Single-copy Sequences Research Articles

Characterization of Two Transposable Elements and an Ultra-Conserved Element Isolated in the Genome of Zootoca vivipara (Squamata, Lacertidae).

Transposable elements (TEs) constitute a considerable fraction of eukaryote genomes representing a major source of genetic variability. We describe two DNA sequences isolated in the lizard Zootoca vivipara, here named Zv516 and Zv817. Both sequences are single-copy nuclear sequences, including a truncation of two transposable elements (TEs), SINE Squam1 in Zv516 and a Tc1/Mariner-like DNA transposon in Zv817. FISH analyses with Zv516 showed the occurrence of interspersed signals of the SINE Squam1 sequence on all chromosomes of Z. vivipara and quantitative dot blot indicated that this TE is present with about 4700 copies in the Z. vivipara genome. FISH and dot blot with Zv817 did not produce clear hybridization signals. Bioinformatic analysis showed the presence of active SINE Squam 1 copies in the genome of different lacertids, in different mRNAs, and intronic and coding regions of various genes. The Tc1/Mariner-like DNA transposon occurs in all reptiles, excluding Sphenodon and Archosauria. Zv817 includes a trait of 284 bp, representing an amniote ultra-conserved element (UCE). Using amniote UCE homologous sequences from available whole genome sequences of major amniote taxonomic groups, we performed a phylogenetic analysis which retrieved Prototheria as the sister group of Metatheria and Eutheria. Within diapsids, Testudines are the sister group to Aves + Crocodylia (Archosauria), and Sphenodon is the sister group to Squamata. Furthermore, large trait regions flanking the UCE are conserved at family level.

The Precise Breakpoint Mapping in Paracentric Inversion 10q22.2q23.3 by Comprehensive Cytogenomic Analysis, Multicolor Banding, and Single-Copy Chromosome Sequencing.

Detection and precise genomic mapping of balanced chromosomal abnormalities in patients with impaired fertility or a clinical phenotype represent a challenge for current cytogenomics owing to difficulties with precise breakpoint localization in the regions enriched for DNA repeats and high genomic variation in such regions. Here, we present a comprehensive cytogenomic approach to breakpoint mapping in a rare paracentric inversion on 10q (in a patient with oligoasthenoteratozoospermia and necrozoospermia) that does not affect other phenotype traits. Multicolor banding, chromosomal microarray analysis, chromosome microdissection with reverse painting, and single-copy sequencing of the rearranged chromosome were performed to determine the length and position of the inverted region as well as to rule out a genetic imbalance at the breakpoints. As a result, a paracentric 19.251 Mbp inversion at 10q22.2q23.3 was described. The most probable location of the breakpoints was predicted using the hg38 assembly. The problems of genetic counseling associated with enrichment for repeats and high DNA variability of usual breakpoint regions were discussed. Possible approaches for cytogenomic assessment of couples with balanced chromosome rearrangements and problems like reproductive failures were considered and suggested as useful part of effective genetic counseling.

Complex host/symbiont integration of a multi-partner symbiotic system in the eusocial aphid Ceratovacuna japonica.

Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid, Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286bp and 853,149bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera-Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.

Morphological and molecular evidence gives insight into the taxonomic position of Peucedanumpubescens (Apiaceae, Selineae).

In this study, morphological and molecular evidences were combined to determine the taxonomic position of Peucedanumpubescens Hand.-Mazz. Morphologically, Peucedanumpubescens is similar to the species of the genus Ligusticopsis in having fibrous remnant sheaths at the stem base, pinnate and linear coexisted bracts, strongly compressed dorsally mericarps, filiform median and lateral ribs, winged marginal ribs, numerous vittae in each furrow and commissure, but can also be easily distinguished from members of Ligusticopsis by its hispid fruit and linear-lanceolate bracteoles. Molecular phylogenetic analyses based on the single-copy protein-coding sequences (CDS) of plastomes and internal transcribed space (ITS) region showed that Peucedanumpubescens nested in the genus Ligusticopsis. As both morphological and molecular data supported the inclusion of Peucedanumpubescens within Ligusticopsis, the species is here transferred and the new combination, Ligusticopsispubescens (Hand.-Mazz.) J.J.Deng, C.K.Liu & X.J.He, made.

Anaeromyxobacter oryzae sp. nov., Anaeromyxobacter diazotrophicus sp. nov. and Anaeromyxobacter paludicola sp. nov., isolated from paddy soils

Three bacterial strains (Red232T, Red267T and Red630T) were isolated from paddy soils sampled in Japan. Cells of these strains were Gram-stain-negative, facultative anaerobic, long rod-shaped with monotrichous flagella or pilus-like structures for motility, and formed red colonies on agar plates. Phylogenetic trees based on 16S rRNA gene and multiple single-copy gene sequences showed that the three strains formed a cluster with the type strains of Anaeromyxobacter species, independent from any other strain genera. Similarity values of the 16S rRNA gene sequences and genomes among the three isolated strains and the type strain of Anaeromyxobacter, Anaeromyxobacter dehalogenans 2CP-1T, were 95.4-97.4% for 16S rRNA gene sequence, 75.3-79.5% for average nucleotide identity, 19.6-21.7% for digital DNA-DNA hybridization and 64.1-72.6% for average amino acid identity, all of which are below the species delineation thresholds. Nitrogenase genes were observed in the genomes of the three novel strains, but not in A. dehalogenans 2CP-1T. Moreover, multiple genomic, physiological and chemotaxonomic features supported the discrimination between these three strains. Based on the evidence in this study, the three isolates represent three novel independent species for which the following names are proposed: Anaeromyxobacter oryzae sp. nov., Anaeromyxobacter diazotrophicus sp. nov. and Anaeromyxobacter paludicola sp. nov. The type strains are Red232T (=NBRC 114074T=MCCC 1K03954T), Red267T (=NBRC 114075T=MCCC 1K04211T), and Red630T (=NBRC 114076T=MCCC 1K03957T), respectively.

Development and applications of a collection of single copy gene-based cytogenetic DNA markers in garden asparagus.

Garden asparagus (Asparagus officinalis, 2n = 2x = 20 chromosomes) is an important dioecious vegetable crop and a model species for studying sex chromosome formation and evolution. However, few molecular cytogenetic studies on garden asparagus have been reported because of its small metaphase chromosomes, the scarcity of distinguished cytogenetic markers, and the high content of repetitive sequences. In this study, a set of single copy genes free of repetitive sequences with sizes ranging from 4.3 kb to 8.2 kb were screened and used as probes for fluorescence in situ hybridization (FISH) to identify individual chromosomes of garden asparagus. The chromosome-specific signal distribution patterns of these probes enabled the distinguishment of each pair of chromosomes. The sequence assembly and cytogenetic map were successfully integrated, and the results confirmed that the chromosome 1 representing the sex chromosome in the genome assembly is chromosome 5 in the karyotype analysis. The cytogenetic identification of the male-specific region of the Y chromosome (MSY) was implemented using a mixed probe derived from a number of MSY-specific single copy sequences. In addition, the chromosome orthologous relationship between garden asparagus (A1–A10, karyotypic analysis) and its hermaphrodite close relative, A. setaceus (B1–B10, karyotypic analysis), was analyzed using this collection of chromosome-specific cytological markers. The results showed that B3 is the ortholog of sex chromosome A5 and thus may represent the ancestral autosome of the current sex chromosome in garden asparagus. Chromosomes B5, B4, B1, B8, B7, and B9 are the orthologs of A2, A3, A4, A7, A8, and A10, respectively. The chromosome identification, cytogenetic recognition of MSY, and the orthologous relationship analysis between garden asparagus and A. setaceus are valuable for the further investigation of the sex chromosome emergence and evolutionary mechanism of garden asparagus and genome structure evolution in the Asparagus genus.

Characterization of the Chloroplast Genome of Argyranthemum frutescens and a Comparison with Other Species in Anthemideae.

Argyranthemum frutescens, which belongs to the Anthemideae (Asteraceae), is widely cultivated as an ornamental plant. In this study, the complete chloroplast genome of A. frutescens was obtained based on the sequences generated by Illumina HiSeq. The chloroplast genome of A. frutescens was 149,626 base pairs (bp) in length, containing a pair of inverted repeats (IR, 24,510 bp) regions separated by a small single-copy (SSC, 18,352 bp) sequence and a large single-copy (LSC, 82,254 bp) sequence. The genome contained 132 genes, consisting of 85 coding DNA sequences, 37 tRNA genes, and 8 rRNA genes, with nineteen genes duplicated in the IR region. A comparison chloroplast genome analysis among ten species from the tribe of Anthemideae revealed that the chloroplast genome size varied, but the genome structure, gene content, and oligonucleotide repeats were highly conserved. Highly divergent regions, e.g., ycf1, trnK-psbK, petN-psbM intronic, were detected. Phylogenetic analysis supported Argyranthemum as a separate genus. The findings of this study will be helpful in the exploration of the phylogenetic relationships of the tribe of Anthemideae and contribute to the breeding improvement of A. frutescens.

Dilemmas in generic delimitation of Senegalia and allies (Caesalpinioideae, mimosoid clade): how to reconcile phylogenomic evidence with morphology and taxonomy?

Senegalia comprises 219 species distributed in tropical and subtropical regions of North and South America, Africa, Asia and Australia. Two sections are currently recognised within Senegalia and these are most readily distinguished by the differences in disposition of their cauline prickles, i.e. sect. Senegalia with prickles at or near leaf nodes and sect. Monacanthea with mostly internodal prickles. Previous phylogenetic studies, based primarily on small numbers of plastid DNA loci, found Senegalia to be monophyletic with two large subclades corresponding to the sections. Here, we present new phylogenomic evidence from 997 single-copy nuclear gene sequences for a small, but representative set of species. These new analyses show that Senegalia is non-monophyletic, but instead, forms a grade that is paraphyletic with respect to the remainder of the ingoid clade (i.e. Ingeae + Acacia s.s. + Acaciella), comprising two well-supported subclades most likely representing the same clades as found in previous phylogenetic studies of the genus and, interspersed between these, a third, moderately supported clade, comprising the genera Mariosousa, Pseudosenegalia and Parasenegalia. In marked contrast to the nuclear phylogeny, the two Senegalia clades are sister groups in the plastid phylogeny, based on analyses of 72 chloroplast genes, rendering the genus monophyletic, based on plastid data alone. We discuss this new evidence that Senegalia is non-monophyletic in relation to the marked cytonuclear discordance, high gene tree conflict and lack of resolution across this senegalioid grade and review the consistency of the key morphological characters distinguishing the two sections of Senegalia. We conclude that it is likely that Senegalia will need to be split into two (or possibly more) genera: a re-circumscribed Senegalia s.s. that corresponds to the existing Senegaliasect.Senegalia plus the S.ataxacantha group (Senegaliasect.Monacanthea s.s.; future studies may show that this group warrants generic status) and a new genus corresponding to the remainder of sect. Monacanthea (here designated as Senegaliasect.Monacanthea p.p.). However, re-delimiting Senegalia now would be premature given that the key morphological characters are not fully congruent with the two sections and pending denser phylogenetic sampling of taxa. A judiciously selected list of critical taxa is presented to facilitate future phylogenomic studies. Finally, we discuss the identity of Albizialeonardii, which is also placed in this senegalioid grade in these new phylogenomic analyses and place it in synonymy with Parasenegaliavogeliana.

Centromere-Specific Single-Copy Sequences of Secale Species.

Single-copy FISH analysis is a useful tool to physically locate a given sequence on chromosome. Centromeric single-copy sequences can be used to locate the position of centromere and disclose the subtle differences among different centromeres. Nine centromeric single-copy sequences 1R1, 3R1, 4R1, 4R2, 5R1, 5R2, 6R2, 6R3, and 7R1 were cloned from Kustro (Secale cereale L.). FISH analysis using these sequences as probes indicated that the signals of 1R1, 3R1, 4R1, 4R2, 5R1, 5R2, 6R1, 6R2, and 7R1 were located in the centromeric regions of rye 1R, 3R, 4R, 4R, 5R, 5R, 6R, 6R, and 7R chromosomes, respectively. In addition, for each of the centromeric single-copy sequences, high sequence similarity was observed among different Secale species. Combined with rye genomic sequence, single-copy FISH analysis indicated that the 1BL.1RS translocations in wheat cultivar CN17 and wheat line 20T363-4 contained the centromeric segment of 1R chromosome from 349,498,361 to 349,501,266 bp, and the 1BL.1RS translocations in the other two wheat cultivars did not contain this segment. The nine sequences are useful in determining the centromere location on rye chromosomes, and they have the potential to disclose the accurate structural differences of centromeres among the wheat-rye centric fusion translocation chromosomes; therefore, more centromeric single-copy sequences are needed.

Hyphomonas sediminis sp. nov., isolated from marine sediment.

A Gram-staining-negative, aerobic and pear-shaped bacterial strain, designated WL0036T, was isolated from coastal sediment sample collected in Nantong city, Jiangsu province of China (120° 51' 13″ E, 32° 6' 26″ N) in October 2020. Strain WL0036T was found to grow at 20-37°C (optimum, 28°C) with 0-9.0% NaCl (optimum, 2.5-4.0%) and displayed alkaliphilic growth with the pH range of pH 6.0-10.0 (optimum, pH 7.0-8.0). The polar lipids profile of strain WL0036T included phosphatidylcholine, phosphatidylethanolamine, glycolipid and an unidentified lipid. The major isoprenoid quinone was determined to be Q-11 and the major fatty acids were C16:0, 11-methyl-C18:1ω7c, and summed features 8 (C18:1ω6c and/or C18:1ω7c). The G + C content of genomic DNA was 61.8%. Phylogenetic trees constructed based on 16S rRNA gene sequence and bac120 gene set (a collection of 120 single-copy protein sequences prevalent in bacteria) indicted that strain WL0036T clustered with strains Hyphomonas neptunium ATCC 15444T and H. polymorpha PS728T. The average nucleotide identities between strain WL0036T and strains H. neptunium ATCC 15444T and H. polymorpha PS728T were 80.7% and 81.2%, respectively. Strain WL0036T showed 22.8% and 23.2% of digital DNA-DNA hybridization identities with H. neptunium ATCC 15444T and H. polymorpha PS728T, respectively. As inferred from the phenotypic and genotypic characteristics and the phylogenetic trees, strain WL0036T ought to be recognized as a novel species in genus Hyphomonas, for which the name Hyphomonas sediminis sp. nov. is proposed. The type strain is WL0036T (= MCCC 1K05843T = JCM 34658T = GDMCC 1.2413T).

Beyond Nuclear Ribosomal DNA Sequences: Evolution, Taxonomy, and Closest Known Saprobic Relatives of Powdery Mildew Fungi (Erysiphaceae) Inferred From Their First Comprehensive Genome-Scale Phylogenetic Analyses.

Powdery mildew fungi (Erysiphaceae), common obligate biotrophic pathogens of many plants, including important agricultural and horticultural crops, represent a monophyletic lineage within the Ascomycota. Within the Erysiphaceae, molecular phylogenetic relationships and DNA-based species and genera delimitations were up to now mostly based on nuclear ribosomal DNA (nrDNA) phylogenies. This is the first comprehensive genome-scale phylogenetic analysis of this group using 751 single-copy orthologous sequences extracted from 24 selected powdery mildew genomes and 14 additional genomes from Helotiales, the fungal order that includes the Erysiphaceae. Representative genomes of all powdery mildew species with publicly available whole-genome sequencing (WGS) data that were of sufficient quality were included in the analyses. The 24 powdery mildew genomes included in the analysis represented 17 species belonging to eight out of 19 genera recognized within the Erysiphaceae. The epiphytic genera, all but one represented by multiple genomes, belonged each to distinct, well-supported lineages. Three hemiendophytic genera, each represented by a single genome, together formed the hemiendophytic lineage. Out of the 14 other taxa from the Helotiales, Arachnopeziza araneosa, a saprobic species, was the only taxon that grouped together with the 24 genome-sequenced powdery mildew fungi in a monophyletic clade. The close phylogenetic relationship between the Erysiphaceae and Arachnopeziza was revealed earlier by a phylogenomic study of the Leotiomycetes. Further analyses of powdery mildew and Arachnopeziza genomes may discover signatures of the evolutionary processes that have led to obligate biotrophy from a saprobic way of life. A separate phylogeny was produced using the 18S, 5.8S, and 28S nrDNA sequences of the same set of powdery mildew specimens and compared to the genome-scale phylogeny. The nrDNA phylogeny was largely congruent to the phylogeny produced using 751 orthologs. This part of the study has revealed multiple contamination and other quality issues in some powdery mildew genomes. We recommend that the presence of 28S, internal transcribed spacer (ITS), and 18S nrDNA sequences in powdery mildew WGS datasets that are identical to those determined by Sanger sequencing should be used to assess the quality of assemblies, in addition to the commonly used Benchmarking Universal Single-Copy Orthologs (BUSCO) values.

A population-level statistic for assessing Mendelian behavior of genotyping-by-sequencing data from highly duplicated genomes

BackgroundGiven the economic and environmental importance of allopolyploids and other species with highly duplicated genomes, there is a need for methods to distinguish paralogs, i.e. duplicate sequences within a genome, from Mendelian loci, i.e. single copy sequences that pair at meiosis. The ratio of observed to expected heterozygosity is an effective tool for filtering loci but requires genotyping to be performed first at a high computational cost, whereas counting the number of sequence tags detected per genotype is computationally quick but very ineffective in inbred or polyploid populations. Therefore, new methods are needed for filtering paralogs.ResultsWe introduce a novel statistic, Hind/HE, that uses the probability that two reads sampled from a genotype will belong to different alleles, instead of observed heterozygosity. The expected value of Hind/HE is the same across all loci in a dataset, regardless of read depth or allele frequency. In contrast to methods based on observed heterozygosity, it can be estimated and used for filtering loci prior to genotype calling. In addition to filtering paralogs, it can be used to filter loci with null alleles or high overdispersion, and identify individuals with unexpected ploidy and hybrid status. We demonstrate that the statistic is useful at read depths as low as five to 10, well below the depth needed for accurate genotype calling in polyploid and outcrossing species.ConclusionsOur methodology for estimating Hind/HE across loci and individuals, as well as determining reasonable thresholds for filtering loci, is implemented in polyRAD v1.6, available at https://github.com/lvclark/polyRAD. In large sequencing datasets, we anticipate that the ability to filter markers and identify problematic individuals prior to genotype calling will save researchers considerable computational time.

Tracing the Evolution of the Angiosperm Genome from the Cytogenetic Point of View

Cytogenetics constitutes a branch of genetics that is focused on the cellular components, especially chromosomes, in relation to heredity and genome structure, function and evolution. The use of modern cytogenetic approaches and the latest microscopes with image acquisition and processing systems enables the simultaneous two- or three-dimensional, multicolour visualisation of both single-copy and highly-repetitive sequences in the plant genome. The data that is gathered using the cytogenetic methods in the phylogenetic background enable tracing the evolution of the plant genome that involve changes in: (i) genome sizes; (ii) chromosome numbers and morphology; (iii) the content of repetitive sequences and (iv) ploidy level. Modern cytogenetic approaches such as FISH using chromosome- and genome-specific probes have been widely used in studies of the evolution of diploids and the consequences of polyploidy. Nowadays, modern cytogenetics complements analyses in other fields of cell biology and constitutes the linkage between genetics, molecular biology and genomics.

Oligo-FISH Paints in Triticeae.

We developed seven oligonucleotide (oligo) pools based on single-copy sequences, targeting chromosomes 1 to 7 of barley (Hordeum vulgare L.) and wheat (Triticum aestivum) for chromosomal Oligo-FISH painting methods. The probes were applied to high-throughput karyotyping for the Triticeae tribe of over 350 species including 30 genera such as Triticum, Hordeum, Secale, Aegilops, Thinopyrum, and Dasypyrum, as well as several wheat alien-derived lines. In combination with other nondenaturing FISH (ND-FISH) procedures using tandem-repeat oligos, the newly developed Oligo-FISH painting technique provides an efficient tool for the identification of individual chromosomes with homologous linkage groups to establish standard karyotypes, particularly with any wild Triticeae species having nonsequenced genomes for chromosome evolutionary analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Oligo-pool probe development Basic Protocol 2: Nondenaturing FISH Basic Protocol 3: Oligo-FISH painting.

Distribution, Phylogeny and Evolution of Clinical and Environmental Vibrio vulnificus Antibiotic-Resistant Genes.

Vibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the evolution mechanism of antibiotic-resistant genes (ARGs) is still unclear. Twenty-two high-quality complete genomes of V. vulnificus were obtained and grouped into 16 clinical isolates and 6 environmental isolates. Genomic annotations found 23 ARG orthologous genes, among which 14 ARGs were shared by V. vulnificus and other Vibrio members. Furthermore, those ARGs were located in their chromosomes, rather than in the plasmids. Phylogenomic reconstruction based on single-copy orthologous protein sequences and ARG protein sequences revealed that clinical and environmental V. vulnificus isolates were in a scattered distribution. The calculation of non-synonymous and synonymous substitutions indicated that most of ARGs evolved under purifying selection with the Ka/Ks ratios lower than one, while h-ns, rsmA, and soxR in several clinical isolates evolved under the positive selection with Ka/Ks ratios >1. Our result indicated that V. vulnificus antibiotic-resistant armory was not only confined to clinical isolates, but to environmental ones as well and clinical isolates inclined to accumulate beneficial non-synonymous substitutions that could be retained to improve competitiveness.

Differentially accessible, single copy sequences form contiguous domains along metaphase chromosomes that are conserved among multiple tissues

BackgroundDuring mitosis, chromatin engages in a dynamic cycle of condensation and decondensation. Condensation into distinct units to ensure high fidelity segregation is followed by rapid and reproducible decondensation to produce functional daughter cells. Factors contributing to the reproducibility of chromatin structure between cell generations are not well understood. We investigated local metaphase chromosome condensation along mitotic chromosomes within genomic intervals showing differential accessibility (DA) between homologs. DA was originally identified using short sequence-defined single copy (sc) DNA probes of < 5 kb in length by fluorescence in situ hybridization (scFISH) in peripheral lymphocytes. These structural differences between metaphase homologs are non-random, stable, and heritable epigenetic marks which have led to the proposed function of DA as a marker of chromatin memory. Here, we characterize the organization of DA intervals into chromosomal domains by identifying multiple DA loci in close proximity to each other and examine the conservation of DA between tissues.ResultsWe evaluated multiple adjacent scFISH probes at 6 different DA loci from chromosomal regions 2p23, 3p24, 12p12, 15q22, 15q24 and 20q13 within peripheral blood T-lymphocytes. DA was organized within domains that extend beyond the defined boundaries of individual scFISH probes. Based on hybridizations of 2 to 4 scFISH probes per domain, domains ranged in length from 16.0 kb to 129.6 kb. Transcriptionally inert chromosomal DA regions in T-lymphocytes also demonstrated conservation of DA in bone marrow and fibroblast cells.ConclusionsWe identified novel chromosomal regions with allelic differences in metaphase chromosome accessibility and demonstrated that these accessibility differences appear to be aggregated into contiguous domains extending beyond individual scFISH probes. These domains are encompassed by previously established topologically associated domain (TAD) boundaries. DA appears to be a conserved feature of human metaphase chromosomes across different stages of lymphocyte differentiation and germ cell origin, consistent with its proposed role in maintenance of intergenerational cellular chromosome memory.

Identification of Pectobacterium versatile Causing Blackleg of Potato in New York State.

Soft rot bacteria classified in the Pectobacteriaceae (SRP), including Pectobacterium and Dickeya spp., are responsible for soft rot and blackleg diseases of potato. Since 2014, blackleg outbreaks caused by D. dianthicola have increased in the United States and Canada. Our previous study found that the most abundant causal organisms of blackleg disease in New York State were P. parmentieri and D. dianthicola, with the latter being the only Dickeya species reported. In the present study, we identified and characterized pathogenic SRP bacteria from 19 potato samples collected in New York State during the 2017 growing season. We used genome sequence comparison to determine the pathogens' species. We found eight P. versatile, one P. atrosepticum, two P. carotovorum, two P. parmentieri, and six D. dianthicola isolates in our 2017 SRP collection. This is the first time that P. versatile has been reported to cause potato blackleg disease in New York State. We determined the phylogenetic relationships between the SRP strains by using 151 single-copy orthologous gene sequences shared among the set of bacteria in our analysis, which provided better resolution than phylogenies constructed with the dnaX gene.

MOTUs: Profiling Taxonomic Composition, Transcriptional Activity and Strain Populations of Microbial Communities.

The mOTU profiler, or mOTUs for short, is a software tool that enables the profiling of microbial communities in terms of their taxonomic composition, relative abundance of metabolically active members, and diversity of strain populations. To this end, it maintains a database of single-copy phylogenetic marker gene sequences, which are used as a reference to which short read metagenomic and metatranscriptomic reads are mapped for the identification and quantification of microbial taxa. Here, we describe the most common use cases of the mOTU profiler in two basic protocols. Additional supporting protocols provide information on its installation and in-depth guidance on adjusting its settings for increasing or decreasing the stringency with which taxa are detected and quantified, as well as for customizing the output file format. Guidelines for understanding the profiling results are provided, along with additional information on unique features, methodological details, and the development history of the tool. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Metagenomic and metatranscriptomic mOTU profiling Basic Protocol 2: Metagenomic SNV profiling Support Protocol 1: Installing mOTUs Support Protocol 2: Profiling pipeline-step by step Support Protocol 3: The mOTUs profiling routine using advanced parameters Support Protocol 4: Metagenomic SNV calling: advanced parameters.

Rhodococcus spelaei sp. nov., isolated from a cave, and proposals that Rhodococcus biphenylivorans is a later synonym of Rhodococcus pyridinivorans, Rhodococcus qingshengii and Rhodococcus baikonurensis are later synonyms of Rhodococcus erythropolis, and Rhodococcus percolatus and Rhodococcus imtechensis are later synonyms of Rhodococcus opacus

Two novel actinobacterial strains, designated C9-5T and C3-43, were isolated from soil samples of a cave in Jeju Island, Republic of Korea, and subjected to taxonomic study by a polyphasic approach. The organisms exhibited a typical rod-coccus developmental cycle during growth and grew at 10-30 °C, pH 5-9 and 0-3 % (w/v) NaCl. In 92 single-copy core gene sequence analysis, strain C9-5T was loosely associated with Rhodococcus tukisamuensis, albeit sharing low 16S rRNA gene sequence similarity (97.4 %). A combination of morphological and chemotaxonomic characteristics supported assignment with the genus Rhodococcus. With respect to 16S rRNA gene sequence similarity, the novel isolates showed the highest identity to the type strain of Rhodococcus subtropicus (98.7 % sequence similarity), followed by Rhodococcus olei (98.5 %) and Rhodococcus pedocola (98.4 %).The average nucleotide identity and digital DNA-DNA hybridization values between strain C9-5T and members of the genus Rhodococcus were ≤81.5 and ≤37.1 %, respectively. A set of physiological and chemotaxonomic properties together with overall genomic relatedness differentiated the novel isolates from members of the genus Rhodococcus, for which the name Rhodococcus spelaei sp. nov. is proposed. The type strain is C9-5T (=KACC 19822T=DSM 107558T). Based on genome analysis performed here, it is also proposed that Rhodococcus biphenylivorans Su et al. 2015 is a later heterotypic synonym of Rhodococcus pyridinivorans Yoon et al. 2000, Rhodococcus qingshengii Xu et al. 2007 and Rhodococcus baikonurensis Li et al. 2004 are later heterotypic synonyms of Rhodococcus erythropolis (Gray and Thornton 1928) Goodfellow and Alderson 1979 (Approved Lists 1980), and Rhodococcus percolatus Briglia et al. 1996 and Rhodococcus imtechensis Ghosh et al. 2006 are later heterotypic synonyms of Rhodococcus opacus Klatte et al. 1995.

Species Tree Inference Methods Intended to Deal with Incomplete Lineage Sorting Are Robust to the Presence of Paralogs.

Many recent phylogenetic methods have focused on accurately inferring species trees when there is gene tree discordance due to incomplete lineage sorting (ILS). For almost all of these methods, and for phylogenetic methods in general, the data for each locus are assumed to consist of orthologous, single-copy sequences. Loci that are present in more than a single copy in any of the studied genomes are excluded from the data. These steps greatly reduce the number of loci available for analysis. The question we seek to answer in this study is: what happens if one runs such species tree inference methods on data where paralogy is present, in addition to or without ILS being present? Through simulation studies and analyses of two large biological data sets, we show that running such methods on data with paralogs can still provide accurate results. We use multiple different methods, some of which are based directly on the multispecies coalescent model, and some of which have been proven to be statistically consistent under it. We also treat the paralogous loci in multiple ways: from explicitly denoting them as paralogs, to randomly selecting one copy per species. In all cases, the inferred species trees are as accurate as equivalent analyses using single-copy orthologs. Our results have significant implications for the use of ILS-aware phylogenomic analyses, demonstrating that they do not have to be restricted to single-copy loci. This will greatly increase the amount of data that can be used for phylogenetic inference.[Gene duplication and loss; incomplete lineage sorting; multispecies coalescent; orthology; paralogy.]