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     

Phylogenetic analysis of Stylosanthes (Fabaceae) based on the internal transcribed spacer region (ITS) of nuclear ribosomal DNA

 Phylogenetic relationships in Stylosanthes are inferred by DNA sequence analysis of the ITS region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA in 119 specimens, representing 36 species of Stylosanthes and 7 species of the outgroup genera Arachis and Chapmannia. In all examined specimens of any particular diploid and (allo)polyploid species, only a single ITS sequence type was observed. This allowed us to identify a parental genome donor for some of the polyploids. In several diploid and polyploid species, different specimens contained a different ITS sequence. Some of these sequence types were present in more than one species. Parsimony analysis yielded several well-supported clades that agree largely with analyses of the chloroplast trnL intron and partially with the current sectional classification. Discordances between the nuclear and cpDNA analyses are explained by a process of allopolyploidization with inheritance of the cpDNA of one parent and fixation of the ITS sequences of the other. S. viscosa has been an important genome donor in this process of speciation by allopolyploidy. Received August 14, 2001; accepted March 4, 2002 Published online: November 14, 2002 Addresses of the authors: Jacqueline Vander Stappen, Steven Van Campenhout and Guido Volckaert (E-mail: guido.volckaert@agr.kuleuven.ac.be), Katholieke Universiteit Leuven, Laboratory of Gene Technology, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium. Jan De Laet, American Museum of Natural History, Division of Invertebrate Zoology, Central Park West at 79th Street, New York 10024–5192, USA. Susana Gama-López, Universidad Nacional Autónoma de México, Unidad de Biología, Tecnología y Protipos (UBIPRO), FES-Iztacala, Laboratorio de Recursos Naturales, Av. de Los Barrios S/N, Colonia Los Reyes Iztacala, Municipio Tlalnepantla, Estado de México, C.P. 54090, México. Present address: Apartado Postal 154, Cto. Parque No. 3, C.P. 53102, México.     

Phylogeny of Allium L. subg. Melanocrommyum (Webb et Berth.) Rouy based on DNA sequence analysis of the internal transcribed spacer region of nrDNA

 Sequence analysis of the ITS region of nuclear ribosomal DNA from subgeneric representatives of Allium L. produced phylogenetic trees which concurred with previous conclusions based on classical taxonomy. Phylogenetic analysis revealed a closer relationship between Nectaroscordum siculum and Allium cernuum (representing Amerallium) than between A. cernuum and the rest of the Allium species employed in this study. The phylogeny of subg. Melanocrommyum based on ITS sequences largely agreed with inferences made by previous researchers based on morphology or a restriction analysis of chloroplast DNA. However, the phylogenetic positions of Allium protensum and Allium macleanii based on ITS sequences did not correspond to their morphological similarity with Allium schubertii and Allium giganteum, respectively. Received: 15 February 1998 / Accepted: 12 March 1998     

Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP

 In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300–900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon’s index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease. Received: 25 August 1997 / Accepted: 19 September 1997     

A phylogenetic analysis of Primulaceae s.l. based on internal transcribed spacer (ITS) DNA sequence data

 Phylogenetic relationships in Primulaceae were investigated by analysis of nuclear rDNA ITS sequences. Thirty-four species of Primulaceae, two of Myrsinaceae and four outgroup taxa were analyzed. In accordance to the results of recently published papers on the phylogeny of Primulaceae we found the family to be paraphyletic and resolved the positions of some genera. Our results show (a) the rather basal position of Centunculus within Lysimachieae, the genus thus being rather distantly related to Anagallis, (b) the close relationship between Lysimachia sect. Lerouxia, Anagallis, Asterolinon, and Pelletiera, (c) the well-supported monophyly of a group consisting of the four genera Hottonia, Omphalogramma, Bryocarpum, and Soldanella, and (d) the affinity of Stimpsonia to the Myrsinaceae-Lysimachieae-Ardisiandra clade. The ITS sequence data do not provide sufficient information to resolve basal relationships within the Primulaceae s.l. There is evidence against the monophyly of the large genera Primula, Androsace, and Lysimachia. In contrast to the phylogenetic reconstructions based on plastid gene sequences, Cyclamen does not appear as a member of the Myrsinaceae-Lysimachieae clade, but its position remains unclear. Revised July 10, 2002; accepted November 21, 2002 Published online: March 20, 2003     

Molecular phylogenetics of Hippophae L. (Elaeagnaceae) based on the internal transcribed spacer (ITS) sequences of nrDNA

 The genus Hippophae comprises 7 species and 8 subspecies according to the latest classification, and has shown enormous ecological, nutrient and medicinal values. Here we analyzed the phylogenetic relationships among 15 taxa of the genus by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). ITS sequences in Hippophae varied in length from 651 bp to 666 bp. The aligned sequences were 690 bp in length and 269 (39.0%) were variable sites with 150 being parsimony-informative. The amount of polymorphism observed within a taxon was extremely low in most taxa except for two putative hybrid species. The aligned sequences were analyzed by maximum parsimony (MP) and neighbor-joining (NJ) methods. In the strict consensus trees of parsimony analysis, the monophyly of Hippophae was supported by 100% bootstrap value. H. tibetana was at the basal position of the genus, and the remaining taxa formed two clades with high bootstrap support. The first clade included subspecies of H.␣rhamnoides and the other one consisted of remaining species. Parsimony analysis also suggested that the species H. tibetana, H. neurocarpa and H.␣salicifolia were all distinct. Although the sequence divergence among subspecies of H. rhamnoides was also remarkably high, the molecular data supported the monophyly of H. rhamnoides when H. rhamnoides subsp. gyantsensis Rousi was excxluded. The NJ trees showed essentially the same topology. The taxonomical arrangement that divided the genus into two sections was not supported based on the ITS sequences. However, the hybrid origin of H. goniocarpa and H. litangensis proposed previously was supported by the present ITS data. Received January 7, 2002; accepted May 10, 2002 Published online: November 22, 2002 Addresses of the authors: Kun Sun, Xuelin Chen, Ruijun Ma, Qin Wang, Institute of Botany, Northwest Normal University, Lanzhou 730070, China. Changbao Li, Song Ge (e-mail: gesong@ns.ibcas.ac.cn or song_ge@hotmail.com), Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.     

Phylogenetic analysis of Acacia (Mimosaceae) as revealed from chloroplast RFLP data

 Chloroplast DNA of 22 species of Acacia (Tourn.) Miller was digested with ten restriction endonucleases, Southern-blotted and probed with cloned fragments covering the chloroplast genome of tobacco (Nicotiana tabacum L.). Phyletic and phenetic analyses of the resulting 176 polymorphic bands recorded among the 22 species were performed. The phylogram was reconstructed using heuristic search and Wagner parsimony. The resulting most parsimonious consensus phylogram displayed three major phyletic lineages, consistent with the previously established three subgenera of Acacia. The 10 species of subgenus Acacia and the 6 species of subgenus Heterophyllum formed two monophyletic sister clades. The 5 species of subgenus Aculeiferum studied and Acacia albida (Syn. Faidherbia albida) grouped together and were basal to the clades of subgenera Acacia and Heterophyllum. The phylogram indicated that subgenus Heterophyllum diverged earlier from subgenus Aculeiferum than did subgenus Acacia; however, the phenogram indicated the reverse. The study indicated that A. nilotica and A. farnesiana are sister species, though A. nilotica is Afro-Asiatic and A. farnesiana is American. The phenogram separated the three subgenera in agreement with the phylogram, but the two dendrograms differed regarding the topologies of the species and the distance of evolution between subgenera Acacia and Heterophyllum. Received: 8 July 1998 / Accepted: 24 July 1998     

Phylogenetic analysis in the Festuca-Lolium complex using molecular markers and ITS rDNA

 Molecular markers were used to investigate phylogenetic relationships among the eight species of ryegrass (Lolium) and 11 species of fescue (Festuca). RAPD and RFLP analyses were carried out on total bulked DNA from each population. Factorial analysis of a phenetic distance matrix yielded three major groups: (1) fine-leaved fescues, (2) broad-leaved fescues and (3) ryegrasses. Six non-coding regions of chloroplastic DNA were PCR-amplified, then digested by 20 restriction enzymes. Nuclear rDNA sequences, including internal transcribed spacers (ITSs) were used to estimate the average proportion of nucleotide substitutions. The correlation between substitution rate estimated from ITS sequences and that estimated from organelle DNA restriction sites was very high (0.94), and the corresponding UPGMA trees were very similar, with a slightly better resolution of the ITS tree in the Lolium genus. The time-scale inferred from substitution rates indicated that the period since divergence of the broad-leaved fescues from the fine-leaved fescues was four times as long as that since divergence of the genus Lolium from the former. Among the broad-leaved fescues, meadow fescue was closer to the Lolium group, while F. glaucescens and tall fescue were very closely related. North-African fescues were clustered together and giant fescue was the most differentiated species in this group. Our dataset was merged with ITS sequences recovered from the EMBL database, and the neighbor-joining method was used to draw a phylogenetic tree. In this tree, the tribe Poeae was clearly monophyletic, and more closely related to the Aveneae than to the Triticeae or Bromoideae. The genus Festuca appeared somewhat artificial, since Vulpia myuros and Dactylis glomerata were placed between fine-leaved and broad-leaved fescues. Received: 10 January 1997/Accepted: 21 February 1997     

Sequence variations of the first ribosomal internal transcribed spacer of Penaeus species in Thailand

The ribosomal DNA internal transcribed spacer 1 (ITS1) was investigated in the search for additional genetic marker that is suitable for population studies of the penaeid shrimps. The sequence variations of the ITS1 were determined and found to be informative in estimating phylogenies in that they differentiate four species of penaeid shrimps, namely Penaeus merguiensis, Penaeus silasi, Penaeus monodon and Penaeus semisalcatus and the populations of P. merguiensis collected in the Gulf of Thailand and the Andaman Sea. The length of the ITS1 ranged from 499 to 772 bp, with a GC content of 63.30-67.37%. Four microsatellite loci are found in the ITS1 at 5′ end and the middle of region and seem to be associated with sequence divergence and size variation in Penaeus species. Some microsatellites were found in only one specie, (GCGA)₄ in P. semisalcatus and (CGGA)_4-9 in P. monodon. These microsatellite regions are considerably long enough and the level of intragenomic variation in P. merguiensis is less than that between different species, hence, provide a great potential use in the population studies.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Phylogenetic reconstruction of endophytic fungal isolates using internal transcribed spacer 2 (ITS2) region

Endophytic fungi are inhabitants of plants, living most part of their lifecycle asymptomatically which mainly confer protection and ecological advantages to the host plant. In this present study, 48 endophytic fungi were isolated from the leaves of three medicinal plants and characterized based on ITS2 sequence – secondary structure analysis. ITS2 secondary structures were elucidated with minimum free energy method (MFOLD version 3.1) and consensus structure of each genus was generated by 4SALE. ProfDistS was used to generate ITS2 sequence structure based phylogenetic tree respectively. Our elucidated isolates were belonging to Ascomycetes family, representing 5 orders and 6 genera. Colletotrichum/Glomerella spp., Diaporthae/Phomopsis spp., and Alternaria spp., were predominantly observed while Cochliobolus sp., Cladosporium sp., and Emericella sp., were represented by singletons. The constructed phylogenetic tree has well resolved monophyletic groups with >50% bootstrap value support. Secondary structures based fungal systematics improves not only the stability; it also increases the precision of phylogenetic inference. Above ITS2 based phylogenetic analysis was performed for our 48 isolates along with sequences of known ex-types taken from GenBank which confirms the efficiency of the proposed method. Further, we propose it as superlative marker for reconstructing phylogenetic relationships at different taxonomic levels due to their lesser length.     

Species-specific molecular signature of Commiphora species of Saudi Arabia inferred from internal transcribed spacer sequences of nuclear ribosomal DNA

The deciduous habit and tendency to produce flowers prior to developing leaves, and a predominantly dioecious system of breeding in the genus Commiphora leads to difficulties in its taxonomic identification at species level. The characteristics of easy amplification by universal primer, shorter length and higher discrimination power at the species level makes the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) to a smart gene for generating species-specific phylogenetic inferences in most of the plants groups. The present study deals the ITS sequence of nrDNA based molecular genotyping of seven species of the genus Commiphora of Saudi Arabia. The molecular phylogenetic analysis of ITS sequences of nrDNA of Commiphora species distributed in Saudi Arabia reveals the the occurrence of C. madagascariens in Saudi Arabia.     

Identification of astigmatid mites using the second internal transcribed spacer (ITS2) region and its application for phylogenetic study

The second internal transcribed spacer (ITS2) of nuclear ribosomal DNA from 73 specimens of Astigmata was analyzed by PCR amplification and DNA sequencing. The length of the ITS2 region varied from 282 to 592 bp. The interspecific variation based on consensus sequences was more than 4.1%, while the intraspecific or intra-individual variation was from 0 to 5.7%. The variation between geographically separated populations (0–3.2%) was almost the same as the variation within strains. The sequences of the ITS2 region of Astigmata were concluded to be species-specific. The phylogenetic tree inferred from the ITS2 region supported Zachvatkins morphological classification in the subfamily Rhizoglyphinae. The species-specific ITS2 sequence is useful for the species identification of astigmatid mites and for studying low-level phylogenetic relationships.Chemical Ecology of Astigmatid Mites LXXVThis revised version was published online in May 2005 with a corrected cover date.     

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.     

ITS DNA sequence relationships between Lilium concolor Salisb., L. dauricum Ker-Gawl. and their putative hybrid, L. maculatum Thunb.

 Cycle sequencing of the ITS region of nuclear ribosomal DNA of L. concolor, L. dauricum and L. maculatum generated surprisingly homogenous sequences from these three species. Analysis of the few (13 out of 639) polymorphic nucleotide sites in the ITS region produced results that do not support the belief that L. maculatum is a hybrid of the two other species. Neighbor-Joining analysis of the genetic distances calculated using the Kimura 2-parameter model of base substitution confirmed the close relationship between L. dauricum and L. maculatum. The phylogenetic tree, in conjunction with the distribution pattern and morphological similarities of the two species, suggest that L. maculatum is derived from the more widely distributed L. dauricum. The results also revealed that there is sufficient molecular divergence between L. maculatum and L. dauricum to support their status as separate species. Received: 4 June 1998 / Accepted: 15 July 1998