Isolation and characterization of lectin from the surface of <Emphasis Type="Italic">Grifola frondosa</Emphasis> (Fr.) S.F.Gray mycelium

From the surface of the dikaryotic mycelium of the xylotrophic basidiomycete Grifola frondosa 0917 a lectin has been isolated with a molecular mass of 68 ± 1 kDa, consisting of two subunits of 33–34 kDa each. The lectin is a hydrophilic glycoprotein with the protein: glycan ratio of 3: 1. It exhibits high affinity to native rabbit erythrocytes and to human erythrocytes of the 0 blood group, but not to trypsin-treated ones. The hemagglutination (HA) caused by lectin was not blocked by any of the 25 tested mono-, di-, and amino sugars; it was also not blocked by some of glyco derivatives. Only 13.9 μg/ml of the homogeneous preparation of a polysaccharide, a linear D-rhamnan with the structure of the repeated component →2)-β-D-Rhap-(1→3)-α-D-Rhap-(1→3)-α-D-Rhap-(1→2)-α-D-Rhap-(1→2)-α-sD-Rhap-1(→ blocked hemagglutination completely. The analysis of the amino acid composition of the lectin showed the greatest percentage of amino acids with positively charged R groups, arginine, lysine, and histidine, as well as the complete absence of sulfurcontaining amino acids, cysteine, and methionine. D-glucose and D-glucosamine were detected in the carbohydrate part. Original Russian Text ? L.V. Stepanova, V.E. Nikitina, A.S. Boiko, 2007, published in Mikrobiologiya, 2007, Vol. 76, No. 4, pp. 488–493.     

<Emphasis Type="Italic">Malassezia</Emphasis> spp. in Acoustic Meatus of Bats (<Emphasis Type="Italic">Molossus molossus</Emphasis>) of the Amazon Region,Brazil

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, “Rondônia”, Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N = 24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.     

High-throughput sequencing of <Emphasis Type="Italic">Prunus ferganensis</Emphasis> indicates that it is a geographical population of <Emphasis Type="Italic">P. persica</Emphasis>

Peach belongs to the genus Prunus, which includes Prunus persica and its relative species, P. mira, P. davidiana, P. kansuensis, and P. ferganensis. Of these, P. ferganensis have been classified as a species, subspecies, or geographical population of P. persica. To explore the genetic difference between P. ferganensis and P. persica, high-throughput sequencing was used in different peach accessions belonging to different species. First, low-depth sequencing data of peach accessions belonging to four categories revealed that similarity between P. ferganensis and P. persica was similar to that between P. persica accessions from different geographical populations. Then, to further detect the genomic variation in P. ferganensis, the P. ferganensis accession “Xinjiang Pan Tao 1” and the P. persica accession “Xia Miao 1” were sequenced with high depth, and sequence reads were assembled. The results showed that the collinearity of “Xinjiang Pan Tao 1” with the reference genome “Lovell” was higher than that of “Xia Miao 1” and “Lovell” peach. Additionally, the number of genetic variants, including single nucleotide polymorphisms (SNPs), structural variations (SVs), and the specific genes annotated from unmapped sequence in “Xia Miao 1” was higher than that in “Xinjiang Pan Tao 1” peach. The data showed that there was a close distance between “Xinjiang Pan Tao 1” (P. ferganensis) and reference genome which belong to P. persica, comparing “Xia Miao 1” (P. persica) and reference ones. The results accompany with phylogenetic tree and structure analysis confirmed that P. ferganensis should be considered as a geographic population of P. persica rather than a subspecies or a distinct species. Furthermore, gene ontology analysis was performed using the gene comprising large-effect variation to understand the phenotypic difference between two accessions. The result revealed that the pathways of gene function affected by SVs but SNPs and insertion-deletions markedly differed between the two peach accessions.     

Single-strand conformational polymorphism markers associated with a major QTL for fusarium head blight resistance in wheat

A major quantitative trait locus (QTL) associated with resistance to Fusarium head blight (FHB) was identified on chromosome 3BS between simple sequence repeat (SSR) markers Xgwm389 and Xgwm493 in wheat “Ning 7840”, a derivative from “Sumai 3”. However, the marker density of SSR in the QTL region was much lower than that required for marker-assisted selection (MAS) and map-based cloning. The objective of this study was to exploit new markers to increase marker density in this QTL region by using single-strand conformational polymorphism (SSCP) markers developed from wheat-expressed sequence tags (ESTs) on 3BS bin 8-0.78-1.0. Sixty-nine out of 85 SSCP primer pairs amplified PCR (polymerase chain reaction) products from the genomic DNA of “Chinese Spring”. Thirty-four primer pairs amplified PCR products that could form clear ssDNA (single strand DNA) bands through denaturation treatment. Ten SSCP markers had polymorphisms between Ning 7840 and “Clark”. Five of the ten polymorphic SSCP markers were located on chromosome 3B by nullitetrasomic analysis. Three SSCP markers (Xsscp6, Xsscp20, and Xsscp21) were mapped into the region between Xgwm493 and Xgwm533 and possessed a higher coefficient of determination (R²) than Xgwm493 and Xgwm533. The SSCP markers, Xsscp6, Xsscp20, and Xsscp21, can be used for map-based cloning of the QTL and for marker-assisted selection in FHB resistance breeding.     

Phylogenetic relationships of sculpin <Emphasis Type="Italic">Cottus volki</Emphasis> Taranetz, 1933 (Scorpaeniformes,Cottidae) according to the results of analysis of control region in mitochondrial DNA

Sequencing of the fragment of control region in mitochondrial DNA in sculpin Cottus volki and the comparison of obtained data with homologous nucleotide sequences in the other species from genus Cottus demonstrated that C. volki occupies the basal position in the “poecilopus” group, which includes also typical sculpin C. poecilopus Heckel, 1837 from water bodies of Europe, sculpin C. szanaga Dybowski, 1869 from the Amur River basin, and sculpin Cottus cf. poecilopus from the Lena River basin. Early separation of C. volki line from common trunk of “poecilopus” group explains the presence in C. volki of several primitive (plesiomorphic for this group) morphological characters: long interior ray of ventral fin, practically complete lateral body line, and well-developed dentition of palatine bones.     

Genome-wide identification and expression analysis of the molecular chaperone binding protein <Emphasis Type="Italic">BiP</Emphasis> genes in <Emphasis Type="Italic">Citrus</Emphasis>

Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.     

Genetic structure of diploid-polyploid complex of spined loach genus <Emphasis Type="Italic">Cobitis</Emphasis> (<Emphasis Type="Italic">Cypriniformes,Cobitidae</Emphasis>) in the Lower Danube

The extremely high diversity of spined loach biotypes in the Lower Danube has been detected by biochemical genetic investigation and cytometric analysis of 358 specimens collected in the riverbed and shallow channels. Along with two diploid species (C. elongatoides and C. “tanaitica”), six hybrid forms were revealed, namely, diploid C. elongatoides-“tanaitica”; triploid C. 2 elongatoides-“tanaitica,” C. elongatoides-2 “tanaitica,” and C. 2 elongatoides-species-1; and tetraploid C. 3 elongatoides-“tanaitica” and C. elongatoides-species-2-2 “tanaitica.” In addition, specimens with recombinant genotypes were also found. In spite of the apomictic mode of reproduction, the polyploids did not possess clonal structure, but according to the level of polymorphism and the genotype distribution, they were isomorphous to parental diploid species. Thus, in contrast to the polyploidy in Cobitids of the Dnieper, which have appeared in the basin due to the expansion, the polyploids of the Lower Danube are autochthonous and were derived by crossing with local diploid species. The process is apparently proceeds without any limitations.     

Molecular genetic characterization of the yeast <Emphasis Type="Italic">Lachancea kluyveri</Emphasis>

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the α-galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79–100%) of their MEL genes. The phylogenetic analysis of all of the known α-galactosidases in the “Saccharomyces” clade showed that the MEL genes of the yeasts L. kluyveri, L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Inheritance and gene mapping of the white flower trait in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Despite being a unique marker trait, white flower inheritance in Brassica juncea remains poorly understood at the gene level. In this study, we investigated a B. juncea landrace with white petal in China. The white petal phenotype possessed defective chromoplasts with less plastoglobuli than the yellow petal phenotype. Genetic analysis confirmed that two independent recessive genes (Bjpc1 and Bjpc2) controlled the white flower trait. We then mapped the BjPC1 gene in a BC₄ population comprising 2295 individuals. We identified seven AFLP (amplified fragment length polymorphism) markers closely linked to the white flower gene. BLAST search revealed the sequence of AFLP fragments were highly homologous with the Scaffold000085 and Scaffold000031 sequences on the A02 chromosome in the Brassica rapa genome. Based on this sequence homology, we developed simple sequence repeat (SSR) primer pairs and identified 13 SSRs linked to the BjPC1 gene, including two that were co-segregated (SSR9 and SSR10). The two closest markers (SSR4 and SSR11) were respectively 0.9 and 0.4 cM on either side of BjPC1. BLAST analysis revealed that these marker sequences corresponded highly to A02 in B. juncea. They were mapped within a 33 kb genomic region on B. rapa A02 (corresponds to a 40 kb genomic region on B. juncea A02) that included three genes. Sequence BjuA008406, homologous to AtPES2 in Arabidopsis thaliana and Bra032956 in B. rapa, was the most likely candidate for BjPC1. These results should accelerate BjPC1 cloning and facilitate our understanding of the molecular mechanisms controlling B. juncea petal color.     

Development and validation of genomic simple sequence repeat markers in <Emphasis Type="Italic">Erianthus arundinaceus</Emphasis>

Erianthus arundinaceus, a member of the Saccharum complex, is of interest as a potential resource for sugarcane improvement and as a bioenergy crop. Genetic analyses of germplasm collections of E. arundinaceus are being used increasingly. To expand the genomic resources in E. arundinaceus, we aimed at developing simple sequence repeat markers. Using pyrosequencing on the 454 GS FLX system, we sequenced genomic DNA from “JW630” collected in Japan. A total of 1682 candidate loci were used to design the primers, and 1234 primer pairs amplified fragments of the expected size in the primer screening with three wild E. arundinaceus accessions (JW630, “JW4,” and “IJ76-349”). The efficiency of genotyping was validated with a subset of 174 primer pairs and 8 E. arundinaceus accessions. Of these primer pairs, 171 amplified fragments in all accessions tested and 162 detected polymorphic loci. The average values of genetic parameters were estimated as 0.30 (range, 0.09–0.49) for polymorphic information content, 1.65 (0.00–5.87) for marker index, and 2.78 (0.00–8.75) for resolving power. Using these parameters, we selected 61 primer pairs with large discriminatory power for the analyzed loci. Of the 174 primer pairs, 45 (25.9%) were also applicable to Saccharum and 33 (19.0%) to Miscanthus species. These markers would provide a valuable tool for estimating genetic diversity and constructing linkage maps in E. arundinaceus, which would be useful for genetic study and breeding.     

Mutual effects of several fouling organisms of the White Sea (<Emphasis Type="Italic">Mytilus edulis,Styela rustica</Emphasis>, and <Emphasis Type="Italic">Hiatella arctica</Emphasis>) on their growth rate and survival

The mutual effects of several fouling species (the bivalves Mytilus edulis and Hiatella arctica and a solitary ascidian Styela rustica) on their growth rate and mortality were studied through field experiments. The interactions between S. rustica and H. arctica appeared to be the least antagonistic. In contrast, the mussel was the most “aggressive” species with regard to both competitors. It was observed that the ascidians died, because they were intensively braided and gummed up with the byssus threads of the mussels. However, in some cases the intraspecific competition was stronger than the interspecific one.     

Ecophysiology and polymorphism of the unicellular extremely natronophilic cyanobacterium <Emphasis Type="Italic">Euhalothece</Emphasis> sp. Z-M001 from Lake Magadi

Strain Z-M001 of a unicellular cyanobacterium, assigned by analysis of the 16S rRNA gene sequence to the phylogenetic group of the generic level Euhalothece, was isolated from soda Lake Magadi. It was shown that strain Z-M001, unlike all other known cultured and uncultured organisms of the Euhalothece group, is extremely natronophilic, and it was named accordingly “Euhalothece natronophila”. In its ecophysiological characteristics, it is comparable to extremely alkaliphilic organotrophic natronobacteria, which is essential for soda ecosystems, because cyanobacteria belong to primary producers. E. natronophila exhibits considerable morphological variability depending on the concentration of carbonates in the medium. The polymorphism of “ E. natronophila” is primarily connected to limitation by utilizable forms of carbon.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.     

Differentiation of petroleum hydrocarbon-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. based on PCR-RFLP of the 16S-23S rDNA intergenic spacer region

Genetic diversity among 43 petroleum hydrocarbon-degrading Pseudomonas belonging to four different species and the type strain Pseudomonas aeruginosa MTCC1034 was assessed by using restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified 16S–23S rDNA intergenic spacer regions (ISRs) polymorphism. PCR amplification from all Pseudomonas species yielded almost identical ISR amplicons of “?” 800 bp and in nested PCR of “?” 550 bp. The RFLP analysis with MboI and AluI revealed considerable intraspecific variations within the Pseudomonas species. The dendrogram constructed on the basis of the PCR-RFLP patterns of 16S–23S rDNA intergenic spacer regions differentiated all the species into seven different clusters.     

Surface Plasmon Resonance Immunosensor for the Detection of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis>

Environmental surveillance of the Gram-negative bacterium Burkholderia pseudomallei is important in order to define human populations at risk of acquiring the infection; hence, in this study, we developed a method for the detection of B. pseudomallei based on surface plasmon resonance (SPR) using 4-mercaptobenzoic acid (4-MBA) modified gold SPR chip by monitoring the interaction of rpGroEL antigen (rpGroEL Ag) with immobilized rabbit antibody (anti-rpGroEL rAb). Affinity constant (K _D ) and maximum binding capacity of analyte (B _max) values for the interaction of rpGroEL Ag with the immobilized anti-rpGroEL rAb were calculated by using kinetic evaluation software and found to be 14.7 7 pM and 105.40 m^o, respectively. In addition, thermodynamic parameters such as ?G (Gibb’s free energy change), ?H (change in the enthalpy), and ?S (change in the entropy) were determined for the interaction between rpGroEL Ag and immobilized anti-rpGroEL rAb, and the values revealed that the interaction is spontaneous, exothermic, and driven by entropy.     

<Emphasis Type="Italic">BcMF11</Emphasis> and its homologous sequences may form a lncRNA family in <Emphasis Type="Italic">Brassica</Emphasis> diploids

BcMF11 is a long non-coding RNA that has been identified in Brassica rapa and shown to be involved in pollen development. Here, when re-cloned the gene sequence, multiple paralogous copies of BcMF11 were identified in B. rapa (A genome). Multiple paralogous copies of BcMF11 were also found in B. nigra (B genome) and Brassica oleracea (C genome), the other two primary diploids of Brassica U triangle. While in the early diverging Brassicaceae lineage including Arabidopsis thaliana, no BcMF11 homolog was found. Phylogenetic analysis showed that the BcMF11 homologous sequences cloned from A genome or C genome could be clustered into a separate branch, respectively. However, there was no distinct cluster defined for BcMF11 homologous sequences cloned from B genome. The expression of BcMF11 in B. rapa was investigated and revealed a different result in the previous study. In addition, 12 expressed sequence tags from B. napus and B. rapa showing high similarities with BcMF11 were identified in the NCBI database, which further verified that rather than the useless repeat fragments in the genome, the BcMF11 homologous genes could transcribe. It is possible that BcMF11 and its homologous sequences may form a large gene family which might be originated in the recent ancestral lineage of Brassica.