Species-specific polymorphisms in transcribed ribosomal DNA of fivePythium species

Twenty-five isolates representing fivePythium species collected from diverse hosts and geographic origins were evaluated using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. DNA regions coding for the small-subunit ribosomal RNA (SrDNA) and the internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The amplified SrDNA was about 1800 bp long and uniform in length among the five species. However, restriction digestion revealed three polymorphic groups. They areP. arrhenomanes andP. graminicola,P. irregulare andP. spinosum, andP. ultimum. The amplified-ITS region showed three different lengths which corresponded to the three polymorphic groups of SrDNA. Each length variant of the ITS showed distinct banding patterns after restriction enzyme digestion. In addition,P. irregulare andP. spinosum each showed distinct banding patterns after digestion with enzymesHinfI andMboI. Physical maps of the restriction sites in the SrDNA and the ITS were determined. Length variation occurred primarily in the spacer between the SrDNA and 5.8 S rDNA; although, it also was detected in the ITS-2 region. Little intraspecific variation was observed in the SrDNA and ITS, and species could be reliably distinguished by RFLP analysis of the amplified rDNA regions. Data presented do not support the maintenance ofP. arrhenomanes andP. graminicola as distinct species. Results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for ecological studies ofPythium species.     

Rapid detection ofRhizoctonia species, causal agents of rice sheath diseases, by PCR-RFLP analysis using an alkaline DNA extraction method

Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR) was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract parasite DNA from diseased rice sheaths. 28S ribosomal DNA (rDNA) derived from fungal genomic DNA extracted by the alkaline method was specifically PCR-amplified. The results of PCR-RFLP analysis for DNA samples from artificially inoculated rice sheath tissues with eachRhizoctonia spp. and the corresponding culture on the medium using two restriction enzymes.HhaI andMspI, showed identical polymorphisms. PCR-RFLP analysis using DNA samples from naturally infected rice sheath tissues also revealed the possibility of direct diagnosis ofR. solani AG 1 IA,R. oryzae andR. oryzae-sativae.     

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.     

PCR-ribotyping of type isolates of currently acceptedExophiala andPhaeococcomyces species

Portion of the ribosomal repeat of the type strains of the generaExphiala andPhaeococcomyces were subjected to RFLP analysis. The amplicon length of the small subunit rRNA, the fragment NS1-NS24, was found to vary between 1800 to 3200 nucleotides. In contrast, the length of the fragment ITS1-ITS4 comprising the internal transcribed spacers (ITS1 and ITS2) was found to be constant at 600 nucleotides. Analysis of restriction profiles confirmed the synonymy ofExophiala dermatitidis andMycotorula schawii. Torula bergeri andSporotrichum gougerotii were found to be identical toPhaeoannellomyces elegans, but different from their alleged synomymE. castellanii. A phenogram is presented.     

Protential use of PCR-amplified ribosomal intergenic sequences for differentiation of varieties and species ofGossypium cotton

Our purpose was to develop a new approach to the identification ofGossypium cotton varieties and species based on polymerase chain reaction (PCR). Species-specific distinctions within the genusGossypium have been detected by the amplification of ribosomal genes, namely theRrn18-Rrn25 internal transcribed spacer (ITS) regions that had sequence differences. Using the primers to the 3′-end ofRrn18 adjacent to ITS1 and the 5′-end ofRrn25 adjacent to ITS2 from tomato, we have obtained amplified fragments of two cotton species,G. barbadense andG. herbaceum. Interspecies distinctions have been revealed by the restriction assay of these amplification products. The restriction patterns are distinguished not only by number but by location and intensity of the bands. Our results illustrate the effective use of differences in ribosomal intergenic sequences for the differentiation of varieties and species ofGossypium.     

Identification ofArmillaria species from hokkaido by analysis of the intergenic spacer (IGS) region of ribosomal DNA using PCR-RFLP

The intergenic spacer (IGS) region, which is located between the 3′ end of 26S ribosomal DNA (rDNA) and the 5′ end of 5S rDNA, of sixArmillaria species from Hokkaido was investigated using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Restriction with onlyAlu I could distinguishA. mellea subsp.nipponica from the other species. WithAlu I andDde I,A. ostoyae andA. gallica could be distinguished from the other species. Digestion withAlu I resulted in two patterns (types A and B) ofA. singula and three patterns (types A, B, and C) ofA. jezoensis. One pattern (type B) of the former species and two patterns (types B and C) of the latter species were each different from those of the other species.Armillaria sinapina gave only oneAlu I digestion pattern, which was identical to that ofA. jezoensis (type A) andA. singula (type A). However, by digestion withDde I,A. singula (type A) could be distinguished fromA. jezoensis (type A) andA. sinapina.     

采用PCR-RFLP和RAPD对球壳孢目真菌系统学的研究

对球壳孢目真菌首次采用ＰＣＲ－ＲＦＬＰ和ＲＡＰＤ进行了系统发育研究。以ＩＴＳ１和ＩＴＳ４为引物对４属１２种２４个菌株的核糖体ＤＮＡ转录间区（ＩＴＳ）进行了ＰＣＲ扩增,４种内切酶（ＡｌｕＩ,Ｈｈａ,ＭｓｐＩ,ＴａｑＩ）酶切,结果表明：主要属的ＩＴＳ区长度不同,同属不同种相同。Ｃｏｎｉｏｔｈｙｒｉｕｍ,Ｐｈｙｌｏｓｔｉｃｔａ,Ａｓｃｏｃｈｙｔａ,ＳｅｐｔｏｒｉａＩＴＳ区长度分别为６３０,５６０,５５０,５４０ｂｐ;酶切图谱属间差别明显,属内种间基本一致,暗示传统的分种可能过细,某些种的成立还有商榷的余地,ＰＣＲ－ＲＦＬＰ对确定疑难种属的地位有重要的意义。３属８种１６个菌株的ＲＡＰＤ结果表明,种内图谱相同或图谱类型相同,而种间明显不同,揭示出过去所划分的种的确存在本质的差别,但是否达到种级的差别还有待于进一步研究     

Molecular divergence of the soil yeasts <Emphasis Type="Italic">Williopsis</Emphasis> sensu stricto

Fifty-three strains of Saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA comprising the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, and Komagataea pratensis and the Williopsis sensu stricto complex. The minisatellite primer M13 was proposed for differentiation between sibling species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of Saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 768–776.Original Russian Text Copyright © 2004 by Naumova, Gazdiev, Naumov.     

Sequence and RFLP analysis of the ITS2 ribosomal DNA in two Neotropical social bees, <Emphasis>Melipona beecheii</Emphasis> and <Emphasis>Melipona yucatanica</Emphasis> (Apidae,Meliponini)

Two stingless bees species of the genus Melipona, M. beecheii and M. yucatanica, are the only ones reported for the Yucatan Peninsula. The natural distribution of M. beecheii ranges from southern Mexico to Costa Rica, that of M. yucatanica from south Mexico to Guatemala. Colonies of both species occur in a variety of habitats and show adaptations to local conditions denoting the occurrence of ecotypes. The ITS2 of ribosomal DNA has been characterized in both species and its utility to discriminate among colonies has been investigated through RFLP experiments. The ITS2 region is unusually long, 1788 bp in M. beecheii and 1845 bp in M. yucatanica (including the 3′ end of the 5.8S gene and partial 5′ of the 28S gene). Mean nucleotide divergence between both ITS2 sequences is 16% (excluding sites with insertions/deletions) and 20% when the insertions/deletions are taken into account. The G+C content in both sequences is close to 53%. The PCR-RFLP assay was performed with 12 restriction enzymes on colonies of M. beecheii from Mexico (Yucatan, Campeche and Chiapas) Costa Rica, El Salvador and Guatemala, and of M. yucatanica from Mexico (Yucatan) and Guatemala. The restriction patterns obtained allow to discriminating colonies of both species with different origins. Both kinds of data are thus useful for assessing intra and interspecific genetic variability and for developing appropriate conservation strategies for these species. Received 20 June 2007; revised 31 August 2007; accepted 12 September 2007.     

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR–RFLP and nested-PCR of ribosomal DNA ITS1 region

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1–PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1–PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.     

PCR-RFLP of the ribosomal DNA internal transcribed spacers (ITS) provides markers for the A and B genomes in<Emphasis Type="Italic"> Musa</Emphasis> L.

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the diploid ancestors of modern bananas that are mostly diploid or triploid cultivars with various combinations of the A and B genomes, including AA, AAA, BB, AAB and ABB. The objective of this study was to identify molecular markers that will facilitate discrimination of the A and B genomes, based on restriction-site variations in the internal transcribed spacers (ITS) of the nuclear ribosomal RNA genes. The ITS regions of seven M. acuminata and five M. balbisiana accessions were each amplified by PCR using specific primers. All accessions produced a 700-bp fragment that is equivalent in size to the ITS of most plants. This fragment was then digested with ten restriction enzymes (AluI, CfoI, DdeI, HaeIII, HinfI, HpaII, MspI, RsaI, Sau3AI and TaqI) and fractionated in 2% agarose gels, stained with ethidium bromide and visualized under UV light. The RsaI digest revealed a single 530-bp fragment unique to the A genome and two fragments of 350-bp and 180-bp that were specific to the B genome. A further 56 accessions representing AA, AAA, AAB, AB and ABB cultivars, and synthetic hybrids, were amplified and screened with RsaI. All accessions with an exclusively A genome showed only the 530-bp fragment, while accessions having only the B-genome lacked the 530-bp fragment but had the 350-bp and 180-bp fragments. Interspecific cultivars possessed all three fragments. The staining intensity of the B-genome markers increased with the number of B-genome complements. These markers can be used to determine the genome constitution of Musa accessions and hybrids at the nursery stage, and, therefore, greatly facilitate genome classification in Musa breeding.Communicated by H.F. Linskens     

Genetic relationship of Tricholoma matsutake and T. nauseosum from the Northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.     

小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性(英文)

应用一对寡核苷酸引物ITS1与ITS4对核DNAG＋C百分数小于30％的小克银汉霉（Cunninghamella）属的核糖体DNA（rDNA）内转录间区（YTS）进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为;第一组4种1变种,小于764碱基对（bp）;第二组2种,765～824bp;第三组2种1变种,大于825bp。所研究的许多种中,特别是第三组,单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了RsaI,TaqI;Tru9I,和HinfI4种限制性内切酶,对所有扩增产物进行了限制性片段长度多型性（RFLPs）的分析。在雅致小克银汉霉（Celegans）、巴西玉蕊小克银汉霉（Cbertholletiae）、和布拉氏霉（Cblakesleeana）各种的限制性酶切图谱,种内非常一致而种与种之间有差别。反之,在某些种其限制性酶切图谱不仅种间互不相同,在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下,只能综合各种资料才能将它们区分开来。本研究肯定了PCR－RFLP在区分小克银汉霉种和变种上的意义,并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。     

ITS sequences and speciation on far easternIndigofera (Leguminosae)

The internal transcribed spacer (ITS) regions of 18–26S nuclear ribosomal DNA was sequenced to address phylogenetic relationships and to measure the extent of differentiation among six species of the Far EasternIndigofera. ITS 1 had 230–240 base pairs (bp) long while ITS 2 had 211–213 bp long. The 5.8S rRNA coding region was 161 bp long. Sequence divergence calculated by Kimura's two parameter method between species ranged from 0.00 to 13.49%. A single most parsimonious tree was produced from 77 variable nucleotide sites, which had a consistency index of 0.97 and a retention index of 0.83. ITS sequence data suggested that the continental species ofI. kirilowii (2n=16) is diverged from the common ancestor of other species at first, and then the island species ofI. decora (2n=48) andI. venulosa and the Korean Peninsular species ofI. grandiflora (2n=16) andI. koreana (2n=32) are diverged from the ancestor. The molecular data supports that the speciation in the Far EasternIndigofera occurred with polyploidization from continental ancestor to peripheral peninsular and island species.     

Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA were analyzed in species belonging to gymnosperms and pteridophytes. The lengths of the ITSs of sixteen species of gymnosperms and seven species of pteridophytes were estimated. The gymnosperms have ITS1 regions larger than those observed in the pteridophytes and angiosperms (ca. 610–3100 bp versus 159–360 bp). On the other hand, the ITS2 regions appear to be of a conserved length (182–370 bp). We have determined the complete nucleotide sequences of ITS regions from four gymnosperm species and five pteridophyte species by cloning the PCR products. Sequence analysis showed the presence of three short tandem arranged subrepeats of about 70 bp in the 1112 bp ITS1 ofEphedra fragilis. Pyrimidine rich (about 90%) DNA segments of 40–50 bp were observed in the ITS1 ofGinkgo biloba. A highly conserved 16 bp long sequence known to be present in the ITS1 of the angiosperm species has been also found in the ITS1 ofCycas revoluta, Taxus baccata andEphedra fragilis. Dedicated to Prof.Emilio Battaglia.     

Chloroplast DNA variation of Panax (Araliaceae) in Nepal and its taxonomic implications

The restriction site and size variation of five PCR amplified fragments of noncoding chloroplast DNA (cpDNA) was examined in material from 13 populations ofPanax from Nepal and China. Fourteen restriction endonucleases produced 81 restriction site and length variations from the large single-copy region of cpDNA, 27 of which are polymorphic. The cpDNA dataset suggests two distinct groups ofPanax from Nepal (clades I and II). Clade I consists of two populations ofP. pseudoginseng subsp.pseudoginseng, and clade II is composed of material referable toP. pseudogingeng subsp.himalaicus (vars.himalaicus, angustifolius, andbipinnatifidus). The three accessions ofP. pseudoginseng subsp.japonicus andP. ginseng studied from China had cpDNA characters that differed from the HimalayanPanax. The highly distinctive cpDNA profile and morphology ofP. pseudoginseng subsp.pseudoginseng sensu Hara (1970) from central Nepal support its status as a separate species, which has an extremely restricted distribution.     

Karyotypic and chloroplast genomic diversity inMedicago sect.Lupularia (Leguminosae)

Studies were made on the chromosome complements and chloroplast genomes ofMedicago lupulina andM. secundiflora, which comprise sectionLupularia ofMedicago. Both types of analyses indicated more substantial differences between these species than suggested by external morphology.Medicago lupulina has a relatively asymmetrical karyotype in terms of centromeric position and relative length. The karyotype ofM. secundiflora is comparatively more asymmetrical in centromeric position and reduced in absolute size but exhibits greater symmetry in relative length. The restriction endonuclease fragmentation patterns of the chloropiast DNA of these two species (with Bam HI, Eco RI, Bgl II, and Xho I) show little similarity, with only 17% of the fragments matching in size. The lack of interspecific congruence among data of morphology, karyology and cpDNA inLupularia is contrary to consistency exhibited among these data inMedicago subsect.Intertextae.     

Actinophages and restriction enzymes fromMicromonospora species (Actinomycetales)

Summary To develop a screening procedure for the detection of restriction endonucleases in micromonosporae and catellatosporae based on efficiency of plating, eight different actinophages were isolated from soils enriched withMicromonospora species and one fromCatellatospora-enriched soil. The lytic actinophages all contained double-stranded DNA and the majority appeared, when examined by electron microscopy, to belong to Ackermann's type B1 since they had isometric heads and noncontractile tails. One actinophage was classified as type C1 because of its isometric head and very short noncontractile tail. The host ranges of the actinophages were determined on strains ofMicromonospora and selected species from other actinomycete genera of cell wall chemotype II. Type II restriction enzymes were isolated fromM. echinospora ssp.echinospora (ATCC 15837),M. purpurea (ATCC 15835) andM. zionensis (LL-100-125) and were designatedMecI,MpuI andMziI, respectively. Restriction enzymesMecI andMpuI are isoschizomers ofXhoI, whileMziI is an isoschizomer ofPvuII.     

Allotetraploid origin ofAllium altyncolicum (Alliaceae, Allium sect.Schoenoprasum) as investigated by karyological and molecular markers

The tetraploidAllium altyncolicum (2n = 4x = 32) is considered to be of hybrid origin, because most of its morphological characters are intermediate between those of its putative parents,A. schoenoprasum andA. ledebourianum. In the present work an attempt has been made to ascertain its parentage by several methods: Giemsa C-banding, genomic in situ hybridization (GISH), PCR-RFLP of cpDNA, restriction enzyme mapping of the rDNA, and RAPDs. C-banding and GISH indicates clearly thatA. altyncolicum is a segmental allopolyploid.Allium schoenoprasum andA. ledebourianum are the most likely the parental species and the larger part of the genome ofA. altyncolicum (26 chromosomes) is derived fromA. schoenoprasum. The low genetic divergence between these three species was confirmed by the lack of sequence variation in the ITS sequences of nuclear rRNA genes and of the plastid rbcL-atpB intergenic spacer. Both parental species andA. altyncolicum could be distinguished by RFLP of the rDNA repeats. The geographic origin of the putative parental species was investigated using RAPDs.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

DNA evidence for multiple origins of intergeneric allopolyploids in annualMicroseris (Asteraceae)

Seventy populations of North American annualMicroseris, Stebbinsoseris, andUropappus species were examined for chloroplast and nuclear ribosomal DNA restriction site variability to determine the origin of the allotetraploid speciesS. heterocarpa andS. decipiens. Previously identified chloroplast DNA restriction site variants were used in concert with restriction site variation forNco I in the nuclear-encoded ribosomal DNA repeat. The presence of two, mutually exclusive restriction site gains were observed in diploid populations ofM. douglasii; these same variants were also found in populations of allotetraploidS. heterocarpa, indicating mutiple origins of this species from different maternal diploid populations ofM. douglasii. Variation in the rDNA repeat between the diploid annual species and the putative paternal genome ofU. lindleyi was found to be additive inS. heterocarpa. A similar relationship was observed for the origin ofS. decipiens; cpDNA restriction site variants found inM. bigelovii andM. douglasii were present inS. decipiens. The rDNANco I variants also were additive in this purported allotetraploid. These results confirm the reticulate evolutionary pattern inStebbinsoseris and provide another example of multiple origins of intergeneric allopolyploids.