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.     

Phylogenetic inferences in Antennaria (Asteraceae: Gnaphalieae: Cassiniinae) based on sequences from Nuclear Ribosomal DNA internal transcribed spacers (ITS)

The phylogenetic relationships among sexually reproducing species of Antennaria (Asteraceae) are poorly understood. An earlier cladistic analysis based on morphology did not fully resolve the phylogeny of these taxa and therefore a different approach using molecular data was explored. The internal transcribed spacer regions (ITS-1 and ITS-2) of nuclear ribosomal DNA were sequenced for 30 species of Antennaria and one species from each of the outgroup genera Anaphalis, Ewartia, Leontopodium, and Pseudognaphalium. The ITS-1 sequence in Antennaria ranged from 253 to 260 base pairs (bp) in length, and the proportion of nucleotide differences between pairs of species of Antennaria ranged from 1 to 14%. For ITS-2, the divergence between pairs of species of Antennaria ranged from 0 to 8%. ITS-2 is shorter than ITS-1, ranging from 213 to 219 bp. Phylogenetic analysis indicates that, relative to the outgroups included, Antennaria is a well-supported monophyletic group. Based on the genera surveyed, Leontopodium appears to be the sister genus of Antennaria. The general topology of the molecular trees agrees with that based on previous morphological analyses and indicates that Antennaria is composed of six clades of equal rank, corresponding to the traditionally recognized informal groups, the Geyeriae, Argenteae, Arcuatae, Dimorphae, Pulcherrimae, and Catipes. Sequence and morphological data indicate that the Alpinae and Dioicae are unnatural, polyphyletic units that should be abandoned and redefined as the monophyletic Catipes group. Phylogenetic analysis of ITS sequences also suggests the dissociation of A. stenophylla from the Dimorphae, where it is traditionally placed, and its affiliation with the Argenteae, as well as the placement of A. arcuata in its own group.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

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.     

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     

Orientobilharzia turkestanicum is a member of Schistosoma genus based on phylogenetic analysis using ribosomal DNA sequences

In the present study, samples representing Orientobilharzia turkestanicum from cattle, sheep, cashmere goat and goat in Heilongjiang Province, China, were characterized and grouped genetically by sequences of internal transcribed spacer (ITS, including ITS-1 and ITS2) and 28S ribosomal DNA (28S rDNA). The ITS and 28S rDNA were amplified by polymerase chain reaction (PCR) and then sequenced and compared with that of other members of the Schistosomatidae available in GenBank™, and phylogenetic relationships between them were re-constructed using the neighbor-joining and maximum parsimony methods. The lengths of ITS-1, ITS-2 and 28S rDNA sequences for all O. turkestanicum samples from different hosts were 384 bp, 331 bp and 1304 bp, respectively. While the ITS-1 sequences of O. turkestanicum from each of the four different hosts, and ITS-2 of O. turkestanicum from cattle, sheep and cashmere goat were identical, respectively, the ITS-2 of O. turkestanicum from goat differed from that of O. turkestanicum from cattle, sheep and cashmere goat by one nucleotide. The 28S rDNA sequences of O. turkestanicum from sheep and cashmere goat were identical, but differed from that of O. turkestanicum from cattle and goat by two nucleotides, with the latter two also having identical 28S rDNA sequence. Phylogenetic analyses based on the combined sequences of the ITS-1 and ITS-2, or the 28S rDNA sequences placed O. turkestanicum within the genus Schistosoma, and it was phylogenetically closer to the African schistosome group than to the Asian schistosome group. These results should have implications for studying the origin and evolution of O. turkestanicum and other members of the Schistosomatidae.     

基于ITS序列分析钩苞大丁草九个居群的亲缘关系

该研究对我国西南地区钩苞大丁草(Gerbera delavayi)9个居群rDNA ITS序列进行PCR的扩增和检测序列,并以非洲菊(G.jamesonii)的ITS序列作为外类群,比较了序列之间的差异,同时分析了钩苞大丁草不同居群在地理距离与遗传距离之间的关系,构建了NJ系统发育树。结果表明:(1)钩苞大丁草9个居群的ITS序列全长介于600~700 bp之间,平均长度约为657 bp,其中,ITS1长度为243~246 bp,(G+C)含量为45.67%~46.80%之间,5.8S长度191~193 bp,(G+C)含量为58.60%~58.61%之间,ITS2长度为220~221 bp,(G+C)含量为57.00%~57.45%之间;ITS序列共有22个变异位点,ITS1序列(17个)、5.8S序列(2个)以及ITS2序列(3个)上均有变异。(2)地理距离与遗传距离有正相关(r2=0.652),序列间遗传分化距离为0.001 1~0.024 3,其中普洱居群与其他居群间遗传距离最大。(3)钩苞大丁草9个居群分成三个分支,普洱居群单独成支,丽江和洱源居群聚为一支,富源、武定、德昌、石林、新平和开远6个居群聚为一支。rDNA ITS序列可以用于钩苞大丁草群体遗传研究的分析,该研究结果为其保护性开发提供了参考依据。     

ITS rDNA sequences of Pomphorhynchus laevis (Zoega in Müller, 1776) and P. lucyi Williams & Rogers, 1984 (Acanthocephala: Palaeacanthocephala)

The internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal RNA gene of Pomphorhynchus laevis (Zoega in Müller, 1776) (Acanthocephala) isolated from various fish species across Central and Southern Europe were compared with those of P. lucyi Williams & Rogers, 1984 collected from the largemouth bass Micropterus salmonoides Boulenger from the USA. The nucleotide sequences of ITS regions of P. laevis from minnows Phoxinus phoxinus (L.) and chub Leuciscus cephalus (L.) from two distant localities in the Slovak Republic were found to be 100% identical. The ITS-1 and ITS-2 of P. laevis from chub from the Czech Republic and Italy were also mutually identical, but significantly different from Slovak worms (88.7% identity for ITS-1, 91.3% identity for ITS-2). A fifth sample collected from Barbus tyberinus Bonaparte from Italy was very similar to the sympatric Italian isolate from chub, possessing four nucleotide substitutions in ITS-1 (98.4% identity). The ITS rDNA sequences of P. lucyi differed significantly from those of P. laevis; the values of identity were 51.8–56.1% for ITS-1 and 63.1–65.3% for ITS-2, and were significantly higher than the range of P. laevis within-species variability. The results based on the ITS sequences confirmed the occurrence of strains in P. laevis from Continental Europe which are well defined by molecules but reveal only slight differences in their morphology.     

MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Internal Transcribed Spacer Sequence Analysis of Puccinia helianthi Schw. and Its Application in Detection of Sunflower Rust

Two basidiomycete‐specific primers ITS1‐F and ITS4‐B were used in identification of the genus Puccinia. The primers showed good specificity for the genus with an 816‐bp product that was amplified exclusively. Twenty sequences of internal transcribed spacer (ITS) regions of Puccinia helianthi isolates from China remain unchanged. The whole ITS length (including ITS1 sequence 194 bp, 5.8S rRNA gene 156 bp, ITS2 sequence 206 bp) was 556 bp. By comparing the aligned ITS sequences of several Puccinia isolates from China, Spain and the United States, ITS homogeneity among these sunflower rust isolates was >99%. Genetic homology and phylogeny of P. helianthi with other Puccinia spp. was investigated. Nineteen sequences of rDNA ITS1 and ITS2 were determined and used as phylogenetic markers. Phylogenetic analysis of ITS regions showed that Puccinia spp. of sunflower was clustered in one clade with P. komarovii and P. violae, divergent from Puccinia spp. of Chrysanthemum, P. tenaceti of tansy (Tanacetum vulgare) and Puccina spp. of big sagebrush (Artemisia tridentate) indicating sunflower rust had distant phylogenetic relationships with other Compositae rusts. With the specified primers SR‐1 and SR‐2, either from purified urediniospores or symptomless (but infected) sunflower leaves could be examined specifically. Therefore, results of this study help in detection and polygenetic study of rust fungi occurring on sunflower.     

Discrimination between <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> and <Emphasis Type="Italic">H. qinghaiensis</Emphasis> based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2)

In the present study, two hard tick species, Haemaphysalis longicornis and H. qinghaiensis from North-western China were characterized genetically by the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and partial 16S rDNA. Based on a fragment within the hypervariable region of 16S rDNA with the length of approximately 453 bp, the phylogenetic trees were constructed by Neighbor-Joining and Maximum-parsimony methods. The results indicated that the phylogenetic status of H. qinghaiensis was distant from that of H. longicornis and closer to H. flava. Furthermore, the ITS-2 rDNA was amplified by PCR and sequenced from individual ticks. The length of ITS-2 is 1,606 bp for H. longicornis and 1,162 bp for H. qinghaiensis. Although sequence variation between the immature stages of H. longicornis was 0.1–0.4%, nucleotide differences between the tested species ranged 2.1–23.2%, indicating that ITS-2 rDNA sequences are genetic markers for the differentiation of the two hard ticks in China. Hence, a PCR-linked restriction fragment length polymorphism (RFLP) approach was developed for their unequivocal differentiation based on ITS-2 rDNA, which provides the foundation for further studies on ticks in China and has implications for studying the population genetic structure of the ticks and for identification and differentiation of closely related ticks.     

Comparative seed resistance in eighty cowpea accessions to the seed bruchid,Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae)

Eighty cowpea accessions comprising both improved and local were assessed for their resistance to Callosobruchus maculatus (F.) attack under laboratory conditions (29 ± 3?°C and 65 ± 5% R.H.). Sixty-nine accessions were derived from the International Institute for Tropical Agriculture breeding programmes, four were local cultivars, while seven were improved cultivars from national research institutes. Results showed that there were significant differences among the accessions in terms of number of initial bruchid eggs laid on them, adult emergence, number of seeds damaged and bruchid-induced seed weight loss at three months after artificial infestation with C. maculatus. On IT04K-334-2, IT04K-343-1, IT06K-141, IT99K-216-48-1, IT99K-494-6 and IT99K-529-2, fewer initial eggs were laid and no adult emergence, seed damage or weight loss and were considered highly resistant to C. maculatus. On the other hand, IT03K-337, IT04K-217-51, IT04K-332-1, IT04K-339-1, IT06K-188-49, IT81D-1137, IT845-2231-15, IT93K-452-1, IT04K-154-1, IT96K-610, IT97K-1069-6, IT97K-390-2, Ife Bpc, IT99K-573-2-1, Ife Modupe, IT845-2246, Drum, Ife Brown and Oloyin were the most susceptible with the highest damage indices values (p?<?0.05). The range of each of the resistance indices measured in the susceptible varieties was: number of initial eggs laid (61–100), adult emergence (40.0–62.10%), number of seeds damaged (53.0–85.5) and seed weight loss (58.0–66.25%).     

Molecular characterization of Fasciola spp. from the endemic area of northern Iran based on nuclear ribosomal DNA sequences

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered as the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the endemic regions of the North of Iran, Fasciola hepatica and Fasciola gigantica have been previously characterized on the basis of morphometric differences, but the use of molecular markers is necessary to distinguish exactly between species and intermediate forms. Samples from buffaloes and goats from different localities of northern Iran were identified morphologically and then genetically characterized by sequences of the first (ITS-1) and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the ITS of the northern Iranian samples with sequences of Fasciola spp. from GenBank showed that the examined specimens had sequences identical to those of the most frequent haplotypes of F. hepatica (n = 25, 48.1%) and F. gigantica (n = 20, 38.45%), which differed from each other in different variable nucleotide positions of ITS region sequences, and their intermediate forms (n = 7, 13.45%), which had nucleotides overlapped between the two Fasciola species in all the positions. The ITS sequences from populations of Fasciola isolates in buffaloes and goats had experienced introgression/hybridization as previously reported in isolates from other ruminants and humans. Based on ITS-1 and ITS-2 sequences, flukes are scattered in pure F. hepatica, F. gigantica and intermediate Fasciola clades, revealing that multiple genotypes of Fasciola are able to infect goats and buffaloes in North of Iran. Furthermore, the phylogenetic trees based upon the ITS-1 and ITS-2 sequences showed a close relationship of the Iranian samples with isolates of F. hepatica and F. gigantica from different localities of Africa and Asia. In the present study, the intergenic transcribed spacers ITS-1 and ITS-2 showed to be reliable approaches for the genetic differentiation of Fasciola spp., providing bases for further studies on F. hepatica, F. gigantica and their intermediate forms in the endemic areas in Asia.     

用ITS序列研究杨属各组之间的系统发育关系

ITS sequences of 15 representative species of five sections inthe genus Populus L. were determined. By using direct sequencing of PCR product, it was found that the fragments of internal transcribed spacers (ITS) are about 594 bp in length. The length of ITS-1 and ITS-2 is about 220 bp and 210 bp, respectively, while that of 5.8s is 164 bp. Its G+C content is about 69.0%. The number of phylogenetically informative loci is higher in ITS-2 than in ITS-1. Transversion and transition are two main factors that drive the ITS evolution, and more insertions and deletions occurred in ITS-2. Taking Salix matsudana Koidz. and Salix suchowensis Cheng as outgroups, phylogenetic analysis of ITS sequences using PAUP 4.0 software indicated that Populus is monophyletic group and can be divided into two main clades. One is the section Leuce, and the other is the remaining sections.     

Phylogenetic Relationship ofPopulusSections by ITS Sequence Analysis

ITS sequences of 15 representative species of five sections in the genus Populus L. were determined. By using direct sequencing of PCR product, it was found that the fragments of internal transcribed spacers (ITS) are about 594 bp in length. The length of ITS-1 and ITS-2 is about 220 bp and 210 bp, respectively, while that of 5.8s is 164 bp. Its G+C content is about 69.0%. The number of phylogenetically informative loci is higher in ITS-2 than in ITS-1. Transversion and transition are two main factors that drive the ITS evolution, and more insertions and deletions occurred in ITS-2. Taking Salix matsudana Koidz. and Salix suchowensis Cheng as outgroups, phylogenetic analysis of ITS sequences using PAUP 4.0 software indicated that Populus is monophyletic group and can be divided into two main clades. One is the section Leuce , and the other is the remaining sections.     

Genetic relationships and population structure of local olive tree accessions from Northeastern Spain revealed by SSR markers

The olive (Olea europaea L. subsp. europaea) is an old traditional crop which was domesticated from the wild at various locations around the Mediterranean basin, resulting in a vast number of accessions worldwide. This is the case of the northeast part of Spain (Aragon), where 163 genotypes were prospected and characterised. The majority of these genotypes (90 %) seem to be autochthonous accessions of this area and the rest are synonyms of accessions from other areas of Spain. In this study, 11 nuclear SSR markers were used to discriminate between the accessions and to understand the genetic relationship among them. All primers produced a successful amplification giving a total of 176 fragments in the genotypes studied, with an average of 16 alleles per SSR, ranging from 5 (DCA13) to 24 (DCA11). Allele size ranged from 120 bp at locus UDO99 to 284 bp at locus DCA15. The dendrogram generated using the variability observed classified most of the genotypes according to their geographical origin (Huesca, Teruel and Zaragoza provinces), confirming the particular evolution of different olive ecotypes. Structure software was used to investigate the genetic population structure among olive accessions, showing that the maximum rate of change in the log probability of the data occurred at K = 2. In addition, the SSR markers have consequently shown their usefulness for cultivar identification in olive, for establishing the genetic closeness among its accessions, and for establishing genealogical relationships.     

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。     

The First Internal Transcribed Spacer (ITS-1) of Ribosomal DNA as a Molecular Marker for Phylogenetic and Population Analyses in Crustacea

The objective of the present study is to explore the feasibility of using the first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for studying the interspecific and intraspecific genetic variations among crustaceans. We designed primers that could amplify ITS-1 from a majority of taxonomic groups of crustaceans. The gene was found to exhibit a high degree of length polymorphism among different groups, ranging from 182 bp in the barnacle Balanus amphitrite to approximately 820 bp in the spiny lobster Panulirus japonicus. With respect to differences between congeneric species, it was found that the ITS-1 sequences of 3 mitten crabs, Eriocheir sinensis, Eriocheir leptognathus, and Eriocheir formosa, exhibit 5.4% to 16.3% nucleotide divergence, suggesting that ITS-1 is informative for phylogenetic analysis at the species level. Yet there are extensive (0.9%–2.3%) variations within individual E. formosa, so that phylogenetic analyses could be obscured. ITS-1 was found to vary between 2 geographical populations of the shrimp Penaeus japonicus. The variations involved substitutions as well as insertions/deletions between shrimp from Australia and South China Sea. These results show that ITS-1 is highly divergent among different crustaceans and could be an appropriate marker for molecular systematic studies at the species and population levels, although the presence of intragenomic variation needs to be taken into consideration. Received August 15, 2000; accepted February 9, 2001     

Sequence variation in ITS spacers and 5.8S rDNA and relationship of E, St, P, Ns, Xm, and H genomes in the genera of Agropyron, Elytrigia, Leymus, Pascopyrum, Psathyrostachys, and Hordeum

Understanding species evolution and improvement requires information of their genome origin and differentiation. Among the species in the family Gramineae, genome identities of Agropyron-Elytrigia-Leymus group are still ambiguous. In order to delineate the genome relationship, nucleotide sequence analysis in the rDNA ITS regions was carried out among the species in the genera Elytrigia, Agropyron, Psathyrostachys, Leymus, and Psacopyrum containing E, St, P, Ns, and Xm genomes. The ITS-1 and ITS-2 showed a narrow range of variation in length except for the presence of a pentanucleotide, TGGGG, in/del in some haplotypes, whereas higher numbers of nucleotide substitutions were observed in most genera. There were 187 variable sites in the ITS-1, 5.8S, and ITS-2 regions, in which a few genome specific mutations were observed. While the level of variation was similar between ITS-1 and ITS-2, the rate of transition mutation versus transversion mutations was different among the ITS-1, 5.8S, and ITS-2 segments. GC contents of the ITS regions ranged between 55–65% between genomes and the haplotypes of P and H genomes were slightly higher than others. In phylogenetic analysis, the ITS haplotypes were classified into two groups; one containing H, Ns, NsXm genomes, and another containing P, St, and E genomes, which are congruous to the genome affinities from other studies. Among the four genomes in Pascopyrum smithii (2n=8x=56, StStNsNsHHXmXm), the haplotypes of H and St genomes were identified with the reference diploid species, but the haplotypes having Ns and Xm genomes were not found in the present analysis.