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.     

Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector,Triatoma dimidiata (Latreille, 1811)

Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5'' end. To overcome these challenges we have designed new primers that amplify only the 3''-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.     

Variability of Ribosomal DNA ITS-2 and Its Utility in Detecting Genetic Relatedness of Pearl Oyster

The objective of this study was to detect interspecific and intraspecific genetic variations of the second internal transcribed spacer of ribosomal DNA (ITS-2), and explore the feasibility of using it as a molecular marker phylogenetic analyses and species identification among pearl oysters. ITS-2 sequences of 6 pearl oysters were amplified via polymerase chain reaction. The amplified DNA fragments were about 500 bp, spanning the partial sequences of 5.8S and 28S rRNA genes. The GC contents of all species used in this study were higher than the AT contents. The variations of sequences involved substitutions as well as insertions/deletions and were mainly concentrated in spacer regions. Sequences of about 30-bp in spacer regions showed no variations among 5 Pincatda species. Intraindividual and intraspecific polymorphisms of ITS-2 sequences were detected in some species; the interspecific variability was significantly larger than the variability within species, and the variability at the genus level was higher than that at the species level. Both neighbor-joining and parsimony analyses of ITS-2 sequences revealed the distinguishable species boundary of 6 pearl oysters, and indicated that P. chemnitzi and P. nigra were the closely related species, as were P. maxima and P. margaritifera. The findings revealed that ITS-2 sequences could be an appropriate tool for phylogenetic study of pearl oysters.     

Ribosomal DNA ITS-1 Intergenic Spacer Polymorphism in Triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.     

Differentiation among three species of bovine Thelazia (Nematoda: Thelaziidae) by polymerase chain reaction–restriction fragment length polymorphism of the first internal transcribed spacer ITS-1 (rDNA)

Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini are nematodes transmitted by some species of Musca (Diptera: Muscidae) which cause ocular infestations in bovines. Differences in the rDNA of these species were determined by a PCR using different sets of relatively conserved oligonucleotide primers. PCR on the first internal transcribed spacer (ITS-1) revealed differences in size in Thelazia species (437 bp for T. gulosa, 370 bp for T. rhodesi and 506 bp for T. skrjabini) while the DNA control of Musca spp. was not amplified. The ITS-1 amplicons of the three species were sequenced and then analysed. The GC contents ranged from 26 to 36% and the level of differences in the nucleotide sequences of ITS-1 was lower between T. skrjabini and T. gulosa (39%) than the latter and T. rhodesi (49–56%). Restriction fragment length polymorphism (RFLP) of ITS-1 amplicons was also carried out and the restriction profiles compared. Clear genetic differences among the three Thelazia examined were demonstrated by using the enzymes HpaII, CpoI and SspI. This PCR–RFLP for the delineation of T. gulosa, T. rhodesi and T. skrjabini offers prospects as a molecular epidemiological tool to study parasite transmission patterns and prevalence.     

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.     

ITS-2 and 18S rRNA Gene Phylogeny of Aplysinidae (Verongida, Demospongiae)

18S ribosomal DNA and internal transcribed spacer 2 (ITS-2) full-length sequences, each of which was sequenced three times, were used to construct phylogenetic trees with alignments based on secondary structures, in order to elucidate genealogical relationships within the Aplysinidae (Verongida). The first poriferan ITS-2 secondary structures are reported. Altogether 11 Aplysina sponges and 3 additional sponges (Verongula gigantea, Aiolochroia crassa, Smenospongia aurea) from tropical and subtropical oceans were analyzed. Based on these molecular studies, S. aurea, which is currently affiliated with the Dictyoceratida, should be reclassified to the Verongida. Aplysina appears as monophyletic. A soft form of Aplysina lacunosa was separated from other Aplysina and stands at a basal position in both 18S and ITS-2 trees. Based on ITS-2 sequence information, the Aplysina sponges could be distinguished into a single Caribbean–Eastern Pacific cluster and a Mediterranean cluster. The species concept for Aplysina sponges as well as a phylogenetic history with a possibly Tethyan origin is discussed.Reviewing Editor: Dr. Martin Kreitman     

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i. e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E. galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the macronucleus and peristomial lip might be the important phylogenetic characteristics within the genus Epistylis.     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

The internal transcribed spacer 1 (ITS-1), a controversial marker for the genetic diversity of Trypanosoma evansi

Seven Trypanosoma evansi isolates from China and a Trypanosoma congolense sp. gifted from Kenya were characterized genetically by the internal transcribed spacer 1 (ITS-1) of nuclear ribosomal DNA (rDNA). The ITS-1 rDNA with the length of 338–342 bp was amplified by polymerase chain reaction (PCR) and sequenced from individual isolates of T. evansi. Although sequence variation between T. evansi isolates from China only was 0.3–3.8%, the constructed phylogenetic tree based on the ITS-1 rDNA sequence by the method of neighbor-joining and maximum parsimony revealed the genetic diversity among T. evansi isolates from China. For T. congolense sp., the most phylogenetically related species was T. congolense IL1180. Although the sequence variation ranged 0.8–14.5% between T. congolense isolates, the phylogenetic tree can not reflected the genetic diversity among T. congolense isolates perhaps because of the fewer number of isolates and sequences. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

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.     

Molecular characterization of larval anisakid nematodes from marine fishes off the Moroccan and Mauritanian coasts

A total of 242 larval forms of Anisakis collected from marine fishes at different sites off the Moroccan and Mauritanian coasts, recognised as belonging to Type I and Type II larvae, were identified by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms) of the ITS (Internal Transcribed Spacers) region (ITS-1, 5.8 subunit rRNA gene and ITS-2), using a previously established molecular key. The Type I larvae were found with a frequency of 98.34% and were identified as belonging to the following species: A. simplex s.str., A. pegreffii, A. simplex s.str/A. pegreffii heterozygote genotypes, A. typica, A. ziphidarum and Anisakis sp. A. The Type II larvae were found to belong to A. physeteris, with the frequency of 1.65%. The results reported in the present study provide further epizootiological and biological data on the Anisakis spp. in marine fishes off the Moroccan and Mauritanian coasts, improving the picture of the occurrence of these species in the central Atlantic coasts.     

Development of a SCAR marker and a strain‐specific genomic marker for the detection of the biocontrol agent strain CPA‐8 Bacillus amyloliquefaciens (formerly B. subtilis)

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA‐8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA‐8 that was sequenced and used to design SCAR (sequence‐characterised amplified regions) primer pairs. The SCAR‐4 marker amplified a semi‐specific fragment of 665 bp not only for the strain CPA‐8 but also for other 12 strains whose morphology was completely different from CPA‐8. Another approach was developed to obtain a strain‐specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA‐8. Our results revealed that these two molecular markers, SCAR‐4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA‐8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA‐8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.     

Little divergence in ribosomal DNA internal transcribed spacer -1 and -2 sequences among non-digitate species of Laminaria (Phaeophyceae) from Hokkaido, Japan

The nuclear ribosomal DNA internal transcribed spacer (ITS-1 and ITS-2) sequences were determined for 10 of 12 Japanese non-digitate Laminaria species, Kjell-maniella gyrata (Kjellman) Miyabe, Costaria costata (Turner) Saunders, Alaria praelonga Kjellman and Chorda filum (L.) Stackhouse collected at Hokkaido. Phyloge-netic analyses (maximum parsimony and distance matrix) of these sequences, including published data for L. sac-charina (L.) Lamouroux from Canada, showed strong nucleotide conservation among these species of Laminaria, but two phylogenetically distinct species groups were recognized. A L. japonica group encompassing L. yapon/ca Areschoug, L. religiosa Miyabe, L. ochotensis Miyabe, L. diabolica Miyabe, L. longipedalis Okamura, L. angustata Kjellman and L. longissima Miyabe; and a L. saccharina group including L. coriacea Miyabe, L. sac-charina, L. cichorioides Miyabe and L. yendoana Miyabe. As to other laminarialean genera, Kjellmaniella gyrata was most closely related to the genus Laminaria, being related to the second Laminaria species group based on both parsimony and distant tree values.     

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.     

Natural occurrence of Wolbachia-infected and uninfected Trichogramma species in tomato fields in Portugal

Minute egg parasitoids of the genus Trichogramma (Hymenoptera; Trichogrammatidae) are promising candidates for biological control of lepidopteran pests in tomato in Portugal. This certainly applies to native Trichogramma strains that have thelytokous reproduction, i.e., produce only daughters. In Trichogramma wasps, thelytoky is mostly induced by the intracellular bacterium Wolbachia. In this study, we carried out a field survey of native Trichogramma species in four locations in Ribatejo, the main processing tomato region of Portugal, and determined the prevalence of Wolbachia in those species. Five Trichogramma species were found to emerge from lepidopteran eggs collected in the field, namely Trichogramma bourarache, Trichogramma cordubensis, Trichogramma evanescens, Trichogramma pintoi, and Trichogramma turkestanica. T. evanescens and T. pintoi were by far the dominating species representing, respectively, 64.9 and 26.4% of the trichogrammatids collected. Total natural parasitism rates of the collected lepidopteran eggs by Trichogramma wasps ranged from 28.2 to 64.6%. Three Trichogramma species were found to be infected with Wolbachia, namely T. cordubensis, T. evanescens, and T. turkestanica. All the wasp broods belonging to T. cordubensis were infected, whereas low infection rates were found in T. evanescens (0.9% of the broods) and T. turkestanica (4.5% of the broods). The latter represents the first record of a Wolbachia infection in T. turkestanica. Sequencing of the Wolbachia surface protein, wsp, revealed this Wolbachia infection to be related to other Wolbachia infections in Trichogramma wasps. As Wolbachia-infected thelytokous strains exist for T. evanescens, the most abundant Trichogramma species naturally occurring in the tomato fields of the Ribatejo region, this species offers interesting and powerful options for biological control of lepidopteran pests in processing tomato in this region.     

Taxonomic reappraisal on<Emphasis Type="Italic">Suaeda australis</Emphasis> (Chenopodiaceae) in korea based on the morphological and molecular characteristics

We used morphological and molecular characteristics to perform a taxonomic reappraisal ofSuaeda australis (Brown) Moquin-Tandon from Korea. Populations of this species are dispersed at the bottoms of sand zones, and usually exhibit a depressed habit. Except for their total heights and leaf lengths, the morphology of these plants does not differ from that ofS. maritima. Molecular traits were examined based on ITS and psbB-psbH spacer region sequences. The former region included ITS-1, 5.8S, and ITS-2, which were 629 nucleotide bases long. Pair-wise distances (p-distance) among KoreanSuaeda species ranged from 1.12 to 17.84. The psbB-psbH spacer region sequences were 618 nucleotide bases long, and were conserved in the alignment of KoreanSuaeda species. In our ML and MrBayesian analysis of ITS sequences aligned with other sequences from GenBank, the plants of KoreanSuaeda made three clades: 1)S. japonica;S. australis, andS. maritima;2)S. malacosperma; and 3) S.glauca. However, the psbB-psbH region sequences could not be resolved amongS. japonica, S. maritima, andS. australis from Korea. Molecular and vegetative characteristics indicated that the plants now classified asS. australis from Korea should instead be named as S.maritima (L.) Dumont.     

Understanding diversity in coral-algal symbiosis: a cluster-based approach to interpreting fine-scale genetic variation in the genus <Emphasis Type="Italic">Symbiodinium</Emphasis>

Reef corals associate with an extraordinary diversity of dinoflagellate endosymbionts (genus Symbiodinium), and this diversity has become critical to understanding how corals respond to environmental changes. A popular molecular marker for Symbiodinium diversity, the Internal Transcribed Spacer-2 (ITS-2) region of ribosomal DNA, has revealed hundreds of distinct variants that are generally interpreted as representing different species, even though many have not been systematically tested for functional or ecological differentiation. Many of these variants are only minimally divergent from one another (1 bp or less), and others occupy basal nodes of traditional species phylogenies (“living ancestors”), indicating that some Symbiodinium ITS-2 diversity may represent intraspecific sequence variation. This hypothesis was tested for Symbiodinium clades A–D (the dominant symbionts of reef corals) through the construction of statistical parsimony networks of ITS-2 sequence diversity, and identification of clusters of closely related sequences within these networks. Initial assessments indicated that ecological differentiation exists between, but not within, these clusters. This approach, although imperfect in its ability to identify species boundaries in all cases, nevertheless dramatically reduces “species” diversity in Symbiodinium (from ~175 to 35). This testable alternative hypothesis indicates that, in Symbiodinium, “species” consist of clusters of closely related ITS-2 sequences diverging from ancestral variants that are typically ecologically dominant. A cluster-based view of Symbiodinium ITS-2 diversity improves our ability to: (1) construct well-supported symbiont phylogenies; (2) establish functional niches for symbiont species; and (3) understand flexibility and specificity within coral-algal symbioses. This cluster-based approach can ultimately be integrated with emerging population-level datasets (microsatellites and microsatellite flanking regions) to improve understanding of species diversity in Symbiodinium. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Communicated by Biology Editor Dr Ruth Gates     

Phylogenetic analysis of restriction-site variation in wild and cultivated Amaranthus species (Amaranthaceae)

Amaranthus includes approximately 60 species, of which three are cultivated as a grain source. Many wild Amaranthus species possess agriculturally desirable traits such as drought and salt tolerance, and pathogen resistance. We examined relationships among wild and cultivated Amaranthus species based upon restriction-site variation in two chloroplast DNA regions and in a nuclear DNA region. The chloroplast regions consisted of (1) an intergenic spacer in transfer RNA genes and (2) the ribulose-1,5-bisphosphate carboxylase gene with a flanking open reading frame. The nuclear region was the internal transcribed spacers ITS-1 and ITS-2 flanking the 5.8S gene in the ribosomal DNA. These regions were amplified by the polymerase chain reaction and digested with a total of 38 restriction endonucleases. We detected 11 potentially informative restriction-site mutations and seven length-polymorphisms among the 28 Amaranthus species. Parsimony analysis was used to find the shortest tree for each separate data set (chloroplast, nuclear, and length) and for two combined matrices (chloroplast/nuclear and all data sets). Overall, there was a low level of variation which generated poorly resolved trees among the 28 species. Congruence analyses revealed that the chloroplast and nuclear data sets were congruent with each other but not to the length data set. The congruence of the chloroplast and nuclear data sets suggested that cytoplasmic gene flow may not be a confounding factor in our analyses. The phylogeny also suggested that drought tolerance evolved independently several times. The molecular phylogeny provides a basis for selection of species pairs for crop development.     

ITS-PCR sequencing approach deciphers molecular phylogeny in chickpea

Twenty chickpea accessions belonging to ten different countries of the world have been subjected to phylogeny and length variations from nuclear ribosomal DNA (nr DNA). ITS1–5.8S–ITS2 regions of Cicer accessions were used for amplification in two sets, each comprising a reverse and forward ITS primers. Lengths of ITS-1 of C. arietinum and C. reticulatum ranged from 340 to 350 bp whereas that of ITS-2 from 400 to 410 bp. In all the 20 accessions investigated, GC content in ITS-1 ranged from 40 to 55% and in ITS-2 from 42 to 55%. Sequencing of polymerase chain reaction product from ITS-1 showed variability in 278 and 290 bp due to adenine and guanine nucleotide base pairs. BLAST search for ITS-1 region revealed highest homology (99%) with four strains of C. arietinum accessions. Whereas, ITS2 showed 100% homology with C. arietinum and 99% homology with that of C. echinospermum.