7.4% in their first and second transcribed spacers, respectively. Some individuals with sequence differences originated from the same host. The levels of difference within C. minutus were higher than that between the morphologically distinct species, Cylicostephanus goldi and Cylicostephanus longibursatus (0.8% for the first internal transcribed spacer and 3.8% for the second internal transcribed spacer). The data provide support for the proposal that C. minutus represents a complex of at least two species. In order to study the population genetic structure of C. minutus, a PCR-linked single-strand conformation polymorphism technique was also established.     

Sequencing of the internal transcribed spacer region ITS1 as a molecular tool detecting variation in the Stylosanthes guianensis species complex

 The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA from Stylosanthes guianensis CIAT 1283 and cv ‘Schofield’ were amplified by polymerase chain reaction using conserved ITS primers from the 18S, 5.8S and 26S ribosomal genes flanking those regions. The entire region of 683 bp long was cloned, and seven clones were sequenced. Comparison of the ITS spacer regions with published DNA sequences of other plant species revealed limited homology only; this was in contrast to their comparison with the 5.8S rDNA sequences. The ITS1 region of 45 S. guianensis accessions was amplified by PCR and sequenced on both strands using the conserved primers ITS2-ITS5. These sequences, ranging from 201 to 204 bp, were aligned to each other to assess intra-specific polymorphism. Within the S. guianensis (Aubl.) Sw. species complex, 11 DNA sequence types could be distinguished based on an insertion/deletion (indel) event and 15 single base-pair substitutions. In 1 of the S. guianensis types, two kinds of ITS1 sequence were observed in each individual, reminiscent of an incomplete homogenization of the repeat structure in this type. Polymorphisms in the sequence of the ITS1 region were used to define molecular markers for S. guianensis on the basis of PCR-restriction fragment length polymorphism and selective PCR. Received: 24 June 1997 / Accepted: 31 October 1997     

Revision of the kelp family Alariaceae and the taxonomic affinities of Lessoniopsis Reinke (Laminariales,Phaeophyta)

The morphologically diverse members of the Laminariales are separated into three families, the Alariaceae, Laminariaceae and Lessoniaceae, on the basis of developmental features exhibited at the stipeblade transition zone. We have investigated the relationships of lessoniacean taxa with those of alariacean and laminariacean affinity. This was done by completing phylogenetic analyses of 3 small-subunit (SSU) rRNA gene (113 bp), first internal transcribed spacer (ITS 1), 5.8S rRNA gene and second internal transcribed spacer (ITS2)(12 bp) sequence data from a variety of taxa. In summary, the Alariaceae and Lessoniaceae are polyphyletic. We present a restricted view of the Alariaceae, including only Alaria and Pterygophora of the genera usually placed in this family, and transfer the monotypic genus Lessoniopsis to this group. Current theories of kelp evolution and biogeography must be reconsidered in view of our data.     

Genotyping of a Miso and Soy Sauce Fermentation Yeast,Zygosaccharomyces rouxii,Based on Sequence Analysis of the Partial 26S Ribosomal RNA Gene and Two Internal Transcribed Spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I–VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type α and type β) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

Identification and typing of miso and soy sauce fermentation yeasts, Candida etchellsii and C. versatilis, based on sequence analyses of the D1D2 domain of the 26S ribosomal RNA gene, and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS sequence) of the miso and soy sauce fermentation yeasts, Candida etchellsii and Candida versatilis, in order to evaluate the usefulness of this sequence analysis for identification and typing of these two species. In the 26S rDNA sequence method, the numbers of base substitutions among C. etchellsii strains were up to 2 in 482 bp (99.6% similarity), and they were divided into three types (types A, B, and C). Those of C. versatilis strains were also up to 2 in 521 bp (99.6% similarity) and they were divided into three types (types 1, 2, and 3). In the ITS sequence method, those of C. etchellsii strains were zero in 433 bp (type a, 100% similarity). Those of C. versatilis were 5 in 409 bp (98.8% similarity), divided into 4 types (types I, II, III and IV). It was found that molecular methods based on the sequences of the 26S rDNA D1D2 domain and the ITS region were rapid and precise compared with the physiological method for the identification and typing of these two species.     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

Molecular characterization of hard tick <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> from China by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of Haemaphysalis longicornis from China were amplified by polymerase chain reaction. The 45 representative amplicons were sequenced, and sequence variation in the ITS was examined. The ITS sequences of H. longicornis were 3644 bp in size, including the part of 18S rDNA, 28S rDNA sequences and the complete ITS-1, 5.8S rDNA and ITS-2 sequences. Sequence analysis revealed that the ITS-1, 5.8S rDNA and ITS-2 of this hard tick were 1582, 152, and 1610 bp in size, respectively. The intra-specific sequence variations of ITS-1 and ITS-2 within H. longicornis were 0–2 and 0–2.2%; however, the inter-specific sequence differences among members of the genus Haemaphysalis were significantly higher, being 35.1–55.2 and 37–52% for ITS-1 and ITS-2, respectively. The molecular approach employed in this study provides the foundation for further studies of the genetic variation of H. longicornis from different hosts and geographical origins in China.     

Construction of genomic phage libraries of the arbuscular mycorrhizal fungi Glomus mosseae and Scutellospora castanea and isolation of ribosomal RNA genes

 Genomic phage libraries of arbuscular mycorrhizal fungi were constructed for the first time, and clones containing ribosomal RNA (rRNA) genes isolated for Glomus mosseae and Scutellospora castanea. The number of rDNA clones per library indicates that these libraries can be also used to isolate genes with low copy numbers. Sequences of the 18S rRNA gene, of the internal transcribed spacer and of the 5.8S rRNA gene were analysed and compared. Differences between the 18S and the 5.8S rRNA genes were few and in the range of variation found for other fungi. In contrast, the internal transcribed spacers of G. mosseae and S. castanea were highly variable, showing the potential of this region for the identification of different species or isolates. Interestingly, nucleotide exchanges were found in this region when the sequence for G. mosseae was compared to those of two other clones of the same isolate. Accepted: 10 November 1995     

奥利亚罗非鱼与尼罗罗非鱼rDNA内转录间隔区序列特征

核糖体DNA内转录间隔区(internal transcribed spacers,ITS)是经常被用作种和种群水平系统研究的分子序列.本文分离了奥利亚罗非鱼(Oreochromis aureus)、尼罗罗非鱼(O.niloticus)内转录间隔区,包括部分185序列,ITS1、5.8S、ITS2全序列及部分28S序列.4尾奥利亚罗非鱼的10个克隆序列分析表明,其存在长度不同的a、b两种类型ITS1.a型长为536 bp,GC含量为69.96%;b型长为520 bp,GC含量为69.04%～69.42%.4尾尼罗罗非鱼的10个克隆序列分析表明,其只存在a型ITS1,长为536～540 bp,GC含量为69.42%～70.19%.与b型ITS1相比,a型ITS1在16～31 nt有16 bp片段(GGCCCGCCTCGGCGC)的插入.奥利亚罗非鱼和尼罗罗非鱼共20条ITS序列中,5.8S长度均为157 bp,GC含量为56.69%～57.96%;ITS2为408 bp,GC含量为72.79%～74.26%.奥利亚罗非鱼和尼罗罗非鱼ITS区序列相似性高达98.2%,表明这两种罗非鱼亲缘关系很近.此外,本文对14尾奥利亚罗非鱼、15尾尼罗罗非鱼以及15尾奥尼罗非鱼[O.aureus(♂)×O.niloticus(♀)]ITS1的扩增结果显示,奥利亚罗非鱼均有a、b两种类型ITS1;15尾尼罗罗非鱼中1尾为a、b两类型ITS1,14尾为a型ITS1;15尾奥尼罗非鱼中则有6尾具有a、b两类型ITS1,9尾为单一的a型ITS1.分析表明,奥利亚罗非鱼在ITS1这个位点一致性高,但尼罗罗非鱼中有1尾混杂了奥利亚罗非鱼的基因,同时也说明分子生物学手段应用于种质鉴定比形态学手段更为精确.     

Marked Intragenomic Heterogeneity and Geographical Differentiation of nrDNA ITS in <Emphasis Type="BoldItalic">Larix</Emphasis><Emphasis Type="BoldItalic">potaninii</Emphasis> (Pinaceae)

Nuclear ribosomal DNA (nrDNA) of gymnosperms, especially Pinaceae, is characterized by slow concerted evolution and exhibits substantial ITS-region length variation (975–3663 bp), in sharp contrast to the narrow range (565–700 bp) in angiosperms. Here we examined intra- and intergenomic heterogeneity of the nrDNA internal transcribed spacer (ITS) region in four varieties of Larix potaninii, a species from the mountainous areas of western China. Two clones with more than a 100-bp deletion in ITS1 were detected in L. potaninii var. chinensis and L. potaninii var. australis, respectively. The deletion resulted in the loss of most part, including the motif sequence, of subrepeat 1 (SR1). Sequence divergence and phylogenetic analyses showed that some clones would be pseudogenes given their low GC content, high substitution rates, unique positions in the phylogenetic trees, or significant length variation. These clones might represent orphons or paralogues at minor loci resulting from large-scale gene or chromosome reorganization. Some recombinants characterized by chimeric structure and discordant phylogenetic positions in partitioned sequence analyses indicate that unequal crossing-over plays an important role in the process of nrDNA concerted evolution. In addition, some varieties of L. potaninii might have experienced an nrDNA founder effect parallel to their geographical isolation.     

Phylogenetic analysis of <Emphasis Type="Italic">Antrodia</Emphasis> species and <Emphasis Type="Italic">Antrodia camphorata</Emphasis> inferred from internal transcribed spacer region

The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.