Variation in the number of copies of ribosomal genes in the genomes of patients with cystic fibrosis

Впервые определено число копий тандемного рибосомного повтора (рДНК) в ДНК лейкоцитов крови больных муковисцидозом различного возраста (от 0 до 66 лет). Показано, что геномы больных муковисцидозом содержат увеличенное количество копий рДНК (289 - 932; среднее: 563 ± 101; N = 203) по сравнению со здоровыми людьми (171-711; среднее 420 ±107 копий; N = 751) того же возраста (p <10-33). В выборке больных и в контрольной выборке наблюдали сужение интервала варьирования числа копий рДНК в старших возрастных группах. В группе больных возраста 30-66 лет интервал варьирования числа копий рДНК в геноме сужен до 430-680 копий (среднее: 544±72 копии; N=27). Потенциально число копий рДНК в геноме больных муковисцидозом можно рассматривать как дополнительный прогностический маркер, отражающий продолжительность жизни больного. The number of copies of tandem ribosomal repeat (rDNA) in the DNA of blood leukocytes of patients with cystic fibrosis of various ages (from 0 to 66 years) was determined for the first time. It was shown that the genomes of patients with cystic fibrosis contain an increased number of rDNA copies (289 - 932; mean: 563 ± 101; N = 203) compared with healthy people (171 - 711; mean 420 ± 107 copies; N = 751) of the same age (p <10-33). In the sample of patients and in the control sample, a narrowing of the range of variation in the rDNA copy number was observed in older age groups. In the group of patients aged 30-66 years, the interval of variation in the number of rDNA copies in the genome was narrowed to 430-680 copies (mean: 544 ± 72 copies; N = 27). Potentially, the number of rDNA copies in the genome of patients with cystic fibrosis can be considered an additional prognostic marker reflecting the patient’s life expectancy.

Complete chloroplast genome comparisons for Pityopsis (Asteraceae)

Pityopsis includes several regionally and one federally endangered species of herbaceous perennials. Four species are highly localized, including the federally endangered P. ruthii. The genus includes several ploidy levels and interesting ecological traits such as drought tolerance and fire-dependent flowering. Results from previous cladistic analyses of morphology and from initial DNA sequence studies did not agree with one another or with the infrageneric taxonomic classification, with the result that infrageneric relationships remain unresolved. We sequenced, assembled, and compared the chloroplast (cp) genomes of 12 species or varieties of Pityopsis to better understand generic evolution. A reference cp genome 152,569 bp in length was assembled de novo from P. falcata. Reads from other sampled species were then aligned to the P. falcata reference and individual chloroplast genomes were assembled for each, with manual gapfilling and polishing. After removing the duplicated second inverted region, a multiple sequence alignment of the cp genomes was used to construct a maximum likelihood (ML) phylogeny for the twelve cp genomes. Additionally, we constructed a ML phylogeny from the nuclear ribosomal repeat region after mapping reads to the Helianthus annuus region. The chloroplast phylogeny supported two clades. Previously proposed clades and taxonomic sections within the genus were largely unsupported by both nuclear and chloroplast phylogenies. Our results provide tools for exploring hybridity and examining the physiological and genetic basis for drought tolerance and fire-dependent flowering. This study will inform breeding and conservation practices, and general knowledge of evolutionary history, hybridization, and speciation within Pityopsis.

Identification and Characterization of Fungi Causing Thread Blight Diseases on Cacao in Ghana

Theobroma cacao (chocolate tree) is currently under serious threat from thread blight disease (TBD), which has been attributed to the causal agent Marasmiellus scandens in other regions of the world. TBD in Ghana has similar symptomology but variable signs. This study sought to determine whether TBD in Ghana was caused by a single agent and whether Marasmiellus scandens was a significant agent of TBD. Forty-eight isolates were collected from eight geographical locations in Ghana for morphological and molecular characterization. Disease signs occurred as vegetative rhizomorphs or hyphal aggregates, which were classified into five morphotypes: A, abundant thin, black, “horse hair”-type rhizomorphs; B, scattered brown rhizomorphs; C, whitish to brownish-white; D, faint cream or dull white; and E, aggregates of shiny or silky white hyphae. Sequencing and analyses of three loci—the internal transcribed spacer region of the nuclear ribosomal repeat, nuclear large subunit, and mitochondrial small subunit—detected four species, all members of the Marasmiaceae, causing TBD-like disease. These were identified as Marasmius crinis-equi (morphotype A), Marasmius tenuissimus (morphotypes B and C), Marasmiellus palmivorus (morphotype E), and Marasmiellus scandens (morphotype D). Marasmius tenuissimus, the most frequently isolated TBD fungus in this study, is primarily an Asian fungus and not previously associated with diseases of cacao. Marasmiellus palmivorus, the second most frequently isolated fungus, is a pan-tropical pathogen with a broad host range; this is the first report of the fungus causing TBD on cacao. Marasmius crinis-equi also has a broad pan-tropical distribution and host range and causes thread blight on several tropical tree crops. Surprisingly, Marasmiellus scandens, the most frequently cited agent of TBD in cacao, made up only 8% of the isolates.

Intragenomic nuclear RNA variation in a cryptic Amanita taxon

Amanita cf. lavendula collections in eastern North America, Mexico, and Costa Rica were found to consist of four cryptic taxa, one of which exhibited consistently unreadable nuclear rDNA ITS1-5.8S-ITS2 (fungal barcode) sequences after ITS1 base 130. This taxon is designated here as Amanita cf. lavendula taxon 1. ITS sequences from dikaryotic basidiomata were cloned, but sequences recovered from cloning did not segregate into distinct haplotypes. Rather, there was a mix of haplotypes that varied among themselves predominantly at 28 ITS positions. Analysis of each of these 28 variable bases showed predominantly two alternate bases at each position. Based on these findings and additional sequence data from the nuclear rDNA 28S, RNA polymerase II subunit 2 (RPB2) and mitochondrial rDNA small subunit (SSU) and 23S genes, we speculate that taxon 1 represents an initial hybridization event between two divergent taxa followed by failure of the ribosomal repeat to homogenize. Homogenization failure may be a result of repeated hybridization between divergent internal transcribed spacer (ITS) types with inadequate time for concerted evolution of the ribosomal repeat or, alternately, a complete failure of the ribosomal homogenization process. To our knowledge, this finding represents the first report of a geographically widespread taxon (Canada, eastern USA, Costa Rica) with apparent homogenization failure across all collections. Findings such as these have implications for fungal barcoding efforts and the application of fungal barcodes in identifying environmental sequences.

Design of plant-specific PCR primers for the ETS region with enhanced specificity for tribe Bromeae and their application to other grasses (Poaceae)

Selective primers were developed for the amplification via polymerase chain reaction (PCR) of the external transcribed spacer (ETS) region in the nuclear ribosomal repeat unit. These primers were intended to be specific to the Poaceae tribe Bromeae but were found to function broadly across subfamily Pooideae. ETS primers previously developed for grasses were unable to amplify tribe Bromeae taxa because of ETS variability and several subrepeats. After detailed analysis, a region was chosen and four candidate primers were designed to amplify and sequence the ETS fragment. This fragment of approximately 800–900 bp is located in the 3′ region of the ETS of 18S–26S nuclear ribosomal DNA. A preliminary study with these primers was conducted in eight samples of Bromus. The best two primers showed strong amplification and successful sequencing for all samples tested. We also tested the specificity of these two primers in samples belonging to all large taxa of Bromeae, including all three genera and five subgenera and samples of eight tribes and 15 subtribes of the subfamily Pooideae, and both worked for most of the taxa tested. Our results demonstrate that the ETS region is more informative for resolving relationships at different taxonomic levels than internal transcribed spacer region. Also, these results indicate the utility of these new primers for studying the ETS region in the tribe Bromeae as well as across the subfamily Pooideae.

Phylogenetic Information Content of Copepoda Ribosomal DNA Repeat Units: ITS1 and ITS2 Impact

The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that theMesocyclops,Thermocyclops,andMacrocyclopsgenera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals.

The taxonomy of Flagellaria (Flagellariaceae)

Flagellariaceae are grass relatives that climb using leaf-tip tendrils in palaeotropical rainforests. As the sister group to Joinvilleaceae, Ecdeiocoleaceae and Poaceae, they could be indicative of the ancestral habitat of the graminid clade. Although four species are usually accepted in this monogeneric family, Flagellaria has never been revised taxonomically and species delimitation is understood poorly, especially for the Pacific. Here, we document the morphological variation in the genus, and explore phylogenetic patterns by maximum likelihood analysis (GARLI) and Bayesian inference (MrBayes) of the internal transcribed spacer (ITS) of the nuclear ribosomal repeat and the plastid matK, psbA–trnH and rps16 regions. Ecological variation was inferred from climatic factors evaluated by MaxEnt analysis. We show that Flagellaria is more diverse than previously thought, and describe one new species in Fiji (Flagellaria collaris Wepfer & H.P.Linder) and three new varieties in F. indica (vars. australiensis, bifurcata, borneensis). Flagellaria is most diverse in the Pacific islands, where Fiji harbours three of the five species.

Molecular identification of mycorrhizal fungi in Dactylorhiza sambucina (Orchidaceae)

We amplified and sequenced internal transcribed spacer (ITS) region of the nuclear ribosomal repeat from fungi in roots of Dactylorhiza sambucina (Orchidaceae) and used the extended database to identify the mycorrhizal fungi. We molecularly identified three ITS recovered from D. sambucina roots, one belonging to Rhizoctonia group, and two to ascomycetes, for the first time in Orchidoidae. In many cases, two sequence types were found from the same orchid root, providing that two taxa may be involved in mycorrhizal formation (multiple mycobiont colonization). Moreover, we demonstrated that D. sambucina plants, irrespective of their colour polymorphism, possess roots containing several fungi belonging to both asco- and basidiomycetes.