Heterogeneity in the ITS of the ribosomal DNA of <Emphasis Type="Italic">Pyrenophora graminea</Emphasis> isolates differing in xylanase and amylase production

Xylanase and amylase have gained increasing interest because of their various biotechnology applications. In this research, the restriction of PCR-amplified internal transcribed spacers (ITS) of ribosomal DNA (rDNA) was used to confirm the genetic variation among 22 isolates of Pyrenophora graminea differing in their xylanase and amylase production. The fingerprints generated from the six restriction digestions of the rDNA ITS region showed high levels of intraspecific variation within the P. graminea population. Neighbour-Joining diagram, based on Nei’s genetic distances, showed that isolates formed two phylogenetic groups. No apparent association could be observed between xylanase and amylase production and genetic diversity among the twenty-two isolates.     

Polymorphism in the ITS region of ribosomal DNA of Cochliobolus sativus isolates differing in xylanase production

The restriction of PCR-amplified internal transcribed spacers (ITS) ofribosomal DNA was used to confirm the genetic variation among 22 isolates of Cochliobolus sativus differing in their xylanase production. Results show a high level of diversity of ITS-RFLP markers among the isolates. The molecular parameter used showed that C. sativus isolates reside in three phylogenetic groups. There was observed the resolution between clustering of isolates and their xylanase production level.     

Molecular and xylanolytic variation identified among strains of Pyrenophora graminea

The inter-retrotransposon amplified polymorphism (IRAP) was used to confirm the genetic variation among 22 strains of Pyrenophora graminea differing in their xylanase production. A total of 162 bands were scored of which 151 (93.21%) were polymorphic. The molecular parameter used showed that P. graminea strains reside in four phylogenetic groups. There was observed the resolution between clustering strains and their xylanase production. Hence, the described approach presented here constitutes no prior assumption about the characterization of P. graminea strains differing in xylanase production.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Random Amplified Polymorphic DNA and Polymerase Chain Reaction Markers for the Differentiation and Detection of Stenocarpella maydis in Maize Seeds

The genetic relationship of 34 isolates of Stenocarpella maydis from different geographic regions in South Africa was analysed by random amplified polymorphic DNA (RAPD) and ribosomal DNA markers. Two genetic groups were differentiated by using three RAPD primers and correlated to the cultural morphology of the isolates. Of all the isolates tested, 79.4% were clustered into RAPD group I (RG I), which did not sporulate when cultured on potato dextrose agar (PDA) at 25°C for 10 days. The rest of the isolates designated as RG II sporulated on PDA medium and showed a higher genetic variation. Ribosomal DNA (rDNA) was amplified using polymerase chain reaction (PCR) with the universal primers, internal transcribed spacer (ITS) 1 and ITS 4. Restriction digestion of PCR products displayed three types (RF A, RF B and RF C) of profiles. RF A was in accordance with RG I. RF B was consistent with RG II except for one isolate, U5. However, U5 displayed a unique profile and had no restriction sites for Hpa II and Hae III. The results indicate that two distinct genetic groups exist among S. maydis isolates from maize in S. Africa. The ITS1 and ITS2 regions of rDNA were sequenced and primers were designed. The designed primer pair P1/P2 permitted a sensitive and specific detection of S. maydis .     

大豆疫霉根腐病菌的rDNA ITS序列分析

采用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增了大豆疫霉根腐病菌具有差异的17个菌株的ITSI与ITS2,经过与DL2000的标准分子量DNA进行比较,得到了大约800~1000bp左右的片段,并对PCR产物进行了序列测定。以USA为外类群利用最大简约法构建了大豆疫霉根腐病菌的系统发生树,并分析了菌株之间的遗传进化关系。结果表明:不同菌株ITS1和ITS2在碱基构成上有很大差异,17个菌株大致分为4个谱系中,且来自于同一地区的菌株大都分布在同一谱系中,显示出地理上的差异。     

黑龙江省大豆菌核病菌核盘菌遗传多样性分析

了解黑龙江省不同地区侵染大豆核盘菌菌株分离物间的主要特性差异,利用PDA培养基对核盘菌进行分离和纯化,同时利用RAPD和rDNA-ITS标记方法对核盘菌进行遗传多样性分析,获得了50株纯化的核盘菌,用RAPD标记确定的遗传相似系数范围为0.54-0.98,平均相似系数为0.76,说明供试的核盘菌菌株的基因型具有一定的差异。对50个测定序列有差异的32个核盘菌ITS和5.8S rDNA片段的多序列对位分析,在ITS1区域的1-40bp种间变化较大,主要以碱基颠换和转换为进化形式。ITS2区域非常保守没有变异位点。黑龙江省核盘菌菌株在DNA水平上和ITS间隔区上具有较显著的遗传变异,显示出丰富的遗传多样性。     

日本血吸虫中国大陆株基因多态性研究

对日本血吸虫中国大陆株湖南、湖北、江西、安徽、四川、云南隔离群以及一个实验室传代品系从基因水平进行了多态性研究。首先,在用ＰＣＲ－ＳＳＷＣＰ技术分析了２８Ｓ ｒＤＮＡ－Ｄ２高变区基础上,测定了该区安徽和云南隔离群的ＤＮＡ序列;其次,用ＰＣＲ获得了含有ＩＴＳ的ｒＤＮＡ片段,并对其进行了酶切点重复序列的多态性分析;最后,用ＲＡＰＤ技术分析了全基因组ＤＮＡ的多态性。结果表明,安徽与云南隔离群的２８Ｓ ｒ     

PCR-RFLP patterns of four isolates of Trichinella for rDNA ITS1 region

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITS1 specific primers and digested with restriction endonucleases. The PCR product of ITS1 was confirmed using Southern blot analysis to be a 910 bp fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa 1 only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Trichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITS1 region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITS1 region.     

Physical localization of NORs and ITS length variants in old Portuguese durum wheat cultivars

The variation at the internal transcribed spacer (ITS) region of the ribosomal DNA has been correlated with the number of nucleolar organizer regions (NORs) in some plant species. Besides, the number of NORs might influence the rate of homogenization of the rDNA repeats. In recent studies, ITS length variants were detected in bread wheat cultivars but no reports about their presence in durum wheat were found. In the present study, we localized and identified the NORs of 51 old Portuguese durum wheat cultivars by using sequential silver staining and fluorescence in situ hybridization performed with the pTa71 rDNA probe. We also detected ITS length variants by PCR-RFLP. No variation at the number of Ag-NORs per metaphase was found among the 51 durum wheat cultivars, but the PCR-RFLP technique carried out with the restriction enzyme HpaII, allowed the detection of ITS length variants among them. The molecular data was used in order to establish the genetic relationships among cultivars and botanical varieties of durum wheat. The knowledge of this feature could be useful for future design of breeding strategies, involving this collection that constitutes an excellent repository of germplasm in Portugal.     

Molecular identification of the edible ectomycorrhizal fungus Lactarius deliciosus in the symbiotic and extraradical mycelium stages

Specific rDNA ITS amplifications, microsatellite-primed PCR and ITS-SSCP analysis were applied to identify and characterize pre-selected isolates of the edible ectomycorrhizal fungus Lactarius deliciosus in different stages of the life cycle. Sampling was performed from pure cultures, mycorrhizas and soil from experimental plots established with nursery-inoculated pine seedlings. A newly-designed reverse primer (LDITS2R) combined with the universal forward ITS1 allowed to perform specific amplifications of L. deliciosus from all the samples. Microsatellite-primed PCR using the (GTG)5 oligonucleotide as a primer showed clear polymorphisms among the different L. deliciosus isolates. The patterns of mycorrhiza samples showed additional bands corresponding to the plant DNA. Single strand conformation polymorphism (SSCP) analysis of the specific rDNA ITS fragment amplified from 18 L. deliciosus isolates showed nine clearly different patterns. Mycorrhiza and soil samples showed coincident patterns with their respective fungal isolates. Specific rDNA ITS amplifications had not been previously used for SSCP analysis of ectomycorrhizas and extraradical mycelium. This relatively simple and inexpensive technique allows tracking L. deliciosus isolates in different stages of the fungus development. Specific ITS-SSCP analysis is promising in studies of the persistence of inoculated L. deliciosus isolates and their competitiveness with native ectomycorrhizal fungi, especially at the extraradical mycelium stage.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Heterogeneity of ITS1 sequences in the biting midge Culicoides impunctatus (Goetghebuer) suggests a population in Argyll, Scotland, may be genetically distinct.

Ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, variation in ITS1 from eight Culicoides species was analysed by PCR, DNA restriction analysis, cloning, and sequencing. ITS1 variants were essentially homogenized within a species, as sequences were identical or closely related. However, Culicoides impunctatus ITS1 sequences derived from one (Argyll) of five populations contained considerable genomic diversity. The secondary structure of each ITS1 was computed. The structure aided the production of an accurate alignment and the identification of a large indel. A phylogenetic analysis was performed. Some of the sequences from the diverse Argyll C. impunctatus population were more related to Culicoides imicola, a vector of animal pathogens in the Old World, than they were to the other C. impunctatus sequences. Thus, the rDNA ITS1 regions of individuals in the Argyll C. impunctatus population were not conforming to the general theory of rDNA homogenization through molecular drive.     

Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.     

土生空团菌的遗传多样性分析

为了研究土生空团菌的遗传多样性,对来自中国、美国、瑞士和法国等菌株的rDNA ITS区进行序列分析,利用Popgene32和phylip软件进行数据计算和聚类分析。序列分析表明土生空团菌rDNA ITS区的序列长度为422–447bp,遗传距离在0.000–0.051之间。居群结构和聚类分析结果表明:(1)土生空团菌有一定的遗传多样性,且遗传差异主要来自于居群内;(2)基因流Nm>1,遗传漂变不是导致土生空团菌居群遗传分化的主要因素;(3)土生空团菌的遗传分化受到地理环境的影响,而与宿主来源没有明显相关性。     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Molecular characterization and evaluation of mycorrhizal capacity of Suillus isolates from Central Spain for the selection of fungal inoculants

Suillus fungal specimens of pine forests from a Mediterranean area of central Spain (Madrid region) were studied based on molecular and physiological analysis of sporocarps to obtain fungal native inocula to produce mycorrhizal Pinus halepensis Miller in nursery. Variation within the internal transcribed spacer (ITS) region of the ribosomal RNA genes of Suillus was examined by restriction fragment length polymorphism (RFLP) and direct sequencing of polymerase chain reaction products. Ribosomal DNA (rDNA) spacers were amplified from pure cultures obtained from fruit bodies of a range of Suillus species: Suillus bellinii (Inzenga) Watling, Suillus bovinus (Pers.) Kuntze, Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.) Snell, Suillus mediterraneensis (Jacquet. & Blum) Redeuil, Suillus luteus L. (Gray), and Suillus variegatus (Sw.) Kuntze. Interspecific variation in the length and number of restriction sites of the amplified ITS region was observed. This variation was confirmed by sequencing, which allowed us to identify some isolates. This is the first time that the ITS sequence of S. mediterraneensis is completely described. No intraspecific rDNA variation was observed within isolates of S. collinitus, S. mediterraneensis, and S. luteus. The phylogenetic analysis established the close relationship among these Mediterranean fungal species. As a further step to characterize the different isolates and to understand the relation between genetic and functional diversity, some physiological variables were evaluated. Intraspecific variation in axenic fungal growth and in mycorrhizal capacities was detected, especially within S. collinitus isolates. The fungal isolates stimulated the growth of P. halepensis in different rates. These studies indicated that ITS analysis, in conjunction with mycorrhizal tests, provides suitable combined tools for the analysis of Suillus spp. in a small geographic area for selecting isolates with final afforestation purposes.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA

Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B1 and B2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.     

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.