Littérature scientifique sur le sujet « S –ITS rDNA »

Sugarcane is a major crop for sugar production around the world. The complexity of the sugarcane genome creates challenges for the use of both conventional and molecular breeding methods to improve sugarcane at a genetic level. DNA sequencing is an important tool to assess how the genus Saccharum and the genera of the Saccharum complex are interrelated. Here, we identify the kinship of Nepal2013-6 (Saccharum spontaneum, x = 10) using a tetra-primer amplification refractory mutation system (ARMS) PCR. Based on rDNA-ITS sequence analysis, the accession Nepal2013-6 falls within a single cluster with S. spontaneum (Yunnan82-114 and SES208), which is consistent with the previous results. Moreover, fluorescence in situ hybridization (FISH) results indicate that the 5S rDNA spots are consistent with the chromosomal ploidy in the analytical Saccharum materials, whereas 35S rDNA has similar or fewer sites than the ploidy. Therefore, 5S rDNA FISH patterns would be more suitable than 35S rDNA for chromosomal ploidy analysis in S. spontaneum with varied basic chromosome number x = 8, 9, 10. Altogether, these results indicate that the rDNA sequences will be a useful marker for further rapidly identifying the relationship and ploidy of S. spontaneum in sugarcane breeding.

During surveys on yeast diversity in forest soils from Taiwan and Thailand, ten yeast strains isolated from different samples were found to have similar molecular and physiological characteristics. Sequence analysis of small subunit (SSU) rDNA, the D1/D2 domain of large subunit (LSU) rDNA and internal transcribed spacer (ITS)-5.8S rDNA demonstrated that these strains were closely related to Scheffersomyces spartinae. The novel strains could be differentiated from S. spartinae by a 0.9 % sequence divergence (5 substitutions, 0 gaps) in the D1/D2 domain of LSU rDNA, a 1.5 % divergence (8 substitutions, 0 gaps) in the ITS-5.8S rDNA and a 0.7 % divergence (12 substitutions, 2 gaps) in the SSU rDNA. The novel strains also showed specific patterns of electrophoretic karyotypes that differed from that of S. spartinae. Therefore, a novel yeast species, Candida gosingica sp. nov., is proposed to accommodate these strains. The type strain SJ7S11T (=BCRC 23194T=CBS 11433T) was assigned and deposited in the Bioresource Collection and Research Center (BCRC), Food Industry Development and Research Institute, Hsinchu, Taiwan, and Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands.

Fluorescence in situ hybridization was used to characterize loci encoding ribosomal RNA (rDNA) among parents and progeny of a somatic fusion between tetraploid Solanum tuberosum (potato) and diploid Solanum brevidens. As expected, four major sites of hybridization to rDNA loci were evident in the tetraploid parent species, two in the diploid parent species, and six in the hexaploid somatic fusion plant. Two of the loci in the somatic fusion plant showed differential signals relative to the other four, which were interpreted as a delayed condensation of sites harboring the rDNA loci. This delayed condensation was heritable to the first backcross generation of the somatic hybrid crossed with potato. In the second backcross generation, differential condensation was not evident. However, a heterochromatic isochromosome was observed whose presence was correlated with a S. brevidens specific marker linked with the rDNA locus. It is suggested that the S. brevidens rDNA loci are preferentially affected in the somatic hybrid and its progeny, and that the delayed condensation may have contributed to the formation of the isochromosome.Key words: introgression, recombination, isochromosome, in situ hybridization.

A strain of Acidithiobacillus ferrooxidans designated S t1 was isolated and identified from the acid mine drainage of Tianmashan Coal Mine, Tongling county, Anhui province. Its morphological, physiological and biochemical characters, growth conditions of pH value and temperature, 16S rDNA sequence and the phylogenetic tree was studied. Results showed that the bacteria strains from different sites have divergence at the curve of pH and Eh values during the enriching culture. The isolated strain S t1 grew in a pH rang from 2.0 to 3.0 and temperature 25 to 35°C with over 98％similarity toAcidithiobacillus ferrooxidansin 16S rDNA sequence.

Only 18S rDNA sequences of Sarcocystis spp. in South American camelids (SACs) are deposited in GenBank as references, and the definitive host of S. masoni in SACs is still unclear. Here, S. masoni sarcocysts detected in an alpaca (Vicugna pacos) in China were investigated with the aid of light (LM) and transmission electron (TEM) microscopy, and characterized using four genetic markers, i.e., 18S rDNA, 28S rDNA and ITS, and the mitochondrial cox1. Additionally, the life cycle of the parasite was completed via experimental animal infection. Under LM, S. masoni sarcocysts exhibited numerous 1.3–2.1 μm conical protrusions. Under TEM, the sarcocyst wall contained conical, cylindrical, or irregular-shaped villar protrusions, similar to type 9j. Two dogs (Canis familiaris) fed S. masoni sarcocysts shed sporocysts with a prepatent period of 8–9 days. The newly obtained 18S rDNA sequences showed 98.4–100% identity with those of S. masoni in SACs previously deposited in GenBank. Interestingly, the newly obtained sequences of 18S rDNA and mitochondrial cox1 shared 99.6–100% and 98.2–98.5% identity, respectively, with those of S. cameli in dromedary camels (Camelus dromedaries). Phylogenetic analysis based on sequences of 18S rDNA, 28S rDNA, or mitochondrial cox1 revealed that S. masoni has a close relationship with Sarcocystis spp. in ruminants. The relationship between S. masoni and S. cameli deserves to be further clarified in the future.

Nuclear ribosomal internal transcribed spacer (ITS) sequences were sequenced for 23 species and subspecies of Elymus sensu lato collected in Russia. The Neighbor-Net analysis of ITS sequences suggested that there are four ribotypes called Core Northern St-rDNA, Core Southern St-rDNA, Northern dahuricus St-rDNA and Southern dahuricus St-rDNA. The Core Southern variant of St-rDNA is closely related to rDNA of diploid Pseudoroegneria stipifolia (PI 313960) and P. spicata (PI 547161). The Core Northern St-rDNA is closely related to rDNA of P. cognata (PI 531720), a diploid species of Kyrgyzstan carrying StY variant of the St genome. The Core Northern St-rDNA is widespread among the Elymus species of Siberia and the Far East, including Yakutia and Chukotka. The Core Southern St-ribotype is typical of southern Elymus and Pseudoroegneria of the South Caucasus, Primorye, Pakistan, and South Korea. The Northern dahuricus St-ribotype and Southern dahuricus St-ribotype are derivatives of the Core Northern and Core Southern St-ribotypes, correspondingly. Both of them were found in all four studied species of the E. dahuricus aggregate: E. dahuricus Turcz. ex Griseb., E. franchetii Kitag., E. excelsus Turcz. ex Griseb. and Himalayan E. tangutorum (Nevski) Hand.-Mazz. In other words, there are at least two population groups (two races) of the Elymus dahuricus aggregate species that consistently differ in their ITS-sequences in Siberia, the Far East and Northern China. Each contains all morphological forms, which taxonomists now attribute either to different species of E. dahuricus aggr. (E. dahuricus sensu stricto, E. franchetii, E. tangutorum, E. excelsus) or subspecies of Campeiostachys dahurica (Turcz. ex Griseb.) B.R. Baum, J.L. Yang et C.C. Yen. At the moment it is unknown if there are any morphological differences between plants carrying either Northern or Southern dahuricus rDNA. Probably, they are cryptic species, but it is certain that if differences in morphology between the two races exist, they are not associated with signs that are now considered taxonomically significant and are used to separate E. dahuricus s. s., E. franchetii, E. tangutorum, and E. excelsus.

Potato wart, caused by the fungal pathogen Synchytrium endobioticum, is a serious disease with the potential to cause significant economic damage. The small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA) were sequenced for several Synchytrium spp., showing a high rate of variability for both of these markers among the different species and monophyly of the genus within phylum Chytridiomycota. The intergenic nontranscribed spacer (IGS) of rDNA was sequenced for different pathotypes and showed no intraspecific variation within S. endobioticum, similar to the other rDNA markers from this study. To facilitate screening for the pathogen in soil, three TaqMan polymerase chain reaction (PCR) assays were developed from SSU, ITS, and IGS rDNA sequences to detect S. endobioticum sporangia in the chloroform-flotation fraction of sieved soil extracts. In the screening portion of the method, a first TaqMan assay targeting the SSU rDNA was developed with positive results that were further confirmed with amplicon melt analysis. A synthetic reaction control cloned into a plasmid was incorporated into the procedure, facilitating the validation of negative results. The presence of the reaction control did not adversely affect the efficiency of the SSU target amplification. A second TaqMan assay targeting the ITS-1 region was developed as a confirmatory test. There was 100% accordance between the SSU and ITS-1 TaqMan assays. Utilizing these two assays in tandem achieved good specificity for S. endobioticum, generating negative results with the cloned SSU and ITS-1 regions from all 14 other Synchytrium spp. considered. Spike recovery experiments indicated that these assays, targeting the SSU and ITS-1 rDNA regions, developed from a phylogeny dataset of the genus, could reliably detect a single sporangium in the chloroform flotation fraction of a soil extract. Good correlation between microscopic detection of sporangia and PCR results in both positive and negative soil samples was dually demonstrated for both the SSU and ITS-1 assays.

One of the emerging technologies is the use of microbial agents for the control of postharvest diseases of fruits and vegetables. A number of antagonistic microorganisms have been discovered which have the potential to effectively control postharvest diseases. Some of this technology has been patented and commercial products such as AspireTM (Ecogen Corporatin, Langhorne, PA, USA), Biosave 10TM and Biosave 11TM (Ecoscience Inc., Worchester, MA, USA) have been registered for commercial use. The principal investigator of this project was involved in developing the yeast-based biofungicide-AspireTM and testing its efficacy under commercial conditions. This research project was initiated to fill the gap between the knowledge available on development and commercial implementation of yeast biocontrol agents and basic understanding of various aspects related to introducing yeast antagonists to fruit surfaces, along with verification of population genetics. The main objectives of this study were: Study ecology, population dynamics and genetic diversity of the yeast antagonists Candida guilliermondii, C. oleophila, and Debaryomyces hansenii, and study the effect of preharvest application of the yeast antagonist C. oleophila naturally occurring epiphytic microbial population and on the development of postharvest diseases of citrus fruit during storage. Our findings, which were detailed in several publications, have shown that an epiphytic yeast population of grapefruit able to grow under high osmotic conditions and a wide range of temperatures was isolated and characterized for its biocontrol activity against green mold decay caused by Penicillium digitatum. Techniques based on random amplified polymorphic DNA (RAPD) and arbitrary primed polymerase chain reaction (ap-PCR), as well as homologies between sequences of the rDNA internal transcribed spacers (ITS) and 5.8S gene, were used to characterize the composition of the yeast population and to determine the genetic relationship among predominant yeast species. Epiphytic yeasts exhibiting the highest biocontrol activity against P. digitatum on grapefruit were identified as Candida guilliermondii, C. oleophila, C. sake, and Debaryomyces hansenii, while C. guilliermondii was the most predominant species. RAPD and ap-PCR analysis of the osmotolerant yeast population showed two different, major groups. The sequences of the ITS regions and the 5.8S gene of the yeast isolates, previously identified as belonging to different species, were found to be identical. Following the need to develop a genetically marked strain of the yeast C. oleophila, to be used in population dynamics studies, a transformation system for the yeast was developed. Histidine auxotrophy of C. oloephila produced using ethyl methanesulfonate were transformed with plasmids containing HIS3, HIS4 and HIS5 genes from Saccharomyces cerevisiae. In one mutant histidin auxotrophy was complemented by the HIS5 gene of S. cerevisiae is functionally homologous to the HIS5 gene in V. oleophila. Southern blot analysis showed that the plasmid containing the S. cerevisiae HIS5 gene was integrated at a different location every C. oleophila HIS+ transformant. There were no detectable physiological differences between C. oleophila strain I-182 and the transformants. The biological control ability of C. oleophila was not affected by the transformation. A genetically marked (with b-glucuronidase gene) transformant of C. oleophila colonized wounds on orange fruits and its population increased under field conditions. Effect of preharvest application of the yeast C. oleophila on population dynamics of epiphytic microbial population on wounded and unwounded grapefruit surface in the orchard and after harvest was also studied. In addition, the effect of preharvest application of the yeast C. oleophila on the development of postharvest decay was evaluated. Population studies conducted in the orchard showed that in control, non-treated fruit, colonization of wounded and unwounded grapefruit surface by naturally occurring filamentous fungi did not vary throughout the incubation period on the tree. On the other hand, colonization of intact and wounded fruit surface by naturally occurring yeasts was different. Yeasts colonized wounded surface rapidly and increased in numbers to about two orders of magnitude as compared to unwounded surface. On fruit treated with the yeast and kept on the tree, a different picture of fungal and yeast population had emerged. The detected fungal population on the yeast-treated intact surface was dramatically reduced and in treated wounds no fungi was detected. Yeast population on intact surface was relatively high immediately after the application of AspireTM and decreased to than 70% of that detected initially. In wounds, yeast population increased from 2.5 x 104 to about 4x106 after 72 hours of incubation at 20oC. Results of tests conducted to evaluate the effect of preharvest application of AspireTM on the development of postharvest decay indicated the validity of the approach.

Within the project "Pathogenic Streptococcus in Tilapia", gram positive cocci pathogens of fish in Israel and in the United States were characterized. We showed that Streptococcus shiloi, the name for an agent causing septicemic infection in fish, is a junior synonym of Streptococcus iniae and that Enterococcus seriolicida is a junior synonym of Lactococcus garvieae, a causative agent of septicemia and meningo-encephalitis in fish. Molecular epidemiology studies on these two pathogens, based on 16S rDNA sequences and ribotyping showed that although each country had specific clones, S. iniae originated probably from the U.S. and L. garvieae from Japan. PCR assays were developed for both pathogens and applied to clinical samples. S. agalactiael S. difficile was also recognized for the first time in the U.S. in tilapia. Our histopathological studies explained the noted paradox (abundant in vitro growth often accompanied by scant to small numbers of organisms within the meninges in histologic sections of brain) in diagnostic of fish streptococcus. The greatest concentration of cocci were consistently observed within macrophages infiltrating the extrameningeal fibroadipose tissue surrounding the brain within the calvarium. These results also suggests that the primary route of meningeal infection may be extension from the extrameningeal connective tissue rather than meningeal vascular emigration of cocci-containing macrophages. Our work has resulted in a cognizance of streptococcus as fish pathogen which goes beyond the pathology observed in tilapia and is already extended to many aquaculture fish species in Israel and in the United States. Finally, our data suggest that vaccines (bivalent or trivalent) could be developed to prevent most of the damages caused by streptococcus in aquaculture.