Comparative Sequence Analysis Research Articles

The Site/Group Extended Data Format and Tools.

Comparative sequence analysis permits unraveling the molecular processes underlying gene evolution. Many statistical methods generate candidate positions within genes, such as fast or slowly evolving sites, coevolving groups of residues, sites undergoing positive selection, or changes in evolutionary rates. Understanding the functional causes of these evolutionary patterns requires combining the results of these analyses and mapping them onto molecular structures, a complex task involving distinct coordinate referential systems. To ease this task, we introduce the site/group extended data format, a simple text format to store (groups of) site annotations. We developed a toolset, the SgedTools, which permits site/group extended data file manipulation, creating them from various software outputs and translating coordinates between individual sequences, alignments, and three-dimensional structures. The package also includes a Monte-Carlo procedure to generate random site samples, possibly conditioning on site-specific features. This eases the statistical testing of evolutionary hypotheses, accounting for the structural properties of the encoded molecules.

Complete chloroplast genome of the Malus baccata var. gracilis provides insights into the evolution and phylogeny of Malus species.

Malus baccata (L.) var. gracilis (Rehd.) has high ornamental value and breeding significance, andcomparative chloroplast genome analysis was applied to facilitate genetic breeding for desired traits andresistance and provide insight into the phylogeny of this genus. Using data from whole-genomesequencing, a tetrameric chloroplast genome with a length of 159,992 bp and a total GC content of36.56% was constructed. The M. baccata var. gracilis chloroplast genome consists of a large single-copy sequence (88,100 bp), a short single-copy region (19,186 bp), and two inverted repeat regions, IRa(26,353 bp) and IRb (26,353 bp). This chloroplast genome contains 112 annotated genes, including 79protein-coding genes (nine multicopy), 29 tRNA genes (eight multicopy), and four rRNA genes (allmulticopy). Calculating the relative synonymous codon usage revealed a total of 32 high-frequencycodons, and the codons exhibited a biased usage pattern towards A/U as the ending nucleotide.Interspecific sequence comparison and boundary analysis revealed significant sequence variation in thevast single-copy region, as well as generally similar expansion and contraction of the SSC and IRregions for 10 analyzed Malus species. M. baccata var. gracilis and Malus hupehensis were groupedtogether into one branch based on phylogenetic analysis of chloroplast genome sequences. Thechloroplast genome of Malus species provides an important foundation for species identification, geneticdiversity analysis, and Malus chloroplast genetic engineering. Additionally, the results can facilitate theuse of pendant traits to improve apple tree shape.

Identification and characterization of integrated prophages and CRISPR-Cas system in Bacillus subtilis RS10 genome.

Bacteriophages have been extensively investigated due to their prominent role in the virulence and resistance of pathogenic bacteria. However, little attention has been given to the non-pathogenic Bacillus phages, and their role in the ecological bacteria genome is overlooked. In the present study, we characterized two Bacillus phages with a linear DNA genome of 33.6kb with 44.83% GC contents and 129.3kb with 34.70% GC contents. A total of 46 and 175 putative coding DNA sequences (CDS) were identified in prophage 1 (P1) and prophage 2 (P2), respectively, with no tRNA genes. Comparative genome sequence analysis revealed that P1 shares eight CDS with phage Jimmer 2 (NC-041976), and phage Osiris (NC-028969), and six with phage phi CT9441A (NC-029022). On the other hand, P2 showed high similarity with Bacill_SPbeta_NC_001884 and Bacillus phage phi 105. Further, genome analysis indicates several horizontal gene transfer events in both phages during the evolution process. In addition, we detected two CRISPR-Cas systems for the first time in B. subtilis. The identified CRISPR system consists of 24 and 25 direct repeats and integrase coding genes, while the cas gene which encodes Cas protein involved in the cleavage of a target sequence is missing. These findings will expand the current knowledge of soil phages as well as help to develop a new perspective for investigating more ecological phages to understand their role in bacterial communities and diversity.

Auxotrophy of <I>Yersinia pestis</I> Strains of Antiqua Biovar and Its Genetic Basis

The aim of the work was to study the growth dependence on amino acids in Yersinia pestis strains of different phylogenetic branches of the antiqua biovar and to determine the genetic basis of auxotrophy of these strains.Materials and methods. We used 38 strains of Y. pestis, the main subspecies of the antiqua biovar, isolated in various foci of the world in the period of 1928–2020. The nutritional requirements of the strains were determined by incubation on Difco minimal agar with different sets of amino acids. Phylogenetic analysis of the strains was carried out using the Wombac 2.0 and SeaView 5.0.5 programs. Comparative analysis of the nucleotide sequences of the genes was performed using the BLAST algorithm and the Mega 7.0 program.Results and discussion. The phylogenetic appurtenance of the investigated Y. pestis strains of antiqua biovar to the phylogenetic branches 0.ANT3, 0.ANT5, 1.ANT, 2.ANT3, 3.ANT2, 4.ANT has been determined. It is established that all studied strains have a common dependence of growth on three amino acids – phenylalanine, threonine, and methionine. In the majority of the strains of antiqua biovar of all phylogenetic branches, growth is also dependent on the presence of cysteine in the medium, except for a part of the strains of the phylogenetic branch 4.ANT. In 14 genes of sulfur and cysteine metabolism, 19 mutations have been identified. Each phylogenetic group of the antique biovar has a distinct profile of mutations in genes involved in cysteine biosynthesis. The leucine requirement of the strains belonging to phylogenetic branch 0.ANT5 has been established, possibly caused by a frame shift in the leuA gene. Strains of the 1.ANT branch isolated in the Democratic Republic of the Congo demonstrate an additional proline-dependent growth. The defined nutritional requirements in the strains and the genetic causes of auxotrophy complement the phenotypic and genetic characteristics of the phylogenetic branches of the antiqua biovar and can be used as genetic markers for the differentiation of these strains.

Suppression of UVB-Induced MMP-1 Expression in Human Skin Fibroblasts Using Lysate of Lactobacillus iners Derived from Korean Women's Skin in Their Twenties.

The process of skin aging is intricate, involving intrinsic aging, influenced by internal factors, and extrinsic aging, mainly caused by exposure to UV radiation, resulting in photoaging. Photoaging manifests as skin issues such as wrinkles and discoloration. The skin microbiome, a diverse community of microorganisms on the skin's surface, plays a crucial role in skin protection and can be affected by factors like humidity and pH. Probiotics, beneficial microorganisms, have been investigated for their potential to enhance skin health by regulating the skin microbiome. This can be accomplished through oral probiotics, impacting the gut-skin axis, or topical applications introducing live bacteria to the skin. Probiotics mitigate oxidative stress, suppress inflammation, and maintain the skin's extracellular matrix, ultimately averting skin aging. However, research on probiotics derived from human skin is limited, and there is no established product for preventing photoaging. The mechanism by which probiotics shield the skin microbiome and skin layers from UV radiation remains unclear. Recently, researchers have discovered Lactobacillus in the skin, with reports indicating a decrease in this microorganism with age. In a recent study, scientists isolated Lactobacillus iners KOLBM20 from the skin of individuals in their twenties and confirmed its effectiveness. A comparative analysis of genetic sequences revealed that strain KOLBM20 belongs to the Lactobacillus genus and closely relates to L. iners DSM13335(T) with a 99.20% similarity. Importantly, Lactobacillus iners KOLBM20 displayed anti-wrinkle properties by inhibiting MMP-1. This investigation demonstrated the inhibitory effect of KOLBM20 strain lysate on MMP-1 expression. Moreover, the data suggest that KOLBM20 strain lysate may prevent UVB-induced MMP-1 expression by inhibiting the activation of the ERK, JNK, and p38 signaling pathways induced by UVB. Consequently, KOLBM20 strain lysate holds promise as a potential therapeutic agent for preventing and treating skin photoaging.

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation

Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.

Characterisation of the mitochondrial genome and phylogenetic analysis of Toxocara apodemi (Nematoda: Ascarididae).

We first sequenced and characterised the complete mitochondrial genome of Toxocara apodeme, then studied the evolutionary relationship of the species within Toxocaridae. The complete mitochondrial genome was amplified using PCR with 14 specific primers. The mitogenome length was 14303 bp in size, including 12 PCGs (encoding 3,423 amino acids), 22 tRNAs, 2 rRNAs, and 2 NCRs, with 68.38% A+T contents. The mt genomes of T. apodemi had relatively compact structures with 11 intergenic spacers and 5 overlaps. Comparative analyses of the nucleotide sequences of complete mt genomes showed that T. apodemi had higher identities with T. canis than other congeners. A sliding window analysis of 12 PCGs among 5 Toxocara species indicated that nad4 had the highest sequence divergence, and cox1 was the least variable gene. Relative synonymous codon usage showed that UUG, ACU, CCU, CGU, and UCU most frequently occurred in the complete genomes of T. apodemi. The Ka/Ks ratio showed that all Toxocara mt genes were subject to purification selection. The largest genetic distance between T. apodemi and the other 4 congeneric species was found in nad2, and the smallest was found in cox2. Phylogenetic analyses based on the concatenated amino acid sequences of 12 PCGs demonstrated that T. apodemi formed a distinct branch and was always a sister taxon to other congeneric species. The present study determined the complete mt genome sequences of T. apodemi, which provide novel genetic markers for further studies of the taxonomy, population genetics, and systematics of the Toxocaridae nematodes.

Superior Induced Pluripotent Stem Cell Generation through Phactr3-Driven Mechanomodulation of Both Early and Late Phases of Cell Reprogramming

Human cell reprogramming traditionally involves time-intensive, multistage, costly tissue culture polystyrene-based cell culture practices that ultimately produce low numbers of reprogrammed cells of variable quality. Previous studies have shown that very soft 2- and 3-dimensional hydrogel substrates/matrices (of stiffnesses ≤ 1 kPa) can drive ~2× improvements in human cell reprogramming outcomes. Unfortunately, these similarly complex multistage protocols lack intrinsic scalability, and, furthermore, the associated underlying molecular mechanisms remain to be fully elucidated, limiting the potential to further maximize reprogramming outcomes. In screening the largest range of polyacrylamide (pAAm) hydrogels of varying stiffness to date (1 kPa to 1.3 MPa), we have found that a medium stiffness gel (~100 kPa) increased the overall number of reprogrammed cells by up to 10-fold (10×), accelerated reprogramming kinetics, improved both early and late phases of reprogramming, and produced induced pluripotent stem cells (iPSCs) having more naïve characteristics and lower remnant transgene expression, compared to the gold standard tissue culture polystyrene practice. Functionalization of these pAAm hydrogels with poly- l -dopamine enabled, for the first-time, continuous, single-step reprogramming of fibroblasts to iPSCs on hydrogel substrates (noting that even the tissue culture polystyrene practice is a 2-stage process). Comparative RNA sequencing analyses coupled with experimental validation revealed that a novel reprogramming regulator, protein phosphatase and actin regulator 3, up-regulated under the gel condition at a very early time point, was responsible for the observed enhanced reprogramming outcomes. This study provides a novel culture protocol and substrate for continuous hydrogel-based cell reprogramming and previously unattained clarity of the underlying mechanisms via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes.

Helminth fauna of the black goby Gobius niger L. (Gobiiformes: Gobiidae) from the Finnish Archipelago, Baltic sea: Molecular and morphological data

Black gobies (Gobius niger) from the Finnish Archipelago, Baltic Sea, were screened for helminth infections in summer 2020. Helminths were identified morphologically and/or molecularly. Altogether 26 novel sequences were generated and analysed using maximum likelihood estimation. Morphological and phylogenetic analyses based on mitochondrial genes revealed the presence of 8 species belonging to the Digenea (Diplostomum mergi Lineage 3), Cestoda (Bothriocephalus scorpii), Nematoda (Contracaecum rudolphii A, Cucullanus sp. and Hysterothylacium aduncum), and Acanthocephala (a putative new species of Corynosoma, Corynosoma semerme and Neoechinorhynchus sp.). Phylogenetic and comparative sequence analyses revealed that the putative new acanthocephalan species is closely related to C. neostrumosum described from the Caspian seal, Pusa caspica, in the Caspian Sea. The black goby represents a new host record for four parasite species (Diplostomum mergi Lineage 3, Contracaecum rudolphii A, Corynosoma semerme and Corynosoma sp.). The Finnish Archipelago is a novel locality record for three species (Corynosoma sp., Diplostomum mergi Lineage 3 and Bothriocephalus scorpii).

Isolation, Screening, Identification, and Assessment of Laccase-Producing Fungi Isolated From Different Environmental Samples

ABSTRACT: One of the extracellular enzymes involved in ligninolytic is lacasse, secreted by white and brown rot fungus. The class of blue copper proteins that includes laccases consists of N-glycosylated multicopper oxidases. Ascomycetes, Deuteromycetes, and Basidiomycetes are all fungi that contain laccase; many of these are white-rot fungi that break down lignin. Due to their wide range of substrate specificity, laccases have been the focus of extensive research over the past few decades. Their latest applications include anything from the textile pulp and paper industries to culinary applications and bioremediation techniques. Laccases are also used in organic synthesis, where phenols and amines are common substrates. Dimers and oligomers are produced due to the coupling of reactive radical intermediates in these reactions. The current investigation gathered 50 white rot fungi, and the most incredible laccase-producing organisms in submerged fermentation were looked into. Ten of the 21 cultures displayed a reddish-brown color zone. Of these ten isolates (PTD 19, PTD 4, PP2J15, LKT 34, ITC 1, NRL 7, GOJ 7, PTD2, PP2J, and PKT12), only PP2J15 and GOJ 7 displayed the most reddish-brown color zone. The isolation of white rot fungus, their molecular characterization, and testing for laccase production are all covered in this Paper. Talaromyces verruculosus and Cladosporium cladosporioides were identified as the PP2J15 and GOJ 7 strains based on sequence comparison and phylogenetic analysis with reference taxa.

Comparative Analysis of Microsatellite Sequences in Six Gamma-coronaviruses

Microsatellite sequences are DNA or RNA repeat sequences with 1-6 bases as repeat units, also known as simple repeat sequences, that appear in the genome's non-coding, coding, and intergenic regions. Microsatellite sequences are extremely changeable and varied, and their presence encourages genomic variety and evolution. Gamma-coronavirus is a genus in the Coronaviridae subfamily. Microsatellites are extensively dispersed in gamma-coronavirus genomes' coding and non-coding regions. There are currently few papers on the microsatellite analysis of γ-coronaviruses. The distribution frequency, GC content, and nucleotide repeat sequences of γ-coronavirus SSR were studied in this work using MISA online program and SSRhunter, and three results were drawn: 1.Viruses have a short evolutionary period and a lower variety of microsatellites than prokaryotes and eukaryotes. 2.Type II microsatellites have a significant role in cross-species infection and gene expression differences. 3.Evolution might be skewed. This research will contribute to a better understanding of the structure, function, and evolutionary importance of microsatellites in tiny genomes.

Comparative molecular profiling of multidrug-resistant Pseudomonas aeruginosa identifies novel mutations in regional clinical isolates from South India.

We sought to analyse the antibiotic susceptibility profiles and molecular epidemiology of MDR clinical Pseudomonas aeruginosa isolates from South India using non-MDR isolates as a reference. We established a comprehensive clinical strain library consisting of 58 isolates collected from patients across the South Indian state of Kerala from March 2017 to July 2019. The strains were subject to antibiotic susceptibility testing, modified carbapenem inactivation method assay for carbapenemase production, PCR sequencing, comparative sequence analysis and quantitative PCR of MDR determinants associated with antibiotic efflux pump systems, fluoroquinolone resistance and carbapenem resistance. We performed in silico modelling of MDR-specific SNPs. Of our collection of South Indian P. aeruginosa clinical isolates, 74.1% were MDR and 55.8% were resistant to the entire panel of antibiotics tested. All MDR isolates were resistant to levofloxacin and 93% were resistant to meropenem. We identified seven distinct, MDR-specific mutations in nalD, three of which are novel. mexA was significantly overexpressed in strains that were resistant to the entire test antibiotic panel while gyrA and gyrB were overexpressed in MDR isolates. Mutations in fluoroquinolone determinants were significantly associated with MDR phenotype and a novel GyrA Y100C substitution was observed. Carbapenem resistance in MDR isolates was associated with loss-of-function mutations in oprD and high prevalence of NDM (blaNDM-1) within our sample. This study provides insight into MDR mechanisms adopted by P. aeruginosa clinical isolates, which may guide the potential development of therapeutic regimens to improve clinical outcomes.

Antagonistic regulation of homeologous uncx.L and uncx.S genes orchestrates myotome and sclerotome differentiation in the evolutionarily divergent vertebral column of Xenopus laevis.

In anurans, the vertebral column diverges widely from that of other tetrapods; yet the molecular mechanisms underlying its morphogenesis remain largely unexplored. In this study, we investigate the role of the homeologous uncx.L and uncx.S genes in the vertebral column morphogenesis of the allotetraploid frog Xenopus laevis. We initiated our study by cloning the uncx orthologous genes in the anuran Xenopus and determining their spatial expression patterns using in situ hybridization. Additionally, we employed gain-of-function and loss-of-function approaches through dexamethasone-inducible uncx constructs and antisense morpholino oligonucleotides, respectively. Comparative analysis of the messenger RNA sequences of homeologous uncx genes revealed that the uncx.L variant lacks the eh1-like repressor domain. Our spatial expression analysis indicated that in the presomitic mesoderm and somites, the transcripts of uncx.L and uncx.S are located in overlapping domains. Alterations in the function of uncx genes significantly impact the development and differentiation of the sclerotome and myotome, resulting in axial skeleton malformations. Our findings suggest a scenario where the homeologous genes uncx.L and uncx.S exhibit antagonistic functions during somitogenesis. Specifically, uncx.S appears to be crucial for sclerotome development and differentiation, while uncx.L primarily influences myotome development. Postallotetraploidization, the uncx.L gene in X. laevis evolved to lose its eh1-like repressor domain, transforming into a "native dominant negative" variant that potentially competes with uncx.S for the same target genes. Finally, the histological analysis revealed that uncx.S expression is necessary for the correct formation of pedicles and neural arch of the vertebrae, and uncx.L is required for trunk muscle development.

Comparative analysis of the complete chloroplast genome sequences of four camellia species

Researching the photosynthetic characteristics based on the whole chloroplast genome sequence of Camellia osmantha cv ‘yidan’ is important for improving production. We sequenced and analyzed the chloroplast (cp) genomes of C. osmantha cv ‘yidan’. The total cp genome length was 156,981 bp. The cp genomes included 134 genes encoding 81 proteins, 39 transfer RNAs, 8 ribosomal RNAs, and 6 genes with unknown functions. In total, 50 repeat sequences were identified in C. osmantha cv ‘yidan’ cp genomes. Phylogenetic analysis showed that C. osmantha cv ‘yidan’ is more closely related to Camellia vietnamensis cv ‘hongguo’ and Camellia oleifera cv ‘cenruan 3’ than to Camellia semiserrata cv ‘hongyu 1’. Our complete assembly of four Camellia cp genomes may contribute to breeding for high oil content plants and further biological discoveries. The results of this study provide a basis for the assembly of the entire chloroplast genome of C. osmantha cv ‘yidan’.

Development of a Gene-Based Marker Set for Orange-Colored Watermelon Flesh with a High β-Carotene Content.

The fruit flesh of watermelons differs depending on the distinct carotenoid composition. Orange-colored flesh relates to the accumulation of β-carotene, which is beneficial to human health. Canary-yellow-fleshed OTO-DAH and orange-β-fleshed (orange-fleshed with high β-carotene) NB-DAH near-isogenic lines (NILs) were used to determine the genetic mechanism attributed to orange watermelon flesh. For genetic mapping, an F2 population was developed by crossing the two NILs. The segregation ratio of flesh color in the F2 population indicated that the orange-β flesh of the NB-DAH NIL was controlled by a single incompletely dominant gene. Through a comparative analysis of the whole-genome sequences of the parent lines and NILs, a major introgression region unique to the NB-DAH NIL was detected on Chr. 1; this was considered a candidate region for harboring genes that distinguish orange from canary-yellow and red flesh. Among the 13 genes involved in the carotenoid metabolic pathway in watermelons, only ClPSY1 (ClCG01G008470), which encodes phytoene synthase 1, was located within the introgression region. The genotyping of F2 plants using a cleaved amplified polymorphic sequence marker developed from a non-synonymous SNP in ClPSY1 revealed its relationship with orange-β flesh. The insights gained in this study can be applied to marker-assisted breeding for this desirable trait.

Genome-wide identification and expression analysis of growth-regulating factors in Dendrobium officinale and Dendrobium chrysotoxum.

Dendrobium, one of the largest genera in Orchidaceae, is popular not only for its aesthetic appeal but for its significant medicinal value. Growth-regulating factors (GRFs) play an essential role in plant growth and development. However, there is still a lack of information about the evolution and biological function analysis of the GRF gene family among Dendrobiumspecies. Growth-regulating factors from Dendrobium officinale Kimura et Migo and Dendrobium chrysotoxum Lindl. were identified by HMMER and BLAST. Detailed bioinformatics analysis was conducted to explore the evolution and function of GRF gene family in D. officinale and D. chrysotoxum using genomic data, transcriptome data and qRT-PCR technology. Here, we evaluated the evolution of the GRF gene family based on the genome sequences of D. officinale and D. chrysotoxum. Inferred from phylogenetic trees, the GRF genes were classified into two clades, and each clade contains three subclades. Sequence comparison analysis revealed relatively conserved gene structures and motifs among members of the same subfamily, indicating a conserved evolution of GRF genes within Dendrobiumspecies. However, considering the distribution of orthologous DoGRFs and DcGRFs, and the differences in the number of GRFs among species, we suggest that the GRF gene family has undergone different evolutionary processes. A total of 361 cis-elements were detected, with 33, 141, and 187 related to plant growth and development, stress, and hormones, respectively. The tissue-specific expression of GRFs showed that DoGRF8 may have a significant function in the stem elongation of D. officinale. Moreover, four genes were up-regulated under Methyl-jasmonic acid/methyl jasmonate (MeJA) treatment, showing that DoGRFs and DcGRFs play a crucial role in stress response. These findings provide valuable information for further investigations into the evolution and function of GRF genes in D. officinale and D. chrysotoxum.

Variability of <i>Puccinia komarovii</i> var. <i>komarovii</i> isolates from <i>Impatiens parviflora</i> DC. and its occurrence in the Drawa and Wigry National Parks in Poland

Small balsam (<i>Impatiens parviflora</i> DC.), an invasive plant native to central Asia, is currently one of the most extensive invasive plants in Central Europe, including Poland. Its expansion may affect populations of native species, such as touch-me-not balsam (Impatiens noli-tangere). It is particularly dangerous in legally protected areas, where the possibilities of eradicating it are very limited. There is some hope for stopping its expansion by using pathogens as a biological weapon. In this case, useful may be host-specific rust fungus <i>Puccinia komarovii</i> var. <i>komarovii</i>. Our three-year observations of I. parviflora infection severity by <i>P. komarovii</i> var. <i>komarovii</i> carried out in Poland in Drawa National Park (DNP) and Wigry National Park (WNP) showed the higher incidence of rust on leaves in DNP, where the median blade infestation for two years was 51% of leaf coverage. In contrast, for three years in WNP, the median blade infestation amounted to only 25%. The most severe symptoms occurred during the summer months. However the percentage of leaf coverage by pathogen exhibited fluctuations depending on the year, implying an influence of environmental conditions that stabilized the infection each season. Molecular analyses, in addition to confirming the infection of the tested plants by <i>P. komarovii</i> var.<i> komarovii</i>, enabled the characterization of its population based on comparative analysis of ITS and LSU sequences. Clear differences were found between the studied isolates and <i>P. komarovii</i> from <i>I. glanduliferae</i> and <i>I. brachycentra</i>. There was no diversity among our isolates, but they differed from the sequences of Polish isolates originating from <i>I. parviflora</i> available in GenBank, which indicates some diversity of rust in a relatively small area. It probably results from sexual reproduction and may enable rapid adaptation of the pathogen to the region or host plant, or it may indicate certain “entry points” to Poland, where the isolates arrived from their native place of occurrence.

Frankia nepalensis sp. nov., a non-infective non-nitrogen-fixing isolate from root nodules of Coriaria nepalensis Wall

Strains CN4T, CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus. The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N2-fixing vesicles and do not fix nitrogen in vitro. The peptidoglycan of strain CN4T contains meso-diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H4) and MK-9(H6) are the predominant menaquinones (>15 %), and iso-C16 : 0 and C17 : 1ω8c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4T is most closely related to Frankia saprophytica CN 3T. An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3T , Frankia asymbiotica M16386T and Frankia inefficax EuI1cT with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4T, CN6, CN7 and CNm7 had dDDH (98.6–99.6 %) and ANI values that grouped them as representing a single species. CN4T has a 10.76 Mb genome. CN4T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4T (=DSM 114740T = LMG 32595T) to a novel species, with CN4T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.

Strand’s mouse <i>Sicista strandi </i> (Rodentia, Dipodoidea) on the southwestern periphery of its range: Genetic and environmental aspects

The position of the Sicista strandi population from the southwestern periphery of its range (the mountainous Caucasus, the vicinity of the Ekiptsoko cordon, Zol’sky district of Kabardino-Balkaria, the adjacent territory with terra typica of the species) in the species structure was investigated on the basis of a comparative analysis of the nucleotide sequences of a fragment of the gene of nu-clear DNA in this population and in representatives of the species from a number of localities from the Russian Plain for the first time. It was established that according to this molecular feature, the studied Caucasian population belongs to the southern grouping of the species, covering, in addition to the Caucasus, samples from the basins of the Lower (Rostov region) and Middle Don (Lugansk region) and presumably from the Lower Volga region, and differs from samples from the northern parts of the range in the Central Chernozem Zone (Kursk and Belgorod region). In the S. strandi population from the southwestern periphery of the range, a number of environmental and biological characteristics (biotopic confinement, population composition, abundance, diurnal, seasonal activity, reproduction, and nutrition) considered in a comparative aspect in connection with the genetic differentiation of the species were studied for the first time.

Cloning, Identification, and Functional Analysis of the Foxl2 Gene in Procambarus clarkii.

Procambarus clarkii is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 (Foxl2) gene has been found to be involved in the reproductive development of many crustaceans. To understand the role of the Foxl2 gene in the gonad development of P. clarkii, we designed CDS-specific primers for the P. clarkii Foxl2 (PcFoxl2) gene and cloned its CDS sequence using RT-PCR. The nucleotide and protein sequence information was then analyzed through bioinformatics analysis. The expression and subcellular localization of PcFoxl2 in various tissues were detected using qRT-PCR and in situ hybridization. The effects of PcFoxl2 knockdown on gonad development were investigated using RNA interference. The results showed that the CDS length of the PcFoxl2 gene was 1614 bp and encoded 537 amino acids. Protein sequence comparison and phylogenetic analysis showed that PcFoxl2 was the closest relative to Crayfish. qRT-PCR analysis indicated that the expression level of PcFoxl2 in the testis was significantly higher (>40 fold) than that in the ovary (p < 0.01). The in situ hybridization results showed that PcFoxl2 was expressed in both the cytoplasm and the nucleus of egg cells, and that the expression was strongest in egg cells at the early stage of yolk synthesis, while weak in the secondary oocytes. The positive signal was strongest in the spermatocyte nucleolus, while only a trace signal was observed in the cytoplasm. After interfering with the PcFoxl2 gene using dsRNA, the expression of PcFoxl2 in the RNA interference group was significantly lower than that in the control group, and this interference effect lasted for one week. Moreover, the gonad index of the experimental group was significantly lower than that of the control group (p < 0.05) after 10 days of P. clarkii cultivation following PcFoxl2 knockdown. The expression levels of the nanos and S3a genes, which are related to gonad development, decreased significantly after PcFoxl2 gene interference. The results suggest that the Foxl2 gene is involved in the growth and development of gonads, particularly in the development of testis, and is related to the early development of oocytes. This study provides a theoretical basis for the artificial breeding of P. clarkii.