Taxonomic status of the white-head langur (Trachypithecus francoisi leucoscephalus) inferred from allozyme electrophoresis and random amplified polymorphism DNA (RAPD)

Six sample specimens of Trachypithecus francoisi and 3 of T. leucocephalus were analyzed by use of allozyme electrophoresis and random amplified polymorphism DNA (RAPD) in order to clarify the challenged taxonomic status of the white-head langur. Among the 44 loci surveyed, only 1 locus (PGM-2) was found to be polymorphic. Nei's genetic distance was 0.0025. In total, thirty 10-mer arbitrary primers were used for RAPD analysis, of which 22 generated clear bands. Phylogenetic trees were constructed based on genetic distances using neighbor-joining and UPGMA methods. The results show that T. francoisi and T: leucocephalus are not monophyletic. T. francoisi from Guangxi, China and Vietnam could not be clearly distinguished, and they are not divided into 2 clusters. A t-test was performed to evaluate between genetic distances within and between T. leucocephalus and T. francoisi taxa groups. The statistical test shows that the taxa group within T: leucocephalus and T: francoisi does not significantly differ from that between T: leucocephalus and T: francoisi at the 5% level. Our results suggest that the level of genetic differentiation between T, leucocephalus and T. francoisi is relatively low. Recent gene flow might exist between T. francoisi and T. leucocephalus. Combining morphological features, geographical distribution, allozyme data, RAPD data, and mtDNA sequences, we suggest that the white-head langur might be a subspecies of T. francoisi.

Population differentiation of Procypris rabaudi (Tchang) in the upper reaches of the Yangtze River revealed by mitochondrial DNA and RAPD markers

Procypris rabaudi (Tchang) is a cyprinid fish endermic to middle and upper reaches of the Yangtze River. Besides in main stream and large tributaries, there exists an early matured, small-sized ecological type in a small tributary, Tang River. In this study, mitochondrial DNA cytochrome h (cyt b) gene sequence analysis and randomly amplified polymorphic DNA (RAPD) analysis were performed to investigate the differentiation of the Tang River population from the Mudong reach population of the Yangtze River, with the purpose of conservation and exploitation of this fish. In the 1140 bps of cyt b gene sequence surveyed, 20 sites were found polymorphic, which defined 23 haplotypes. Among them, four haplotypes accounted for 54.4% of all individuals, while population-specific haplotypes occurred in low frequencies. Analysis of molecular variation on cyt b data revealed no significant partition existing between Tang River population and Mudong reach population. Analyses of 132 RAPD loci suggested that genetic variation between populations was significant, though values of different F-ST were not very high. The results revealed low genetic diversity and the beginning of population differentiation, suggesting that Tang River population should be designated as a separate Management Unit.

Genetic diversity and structure of natural and managed populations of Cedrus atlantica (Pinaceae) assessed using random amplified polymorphic DNA

Cedrus atlantica (Pinaceae) is a large and exceptionally long-lived conifer native to the Rif and Atlas Mountains of North Africa. To assess levels and patterns of genetic diversity of this species. samples were obtained throughout the natural range in Morocco and from a forest plantation in Arbucies, Girona (Spain) and analyzed using RAPD markers. Within-population genetic diversity was high and comparable to that revealed by isozymes. Managed populations harbored levels of genetic variation similar to those found in their natural counterparts. Genotypic analyses Of Molecular variance (AMOVA) found that most variation was within populations. but significant differentiation was also found between populations. particularly in Morocco. Bayesian estimates of F,, corroborated the AMOVA partitioning and provided evidence for Population differentiation in C. atlantica. Both distance- and Bayesian-based Clustering methods revealed that Moroccan populations comprise two genetically distinct groups. Within each group, estimates of population differentiation were close to those previously reported in other gymnosperms. These results are interpreted in the context of the postglacial history of the species and human impact. The high degree of among-group differentiation recorded here highlights the need for additional conservation measures for some Moroccan Populations of C. atlantica.

Comparison between human and armadillo Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis

Sixty-three Paracoccidioides brasiliensis isolates obtained from three nine-banded armadillos (Dasypus novem-cinctus), one Amazonian armadillo's and 19 clinical isolates were compared by random amplified polymorphic DNA analysis with the primer OPG-19. The isolates were divided into three major clusters, I, II and III. Coincidences between human and armadillo isolates were observed in clusters I and II. Cluster III consisted only of armadillos' isolates. The results suggested that (I) humans may acquire P. brasiliensis infection by contact with armadillo's environment, (II) there may be P. brasiliensis genotypes peculiar to the animal, and (III) individual armadillos may be infected with P brasiliensis cells with different genotypes.

Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce.

Controversy still exists over the adaptive nature of variation of enzyme loci. In conifers, random amplified polymorphic DNAs (RAPDs) represent a class of marker loci that is unlikely to fall within or be strongly linked to coding DNA. We have compared the genetic diversity in natural populations of black spruce [Picea mariana (Mill.) B.S.P.] using genotypic data at allozyme loci and RAPD loci as well as phenotypic data from inferred RAPD fingerprints. The genotypic data for both allozymes and RAPDs were obtained from at least six haploid megagametophytes for each of 75 sexually mature individuals distributed in five populations. Heterozygosities and population fixation indices were in complete agreement between allozyme loci and RAPD loci. In black spruce, it is more likely that the similar levels of variation detected at both enzyme and RAPD loci are due to such evolutionary forces as migration and the mating system, rather than to balancing selection and overdominance. Furthermore, we show that biased estimates of expected heterozygosity and among-population differentiation are obtained when using allele frequencies derived from dominant RAPD phenotypes.

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides(CT)(10) and (GT)(10) served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.

Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD)

Trichophyton rubrum é um importante agente causal de dermatomicose. Os métodos de tipagem molecular têm sido recentemente desenvolvidos para responder questões sobre epidemiologia e auxiliar no esclarecimento de recidivas, após o tratamento. As seqüências aleatórias 1- (5'-d[GGTGCGGGAA]-3') e 6- (5'-d[CCCGTCAGCA]-3') foram usadas para tipagem molecular deste fungo por RAPD produzindo variabilidade intraespecífica. Cinco padrões foram observados entre os 10 isolados de T. rubrum, com ambas as seqüências. Foi concluído que a análise por RAPD pode ser utilizada para estudos epidemiológicos.

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pbl 8) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pbl Ba by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

Cytogenetic and random amplified polymorphic DNA analysis of Leptodactylus species from rural and urban environments (Anura, Amphibia)

Cytogenetic and random amplified polymorphic DNA analyses carried out in the species Leptodactylus podicipinus, L. ocellatus, L. labyrinthicus, and L. fuscus from rural and urban habitats of the northwest region of São Paulo State, Brazil, showed that the karyotypes (2n = 22), constitutive heterochromatin distribution and nucleolus organizer region (NOR) location did not differ between the populations from the two environments. The in situ hybridization with an rDNA probe confirmed the location of the NORs on chromosome 8 revealing an in tandem duplication of that region in one of the chromosomes of L. fuscus. DAPI showed that part of the C-band-positive heterochromatin is rich in AT, including that in the proximity the NORs in L. podicipinus and L. ocellatus. The molecular analyses showed that the two populations (urban and rural) of L. podicipinus and L. fuscus are similar from a genetic point of view. The urban and rural populations of species L. ocellatus and L. labyrinthicus showed differences in genetic structures, probably due to urbanization which interferes with the dispersion of those frogs. The marked differences observed between the two populations of L. ocellatus can be representing the cryptic condition of the species. Unweighted pair-group method of analysis and genetic distance analysis detected the genetic proximity between L. ocellatus and L. fuscus. The results indicate that there was no reduction in the genetic diversity in the populations from the urban environment; however, the survival of these frogs would not be guaranteed in the case of an increase in human impact especially for populations of L. labyrinthicus and L. ocellatus. ©FUNPEC-RP.

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

An Improved molecular tool for distinguishing monoecious and dioedious Hydrilla

Two biotypes of hydrilla [Hydrilla verticillata(L.f.) Royle] occur in the United States, a dioecious type centered in the southeast and a monoecious type in the central Atlantic and northeastern states. Ecosystem managers need tools to distinguish the types as the ranges of each type expand and begin to overlap. A molecular tool using the randomly amplified polymorphic DNA (RAPD) procedure is available but its use is limited by a need for reference samples. We describe an alternative molecular tool which uses “universal primers” to sequence the trnL intron and trnL-F intergenic spacer of the chloroplast genome. This sequence yields three differences between the biotypes (two gaps and one single nucleotide polymorphism). A primer has been designed which ends in a gap that shows up only in the dioecious plant. A polymerase chain reaction (PCR) using this primer produces a product for the monoecious but not the dioecious plant.

Rapid establishment of pure lines of silver carp (Hypophthalmichthys molirix) and bighead carp (Aristichthys nobilis)

The diversity of gynogenetic, artificial sex reversal and natural silver carp and bighead carp is examined using randomly amplified polymorphic DNA (RAPD) method. All of the 187 bands are obtained and 19 (10.16%) of them are polymorphic in gynogenetic silver carp. Meanwhile 32 (15.61%) out of 205 bands are polymorphic in control group. In gynogenetic bighead carp a total of 232 bands are identified and 11 (4.74%) out of them are polymorphic, while 25 (10.37%) out of 241 bands are polymorphic in control group. The genetic distance of four populations is calculated and it is 0.102 and 0.023 for gynogenetic silver carp and gynogenetic bighead carp respectively. The values of natural silver carp and bighead carp are 0.161 and 0.104. From the UPGMA trees constructed based on genetic distance, the sex reversal individuals that match with the gynogenetic female individuals are picked out. A new breeding process of establishing a pure line is developed.