Intergenic Spacer Sequences Research Articles

First report of saxitoxins and anatoxin-a production by cyanobacteria from Lithuanian lakes

ABSTRACT This study describes the first report on saxitoxins (STXs) and anatoxin-a (ANTX-a) production by cyanobacteria in Lithuanian lakes. Seasonal fluctuation patterns for neurotoxins were determined in samples collected from eutrophic lakes during July–September. Strains of Aphanizomenon gracile, A. flos-aquae, Cuspidothrix issatschenkoi, Sphaerospermopsis aphanizomenoides and Anabaenopsis cf. elenkinii were also isolated from the lakes and screened for the occurrence of neurotoxic STXs, ANTX-a and for the cytotoxin cylindrospermopsin (CYN). The highest concentration of STX (up to 1.06 µg l–1 in Lake Jieznas) was detected during July‒August and coincided with a predominance of A. gracile in the phytoplankton. Of the isolated strains, only A. gracile was confirmed to be a producer of STXs by chemical (LC-MS/MS) and genetic (sxtA) methods. Sequencing of the phycocyanin intergenic spacer and flanking regions (cpcBA) revealed that A. gracile formed a genetically homogenous group. Only trace amounts of ANTX-a were detected in field samples (up to 0.31 µg l–1 in Lake Širvys). CYN was detected in neither the bloom samples nor in the isolated strains.

Gene transfer and nucleotide sequence evolution by Gossypium cytoplasmic genomes indicates novel evolutionary characteristics.

The DNA fragments transferred among cotton cytoplasmic genomes are highly differentiated. The wild D group cotton species have undergone much greater evolution compared with cultivated AD group. Cotton (Gossypium spp.) is one of the most economically important fiber crops worldwide. Gene transfer, nucleotide evolution, and the codon usage preferences in cytoplasmic genomes are important evolutionary characteristics of high plants. In this study, we analyzed the nucleotide sequence evolution, codon usage, and transfer of cytoplasmic DNA fragments in Gossypium chloroplast (cp) and mitochondrial (mt) genomes, including the A genome group, wild D group, and cultivated AD group of cotton species. Our analyses indicated that the differences in the length of transferred cytoplasmic DNA fragments were not significant in mitochondrial and chloroplast sequences. Analysis of the transfer of tRNAs found that trnQ and nine other tRNA genes were commonly transferred between two different cytoplasmic genomes. The Codon Adaptation Index values showed that Gossypium cp genomes prefer A/T-ending codons. Codon preference selection was higher in the D group than the other two groups. Nucleotide sequence evolution analysis showed that intergenic spacer sequences were more variable than coding regions and nonsynonymous mutations were clearly more common in cp genomes than mt genomes. Evolutionary analysis showed that the substitution rate was much higher in cp genomes than mt genomes. Interestingly, the D group cotton species have undergone much faster evolution compared with cultivated AD groups, possibly due to the selection and domestication of diverse cotton species. Our results demonstrate that gene transfer and differential nucleotide sequence evolution have occurred frequently in cotton cytoplasmic genomes.

Evolutionary Diversity in the Intracellular Microsporidian Parasite Nosema sp. Infecting Wild Silkworm Revealed by IGS Nucleotide Sequence Diversity.

Intracellular microsporidian Nosema mylitta infects Indian wild silkworm Antheraea mylitta causing pebrine disease. Genetic structure and phylogeny of N. mylitta are analysed using nucleotide variability in 5S ribosomal DNA and intergenic spacer (IGS) sequence from 20 isolates collected from Southern, Northern and Central regions of Jharkhand State. Nucleotide diversity (π) and genetic differentiation Gst were highest in the Central isolates whereas lowest in the North. Among the isolates, absence of nucleotides, transitions and transversions were observed. Haplotyping showed nucleotide variability at 83 positions in IGS and 13 positions in 5S rDNA. Haplotype-based genetic differentiation was 0.96 to 0.97 whereas nucleotide sequence-based genetic differentiation was higher (Ks = 22.29) between Southern and Central isolates. Bottleneck analysis showed negative value for Tajima's D and other summary statistics revealing induction of loss of rare alleles and population explosion. From IGS, 17 ancestral sequences were inferred by Network algorithm. Core of nine closely related nodes having ancient nucleotides and peripheral nodes with highly divergent nucleotides were derived. Most diverged peripheral haplotype was Bero (H11) from the Central region whereas Deoghar (H3) of the Northern region diverged early. Phylogeny of N. mylitta grouped Southern and Northern isolates together revealed weak phylogenetic signal for these locations. Phylogeny of N. mylitta with Nosema sp. infecting other lepidopterans clustered N. mylitta isolates with N. antheraea and N. philosamiae of China indicating genetic similarity whereas other species were dissimilar showing diversity irrespective of country of origin.

Multi-locus phylogeny and taxonomy of an unresolved, heterogeneous species complex within the genus Golovinomyces (Ascomycota, Erysiphales), including G. ambrosiae, G. circumfusus and G. spadiceus

BackgroundPrevious phylogenetic analyses of species within the genus Golovinomyces (Ascomycota, Erysiphales), based on ITS and 28S rDNA sequence data, revealed a co-evolutionary relationship between powdery mildew species and hosts of certain tribes of the plant family Asteraceae. Golovinomyces growing on host plants belonging to the Heliantheae formed a single lineage, comprised of a morphologically differentiated complex of species, which included G. ambrosiae, G. circumfusus, and G. spadiceus. However, the lineage also encompassed sequences retrieved from Golovinomyces specimens on other Asteraceae tribes as well as other plant families, suggesting the involvement of a plurivorous species. A multilocus phylogenetic examination of this complex, using ITS, 28S, IGS (intergenic spacer), TUB2 (beta-tubulin), and CHS1 (chitin synthase I) sequence data was carried out to clarify the discrepancies between ITS and 28S rDNA sequence data and morphological differences. Furthermore, the circumscription of species and their host ranges were emended.ResultsThe phylogenetic and morphological analyses conducted in this study revealed three distinct species named, viz., (1) G. ambrosiae emend. (including G. spadiceus), a plurivorous species that occurs on a multitude of hosts including, Ambrosia spp., multiple species of the Heliantheae and plant species of other tribes of Asteraceae including the Asian species of Eupatorium; (2) G. latisporus comb. nov. (≡ Oidium latisporum), the closely related, but morphologically distinct species confined to hosts of the Heliantheae genera Helianthus, Zinnia, and most likely Rudbeckia; and (3) G. circumfusus confined to Eupatorium cannabinum in Europe.ConclusionsThe present results provide strong evidence that the combination of multi-locus phylogeny and morphological analysis is an effective way to identify species in the genus Golovinomyces.

Sex Chromosome Pairing Mediated by Euchromatic Homology in Drosophila Male Meiosis.

Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the ribosomal DNA (rDNA). Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y) Using fluorescence in situ hybridization to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis, while the autosomal-specific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.

Characterizing the ribosomal tandem repeat and its utility as a DNA barcode in lichen-forming fungi

BackgroundRegions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing.ResultsWe estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of this complex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, intergenic spacer region, and some intronic regions, independently; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny.ConclusionsThe results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.

Polyphasic phenotypic and genetic analysis reveals clonal nature of Xanthomonas axonopodis pv. punicae causing pomegranate bacterial blight

Yellow‐pigmented bacteria isolated from blight‐affected pomegranate leaves and fruit across seven Indian states in epidemics during the years 2008–2016 were characterized and identified using phenotypic and genotypic tools. All bacterial isolates shared phenotypic traits such as colony morphology, NaCl and pH sensitivity and fuscan production, and caused typical lesions on pomegranate plants upon artificial inoculation. Analysis of 16S ribosomal DNA and 16S–23S rDNA intergenic spacer sequences confirmed their identity as Xanthomonas axonopodis pv. punicae. The new isolates collected after 2000 were compared with an old isolate from the 1950s using polyphasic taxonomic approaches including multilocus sequence analysis (MLSA). Nucleotide polymorphism in 24 isolates for nine genomic loci (dnaK, fyuA, gyrB (Young), gyrB (Almeida), rpoD, fusA, gapA, gltA and lepA) showed minor variations in loci fyuA and gyrB. Isolates were grouped into four nearly identical sequence types, ST1, ST2, ST3 and ST4, based on their allelic profiles, ST3 being widespread in Indian states. Molecular phylogenetic analysis of concatenated 5690 bp with other Xanthomonas pathovars revealed its close genetic similarity with the X. citri group. The blight outbreak in diverse geographical locations is attributed to a re‐emerged clonal population of X. axonopodis pv. punicae on a genetically homogenous pomegranate cultivar. The latently infected vegetative planting material of elite pomegranate cultivars contributed to the dissemination of the bacterial inoculum. This study highlights and forewarns of the role played by the clonally propagated elite pomegranate cultivars in disseminating and sustaining clonal populations of this bacterial plant pathogen in many Indian states.

Distribution and Pathogenicity of Colletotrichum Species Associated With Mango Anthracnose in Mexico.

Mango anthracnose, caused by Colletotrichum spp., is the most significant disease of mango (Mangifera indica L.) in almost all production areas around the world. In Mexico, mango anthracnose has only been attributed to C. asianum and C. gloeosporioides. The aims of this study were to identify the Colletotrichum species associated with mango anthracnose symptoms in Mexico by phylogenetic inference using the ApMat marker, to determine the distribution of these species, and to test their pathogenicity and virulence on mango fruits. Surveys were carried out from 2010 to 2012 in 59 commercial orchards in the major mango growing states of Mexico, and a total of 118 isolates were obtained from leaves, twigs, and fruits with typical anthracnose symptoms. All isolates were tentatively identified in the C. gloeosporioides species complex based on morphological and cultural characteristics. The Bayesian inference phylogenetic tree generated with Apn2/MAT intergenic spacer sequences of 59 isolates (one per orchard) revealed that C. alienum, C. asianum, C. fructicola, C. siamense, and C. tropicale were associated with symptoms of mango anthracnose. In this study, C. alienum, C. fructicola, C. siamense, and C. tropicale are reported for the first time in association with mango tissues in Mexico. This study represents the first report of C. alienum causing mango anthracnose worldwide. The distribution of Colletotrichum species varied among the mango growing states from Mexico. Chiapas was the only state in which all five species were found. Pathogenicity tests on mango fruit cultivar Manila showed that all Colletotrichum species from this study could induce anthracnose lesions. However, differences in virulence were evident among species. C. siamense and C. asianum were the most virulent, whereas C. alienum and C. fructicola were considered the least virulent species.

First Report of Crown and Root Rot Caused by Fusarium oxysporum on Sweet Pepper (Capsicum annuum) in Italy

During August 2018, sweet pepper (Capsicum annuum L.) cv. Cuneo, grown in 3,000 m² of commercial greenhouses near Cuneo in Piedmont, northern Italy, exhibited symptoms of reduced growth and sudden wilting. Crown and roots were severely affected, showing a brown necrosis and slight rot. No internal discoloration of vascular tissues was observed. Eventually, affected plants collapsed. Symptoms developed on approximately 10% of the plants 3 months after transplanting. Tissue fragments of 1 mm² were excised from the crown and roots of 10 symptomatic plants, dipped in a 1% sodium hypochlorite solution, washed in sterile water, and placed on potato dextrose agar. Fungal colonies were isolated from all samples incubated under constant fluorescent light at 22 ± 1°C for 6 days. Thirty-day-old single-spore cultures of four selected isolates (FOC1, FOC2, FOC3, and FOC4) grown on carnation leaf agar generated short monophialides with unicellular, ovoid-elliptical microconidia, measuring 5.6 to 13.5 × 2.5 to 3.5 µm. Sparse macroconidia were 3-septate, slightly curved, with a foot-shaped basal cell and a short apical cell, and measured 19.4 to 38.3 × 2.9 to 4.5 µm. Chlamydospores were terminal and intercalary, single, in pairs, clusters, or in chains, and measured 6.7 to 10.6 µm in diameter. Such characteristics are typical of Fusarium oxysporum (Leslie and Summerell 2006). DNA was extracted from the four isolates and molecular identification was carried out by sequencing the portions of the elongation factor-1 alpha (TEF-1α) gene (O’Donnell et al. 1998) and the intergenic spacer (IGS) region (GenBank accession nos. MK061116–MK061119 for TEF-1α and MK061120–MK061123 for IGS). A BLASTn search showed that the TEF-1α sequences (625 bp) were 99 to 100% similar to the corresponding sequences HG916993–HG916995 of F. oxysporum f. sp. radicis-capsici from sweet pepper (Lomas-Cano et al. 2014), while the IGS sequences (1187–1274 bp) were 99% similar to that of F. oxysporum (FJ985621). The four isolates were used for pathogenicity tests conducted in 2-liter pots filled with steamed peat substrate in a greenhouse at 25 to 28°C. Twenty-day-old sweet pepper plants (cv. Cuneo) at the three true leaf stage were inoculated by dipping roots in a conidial suspension (1 × 10⁶ conidia/ml) obtained from a 10-day-culture in potato dextrose broth. Twenty plants (5 plants/pot) per treatment were used in two repeated trials for each isolate, and 20 noninoculated plants were used as controls. First wilt symptoms developed 8 to 10 days after inoculation, and 70 to 100% of inoculated plants were dead 15 to 20 days after inoculation, showing crown and root rot. Tomato plants (Solanum lycopersicum) cv. Cuore di bue, inoculated with the four isolates under the same conditions, did not develop symptoms after 50 to 60 days. All noninoculated control plants remained healthy. A fungus morphologically identified as F. oxysporum was consistently reisolated from all the symptomatic pepper plants and confirmed by the TEF-1α and the IGS analysis. To our knowledge, this is the first report of F. oxysporum on sweet pepper in Italy. Although F. oxysporum f. sp. capsici is reported on sweet pepper (Pernezny et al. 2003), symptoms observed in this study were very similar to those caused by F. oxysporum f. sp. radicis-capsici described by Lomas-Cano et al. (2014, 2016) on sweet pepper seedlings in Spain. The disease, although limited at present in the greenhouses surveyed in northern Italy, could expand to other areas where sweet pepper is grown intensively.

Analysis of matK, rbcL and trnL-trnF Intergenic Spacer Sequences on Durik-Durik (Syzygium sp)

Syzygium sp. with the local name of durik-durik, that grows in Riau Province, Indonesia, is one of many adapted plants to flooding stress in floodplain ecosystems, and plays a role for survival of the ecosystems. This study reports analysis of three DNA barcodes, i.e. matK, rbcL and trnL-trnF intergenic spacer on this plant. The fresh leaves of this plant were picked up from floodplain ecosystem in Pelalawan, Riau Province for DNA extraction requiring. In this study had been obtained DNA sequences of matK, rbcL and trnL-trnF intergenic spacer with the size of 686 bp, 641 bp and 828 bp, respectively. The BLASTn analysis based on those sequences showed that none of the accessions of the Syzygium genus were 100% similar to Syzygium sp. The phylogenetic tree showed that Syzygium sp. formed one group with the same species of Syzygium and separated from other genera in Myrtaceae family. These results confirmed that durik-durik was a member Syzygium. Those sequences had been deposited in GenBank and could be used for molecular identification of this plant.

Structure of the intergenic spacers in chicken ribosomal DNA

BackgroundRibosomal DNA (rDNA) repeats are situated in the nucleolus organizer regions (NOR) of chromosomes and transcribed into rRNA for ribosome biogenesis. Thus, they are an essential component of eukaryotic genomes. rDNA repeat units consist of rRNA gene clusters that are transcribed into single pre-rRNA molecules, each separated by intergenic spacers (IGS) that contain regulatory elements for rRNA gene cluster transcription. Because of their high repeat content, rDNA sequences are usually absent from genome assemblies. In this work, we used the long-read sequencing technology to describe the chicken IGS and fill the knowledge gap on rDNA sequences of one of the key domesticated animals.MethodsWe used the long-read PacBio RSII technique to sequence the BAC clone WAG137G04 (Wageningen BAC library) known to contain chicken NOR elements and the HGAP workflow software suit to assemble the PacBio RSII reads. Whole-genome sequence contigs homologous to the chicken rDNA repetitive unit were identified based on the Gallus_gallus-5.0 assembly with BLAST. We used the Geneious 9.0.5 and Mega software, maximum likelihood method and Chickspress project for sequence evolution analysis, phylogenetic tree construction and analysis of the raw transcriptome data.ResultsThree complete IGS sequences in the White Leghorn chicken genome and one IGS sequence in the red junglefowl contig AADN04001305.1 (Gallus_gallus-5.0) were detected. They had various lengths and contained three groups of tandem repeats (some of them being very GC rich) that form highly organized arrays. Initiation and termination sites of rDNA transcription were located within small and large unique regions (SUR and LUR), respectively. No functionally significant sites were detected within the tandem repeat sequences.ConclusionsDue to the highly organized GC-rich repeats, the structure of the chicken IGS differs from that of IGS in human, apes, Xenopus or fish rDNA. However, the chicken IGS shares some molecular organization features with that of the turtles, which are other representatives of the Sauropsida clade that includes birds and reptiles. Our current results on the structure of chicken IGS together with the previously reported ribosomal gene cluster sequence provide sufficient data to consider that the complete chicken rDNA sequence is assembled with confidence in terms of molecular DNA organization.

IGS sequences in Cestrum present AT- and GC-rich conserved domains, with strong regulatory potential for 5S rDNA.

The 35S and 5S ribosomal DNA (rDNA) organized in thousands of copies in genomes, have been widely used in numerous comparative cytogenetic studies. Nevertheless, several questions related to the diversity and organization of regulatory motifs in 5S rDNA remain to be addressed. The 5S rDNA unit is composed of a conserved 120bp length coding region and an intergenic spacer (IGS) containing potential regulatory motifs (Poly-T, AT-rich and GC-rich) differing in number, redundancy and position along the IGS. The Cestrum species (Solanaceae) have large genomes (about 10pg/1C) and conserved 2n = 16 karyotypes. Strikingly, these genomes show high diversity of heterochromatin distribution, variability in 35S rDNA loci and the occurrence of B chromosomes. However, the 5S rDNA loci are highly conserved in the proximal region of chromosome 8. Comparison of seventy-one IGS sequences in plants revealed several conserved motifs with potential regulatory function. The AT- and GC-rich domains appeared highly conserved in Cestrum chromosomes. The 5S genic and the GC-rich IGS probe produced FISH signals in both A (pair 8) and B chromosomes. The GC-rich domain presented a strong potential for regulation because it may be associated with CpG islands organization, as well as to hairpin and loop organization. Another interesting aspect was the ability of AT- and GC-rich motifs to produce non-heterochromatic CMA/DAPI signals. While the length of the 5S rDNA IGS region varied in size between the Cestrum species, the individual sequence motifs seem to be conserved suggesting their regulatory function. The most striking feature was the conserved GC-rich domain in Cestrum, which is recognized as a signature trait of the proximal region of chromosome pair 8.

Comparative analysis of the ribosomal DNA repeat unit (rDNA) of Perna viridis (Linnaeus, 1758) and Perna canaliculus (Gmelin, 1791).

Perna viridis and P. canaliculus are economically and ecologically important species of shellfish. In this study, the complete ribosomal DNA (rDNA) unit sequences of these species were determined for the first time. The gene order, 18S rRNA–internal transcribed spacer (ITS) 1–5.8S rRNA–ITS2–28S rRNA–intergenic spacer (IGS), was similar to that observed in other eukaryotes. The lengths of the P. viridis and P. canaliculus rDNA sequences ranged from 8,432 to 8,616 bp and from 7,597 to 7,610 bp, respectively, this variability was mainly attributable to the IGS region. The putative transcription termination site and initiation site were confirmed. Perna viridis and P. canaliculus rDNA contained two (length: 93 and 40 bp) and one (length: 131 bp) repeat motifs, respectively. Individual intra-species differences mainly involved the copy number of repeat units. In P. viridis, three cytosine-guanine (CpG) sites with sizes of 440, 1,075 and 537 bp were found to cover nearly the entire IGS sequence, whereas in P. canaliculus, two CpG islands with sizes of 361 and 484 bp were identified. The phylogenetic trees constructed with maximum likelihood and neighbour-joining methods and based on ITS sequences were identical and included three major clusters. Species of the same genus were easily clustered together.

The ribosomal intergenic spacer (IGS) in the potato and tobacco cyst nematodes, Globodera pallida, G. rostochiensis and G. tabacum

The potato cyst nematodes Globodera pallida and G. rostochiensis (PCN), and tobacco cyst nematode (TCN), G. tabacum, are the most important parasitic nematodes of potato and tobacco worldwide. Ribosomal DNA provides useful molecular data for diagnostics, the study of polymorphisms and for evolutionary research in eukaryotic organisms including nematodes. Here we present data on the structure and organization of a rarely studied part of the intergenic spacer (IGS) region of the PCN and TCN genome of cyst nematodes. This region has shown potential for diagnostic purposes and population studies in other organisms including nematodes. In nematodes, the ribosomal RNA gene cluster comprises three genes: 5.8S, 18S and 28S rRNA, which are separated by spacer regions: the intergenic spacer (IGS), non-transcribed spacer (NTS), externally transcribed spacer (EST) and the internally transcribed spacer (ITS). The intergenic spacer (IGS) region consists of an external transcribed spacer (ETS) and a non-transcribed spacer (NTS) which is located between the 28S of one repeat and the 18S gene of the next repeat within the rRNA genes cluster. In this study, the first flanking portion of the IGS was amplified, cloned and sequenced from PCN and TCN. Primers were then designed to amplify the whole IGS sequence. PCR amplification of IGS from G. tabacum, G. pallida, and G. rostochiensis yielded respectively: a single amplicon of 3 kb, three amplicons sized 2.5, 2.6 and 2.9 kb, and two amplicons sized 2.8 and 2.9 kb. Results showed that Globodera spp. has more than one variant copy of the IGS, with both long and short repetitive DNA elements. An approximately 400 bp long region without any internal repetitive elements, were identified in a position between the two repetitive regions suggesting that there is a 5S gene in the IGS of these species.

Complete mitochondrial genome of the silkworm strain, Chilseongjam Bombyx mori (Lepidoptera: Bombycidae), with a unique larval body marking

Recently, a new silkworm strain with a peculiar larval marking and rare cocoon colour was bred in Korea for educational learning and exhibition. In order to obtain the genetic information of the newly bred strain, Chilseongjam Bombyx mori (Lepidoptera: Bombycidae), its complete mitochondrial genome (mitogenome) was sequenced. The mitogenome is 15,660 bp in length, contains a typical set of genes, and has gene arrangement and composition typical of Lepidoptera. However, the Chilseongjam strain mitogenome is 4–36 bp longer than 19 other strains originating from other countries and 16 bp shorter than the whole genome of a Korean Hukpyobeom strain. In particular, the Chilseongjam strain has an intergenic spacer sequence that is shorter than that of the Hukpyobeom strain at the tRNAHis and ND4 junction as it has fewer microsatellite-like AT repeats. Phylogenetic analyses conducted using a total of 21 silkworm strains originating from nine countries revealed a few subgroups with moderate-to-high nodal support (80–94%). The Korean Chilseongjam strain formed a relatively strong subgroup (85%) with a Japanese strain (J106) instead of the Korean Hukpyobeom strain.

Організація 5S рДНК клена польового (Acer campestre L.)

In recent decades, molecular methods have been widely used to study plant speciation and taxonomy. The 5S rDNA, which belongs to the class of repeated sequences present in the genomes of all eukaryotes, represents a convenient model for studying the patterns of molecular evolution in plants. Each of 5S rDNA repeated units consist of a coding region and an intergenic spacer (IGS). The coding sequence is highly conserved in evolution, whereas the IGS can differ between related species and even between populations of the same species. The molecular organization of the 5S rDNA in representatives of genus Acer is still poorly explored. Accordingly, the aim of the study was to investigate the organization and variability of the 5S rDNA of the European species Acer campestre. Analysis of the obtained sequences showed, that the 5S rDNA clones of A. campestre are identical. The level of IGS sequence similarity between A. сampestre and A. platanoides amounts to 96.1%, whereas the similarity between these species and A. pseudoplatanus is lower, namely – 81,9–82,4%. Therefore, A. campestre and A. platanoides can be considered as closely related species. Only one class of 5S rDNA repeats is present in the genome of A. сampestre. The potential external elements of the RNA polymerase III promoter localized in the IGS differ from those described early for representatives of other families of angiosperms.

A pre-Miocene Irano-Turanian cradle: Origin and diversification of the species-rich monocot genus Gagea (Liliaceae).

The Irano‐Turanian (IT) floristic region is considered an important center of origin for many taxa. However, there is a lack of studies dealing with typical IT genera that also occur in neighboring areas. The species‐rich monocot genus Gagea Salisb. shows a center of diversity in IT region and a distribution in adjacent regions, therefore representing a good study object to investigate spatial and temporal relationships among IT region and its neighboring areas (East Asia, Euro‐Siberia, Himalaya, and Mediterranean). We aimed at (a) testing the origin of the genus and of its major lineages in the IT region, (b) reconstructing divergence times, and (c) reconstructing colonization events. To address these problems, sequences of the ribosomal DNA internal transcribed spacer (ITS) region of 418 individuals and chloroplast intergenic spacers sequences (psbA‐trnH, trnL‐trnF) of 497 individuals, representing 116 species from all sections of the genus and nearly its entire distribution area were analyzed. Divergence times were estimated under a random molecular clock based on nrITS phylogeny, which was the most complete data set regarding the representation of species and distribution areas. Ancestral distribution ranges were estimated for the nrITS data set as well as for a combined data set, revealing that Gagea most likely originated in southwestern Asia. This genus first diversified there starting in the Early Miocene. In the Middle Miocene, Gagea migrated to the Mediterranean and to East Asia, while migration into Euro‐Siberia took place in the Late Miocene. During the Pleistocene, the Arctic was colonized and Gagea serotina, the most widespread species, reached North America. The Mediterranean basin was colonized multiple times from southwestern Asia or Euro‐Siberia. Most of the currently existing species originated during the last 3 Ma.

Molecular organization of 5S rDNA of perennial ryegrass Lolium perenne L.

Aim. Ribosomal DNA (rDNA) is an important tool for molecular taxonomy and represents a convenient model for studying the evolution of repeated sequences. However, for many groups of angiosperms, 5S rDNA remains poorly studied, in particular for such a large group as the Poaceae (Gramineae) family. Accordingly, it was decided to analyze the molecular organization of 5S rDNA in the widespread and economically important species Lolium perenne. Methods. DNA extraction, PCR amplification, cloning and sequencing. Results. Two clones of L. perenne 5S rDNA were sequenced. It was shown that only one variant of 5S rDNA repeats with a 188–189 bp-long intergenic spacer (IGS) is present in the genome of L. perenne. Putative RNA polymerase III promoter elements were detected in the IGS. The level of IGS sequence similarity between representatives of different genera of the tribe Poeae ranges from 46 to 63 %. Conclusions. Comparison of IGS can be used to clarify the phylogenetic relationship between taxa of low ranks, in particular, between species and genera of the tribe Poeae. Keywords: 5S rDNA, intergenic spacer, molecular evolution, Lolium, Poaceae.

Study of mosaicism of Pyropia yezoensis

ABSTRACTCytological observations and sequence analysis of the ribosomal intergenic spacer (IGS) were conducted on conchospores and thalli of Pyropia yezoensis from China. The complete progress of conchospore germination of P. yezoensis was observed, and a tracking study of P. yezoensis mosaics was conducted by microscope, our results showed that the thallus is haploid, with three chromosomes; and the conchocelis is diploid, with six chromosomes. Conchospore germination of P. yezoensis developed in a linear pattern, and cell divisions were not synchronous. The thallus of P. yezoensis was divided into ten parts in our study, IGS was used as a molecular marker to conduct a sequence analysis of different portions of a complete thallus (the reproductive region, nutrient region, and base). P. yezoensis is mosaic of different genetic material based on molecular level differences among the IGS sequences of the different parts of P. yezoensis. The thallus was homozygous; with either two or three mosaics. Chimerism of Pyropia was proven at both the morphological and the molecular level. The results of our study supported the dominant view that meiosis of Pyropia occurs during conchospore germination.

Characterization analysis of the 35S rDNA intergenic spacers in Erianthus arundinaceus

The 35S ribosomal DNA (rDNA) units that occur in tandem repeat are separated by an intergenic spacer (IGS) that plays an important role in rRNA transcription. Moreover, IGS is an important molecular marker for evolutionary research in plants. In the present study, the IGS sequence of Erianthus arundinaceus was isolated and sequenced for the first time. Structure analysis indicated the entire IGS sequence of three typical E. arundinaceus genotypes was highly conserved, with approximately 3087 bp and 67.1% mean GC content. The putative transcription termination, and initiation sites as well as a large number of methylation sites were found to be present in the IGS of E. arundinaceus compared to other plants. The phylogenic tree constructed using the E. arundinaceus IGS sequence showed that Miscanthus sinensis var. glaber was genetically close to Saccharum spp. while E. arundinaceus was close to Imperata cylindrica. Moreover, fluorescent in situ hybridization revealed that IGS and pTa71 probes had the same locus at nucleolar organizer regions. Taken together, this work enhances our current understanding of the organization of IGS in E. arundinaceus and provides a molecular evidence for an evolutionary relationship between Saccharum spp., E. arundinaceus, I. cylindrica and M. sinensis var. glaber.